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WO2023139248A1 - Inhibitors of acyl protein thioesterases against microbial infections - Google Patents

Inhibitors of acyl protein thioesterases against microbial infections Download PDF

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Publication number
WO2023139248A1
WO2023139248A1 PCT/EP2023/051499 EP2023051499W WO2023139248A1 WO 2023139248 A1 WO2023139248 A1 WO 2023139248A1 EP 2023051499 W EP2023051499 W EP 2023051499W WO 2023139248 A1 WO2023139248 A1 WO 2023139248A1
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Prior art keywords
cells
apt
inhibitor
cov
formula
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PCT/EP2023/051499
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French (fr)
Inventor
Gisou VAN DER GOOT
Francisco MESQUITA
Laurence ABRAMI
Caroline TAPPAREL VU
Valeria CAGNO
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Ecole Polytechnique Federale De Lausanne (Epfl)
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Publication of WO2023139248A1 publication Critical patent/WO2023139248A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the present invention relates to inhibitors of acyl protein thioesterases (APT) and method of preparation thereof. It also relates to the use of inhibitors of APT in the treatment and/or prevention of a microbial infection such as viral or bacterial infections or coinfection by virus and bacteria.
  • APT acyl protein thioesterases
  • S-acylation a reversible post- translational lipidation
  • S-acylation consists of the addition of an acyl chain, often palmitate, to cytosolic cysteines in target proteins via a thioester bond. This modification is reversible and therefore acts as a functional switch on target proteins (Zaballa & van der Goot, 2018). S-acylation and deacylation are crucial for membrane trafficking events and impact the cellular distribution, abundance, and function of proteins. During infection, S-acylation modifies numerous viral proteins from clinically relevant viruses (Veit, 2012), as well as multiple host receptors fundamental for internalization, and therefore virulence, of numerous pathogens and pathogenic molecules.
  • Pathogens are highly opportunistic organisms. Viruses often exploit S-acylation of viral proteins or host products to promote pathogenesis.
  • CoVs 3-Coronaviruses (CoVs) have enormous zoonotic potential being responsible for various outbreaks of severe acute respiratory syndrome (SARS) in recent years including the pandemic Coronavirus disease-2019 (COVID-19). CoVs depend on S-acylation to produce fully infectious viruses (Mesquita et al, 2021 ). SARS-CoV-2, the etiological agent of COVID 19, contains several essential pathogenic molecules that are modified by S-acylation.
  • Vaccines, antibodies and pathogen-directed antibiotics or antivirals are effective therapeutic strategies that work by targeting the specific infectious agent(s) they were designed for.
  • Host-directed anti-microbial drugs in the other hand, have the potential to broadly target infectious agents with a lower risk of rapid resistance development (Fernandez-Oliva et al, 2019; Kaufmann et al, 2018).
  • the present invention provides an inhibitor of acyl protein thioesterase (APT) of or a pharmaceutically acceptable salt thereof, wherein Ring A is a thiophene ring or a furane ring;
  • APT acyl protein thioesterase
  • Each RAm is independently selected from the group comprising hydrogen, halogen, Ci- C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups; and m is an integer from 0 to 2;
  • Each R1 , R2, R4, R5 is independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, optionally substituted Ci-Ce alkyl acyl, optionally substituted Ci-Ce alkyl aminocarbonyl, optionally substituted carbonyl, optionally substituted Ci-Ce alkyl carbonyl, optionally substituted Ce-C aryl, ether, C2-C10 alkenyl, C6-C10 heteroaryl, C3- C10 cycloalkyl, C3-C10 heterocycloalkyl, Ci-Ce alkoxy, optionally substituted amino, and/or Ci-Ce alkyl amino; and
  • R3 is selected from the group comprising
  • the invention also provides said inhibitors of APT of formula (II), or pharmaceutically acceptable salts thereof, for use as a medicament.
  • the invention provides said inhibitors of APT of formula (II), or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of said inhibitors of APT, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the invention also provides a kit comprising said inhibitors of APT, or pharmaceutically acceptable salts thereof, and information for use thereof.
  • the invention further provides an inhibitor of APT of formula (ML349), or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • the invention further provides an inhibitor of APT of formula (ML348), or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • Anthrax toxin Protective Antigen (PA) PA
  • (D): MEK2 degradation. Quantification for each time point for cells pre-treated for 2 h and expressed as % of t 0 for each sample. All results are mean ⁇ SEM.
  • Figure 4 (A), (B) and (C): Microscopy-based quantification assay of Vero E6 cells infected with SARS-CoV-2-Spike-pseudotyped particles (VSV-S-CoV2) (A, C) or VSV-G- pseudotyped particles (VSVG) (B).
  • C Dose response curve calculated for ML348, ML349, and hydroxychloroquine. Results are mean ⁇ SEM (A,B), or SD (C).
  • FIG. 5 (A): Infection with SARS-CoV-2 determined using a plaque assay of Vero E6 cells pre-treated with ML348 or ML349 and infected in the presence of increasing concentrations of each drug. Dose-response curves were obtained using non-linear regression.
  • Figure 7 Quantification of viral RNA replication (E, RdRp and E subgenomic) from Vero E6 cells (A) and correspondent supernatants (B) infected with SARS-CoV-2 (MOI- 0.1 for 24 h). Cells were inoculated for 1 h with SARS-CoV-2, washed and subsequently incubated in complete medium with 12.5 pM of ML348 or ML349.
  • C Cells were previously transfected with control or siRNA oligos targeting the expression of APT1 (siAPT) for 72 h.
  • D Quantification of APT1 mRNA levels from the samples used in C.
  • Figure 9 (A): LYPLA1 (APT1 ) and LYPLA2 (APT2) mRNA levels in different cell lines. Values are TAG per Million (TPM) corresponding to 10*6 X (reads mapped to transcript/transcript length)/ SUM (reads mapped to transcript/transcript length).
  • TPM TAG per Million
  • B and C Quantification of viral RNA replication (E, RdRp and E subgenomic) from Calu-3 cells and correspondent supernatants infected with SARS-CoV-2 (MOI-0.1 for 24 h), treated as indicated.
  • D Quantification of APT2 mRNA levels from the samples used in C.
  • each data point represents an individual experiment and results are mean (A) or mean ⁇ SEM (B, C, D). All p values comparing RNA levels in relation to control conditions were obtained by (B, C) one-way ANOVA with Dunnet’s multiple comparison or (D) Paired two- tailed student’s t-test (*p ⁇ 0.05, **p ⁇ 0.01 , ****p ⁇ 0.0001 )
  • Figure 12 (A): dose response inhibitions of SARS-CoV-2 replication (infection at MOI «0.05-01 ) in human lung-derived Calu-3 cells by ML349-derived compounds C5, C6, and C7, 24 h post-viral inoculation, quantified by QPCR of total viral RNA (E, RdRp and E sub-genomic). (B): levels of housekeeping (HK) transcripts during infection, indicative of cell viability. For each concentration, results are mean ⁇ SEM of n >3. Dose inhibition curves were fitted using nonlinear regression, and EC50 ⁇ SD concentrations are indicated.
  • Figure 13 shows replication of SARS-CoV-2 inoculated in human lung-derived Calu-3 cells (MOI «0.5, 1 h), incubated with infection media supplemented with 5 pM of compound C2 and 0.5 pg/ml of P2G3 anti-Spike antibody, from 1 h post viral inoculation.
  • Cells were harvested at the indicated time points and infection was determined by monitoring the levels of intracellular (A): genomic (E and RdRp) and (B): sub-genomic E (E-subgen) RNA transcripts by QPCR. Values were normalised to 1 for the timepoint 1 h post viral inoculation (before drug treatment) and results are mean ⁇ SEM of n>3. Data is presented as equivalent side-by-side plot graphs with log or linear scale representation of viral RNA levels - Fold-infection.
  • Figure 15(A): dose-response inhibition of replication of SARS-CoV-2 variant Omicron BA.5 (infection at MOI 0.05-01 ) in human lung-derived Calu-3 cells by compound 2 (C2), 24 h post-viral inoculation, quantified by QPCR of total viral RNA (E, RdRp and E sub- genomic).
  • B levels of housekeeping (HK) transcripts during infection, indicative of cell viability. For each concentration, results are mean ⁇ SEM of n >3. The dose inhibition curve was fitted using nonlinear regression, and EC50 ⁇ SD concentrations are indicated.
  • Figure 16 toxicity evaluation of inhibiting APT2 with ML349 and C2 in vivo in C57BL/6 mice.
  • A Body weight (g);
  • B blood pressure (Hgmm);
  • C pulse (beats per minute - bpm) were monitored for the indicated time points for every mouse mock- treated or treated with 1 mg of ML349 or C2 per 25 g of body weight for several days through 13 days.
  • Mock-treated control mice were inoculated in parallel with equivalent volumes of vehicle solution used to resuspend ML349 and C2.
  • FIG 17(A) Schematic representation of the APT2-dependent mechanism of synchronized anthrax toxin entry into host target cells.
  • the toxin binds to the S-acylated anthrax receptor (CMG2) at the cell surface; this leads to 2, the recruitment and binding of APT2 to CMG2, which, in turn, promotes, 3, the complete de-acylation of the receptor; and 4, its dissociation from the host cytoskeleton components (actin, vinculin, and talin) and binding to the host GTPase RhoA.
  • CMG2 S-acylated anthrax receptor
  • IP co-immunoprecipitation
  • B western blot analysis of CMG2- immunoprecipitation (IP) fractions to monitor the co-immunoprecipitation (co-IP) of APT2- CMG2 complexes from cell lysates of control (untreated) or anthrax toxin-treated cells.
  • FIG. 18 Western blot analysis of APT2-CMG2 complexes present in CMG2- immunoprecipitation (IP) fractions from cell lysates of control (untreated) or anthrax toxin- treated cells.
  • A RPE-1 cells ectopically expressing APT2-FLAG for 24 h, left untreated or treated with anthrax toxin (protective antigen-PA subunit) in the presence of APT2 inhibitors, ML349 or compound 02, C3, or control drug carrier DMSO. Drugs were added at a final concentration of 2 pM, 4 h before and during toxin treatment.
  • B quantification of APT2-FLAG levels in equivalent CMG2 fractions displayed in A.
  • FIG. 19 Quantification of APT2-FLAG levels in equivalent CMG2 fractions from western blot analysis of APT2-CMG2 complexes present in CMG2-immunoprecipitation (IP) fractions from cell lysates of control (untreated) or PA-toxin-treated cells: RPE-1 cells ectopically expressing APT2-FLAG for 24 h, left untreated or treated with anthrax toxin (protective antigen-PA subunit) in the presence of APT2 inhibitors, ML349 or compound of formula (II) (C9, C12, C13, C16, and C17), or control drug carrier DMSO. Drugs were added at a final concentration of 5 pM, 4 h before and during toxin treatment. Data are normalized to 100% for WT, and results are mean or mean ⁇ SEM, and each data point represents an individual experiment.
  • IP CMG2-immunoprecipitation
  • Figure 20 Quantification of western blot analysis of the dynamics of toxin pore formation (PA-Pore) in RPE-1 mammalian cells treated with anthrax toxin (protective antigen-PA subunit) in the presence of APT2 inhibitors (C2, C9, C12, C13, C16 at 5 pM) for the indicated times (0, 30 and 60 min).
  • Figure 21 Inhibition of SARS-CoV-2 replication in human lung-derived Calu-3 cells (MOI «0.05-0.1 , 1 h), in the presence of the APT2 inhibitors, ML349, C2, C9, C12, C13, C15, C16 and C17 (used at 5 pM), from 1 h post-viral inoculation. Culture supernatants and infected cells were harvested 24 h post-viral inoculation. The levels of viral nucleocapsid protein N: cell lysates, were monitored by Western blot.
  • the present invention relates to an inhibitor of acyl protein thioesterase (APT) of formula (II): or a pharmaceutically acceptable salt thereof, wherein
  • Ring A is a thiophene ring or a furane ring
  • Each RAm is independently selected from the group comprising hydrogen, halogen, Ci- C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups; and m is an integer from 0 to 2;
  • Each R1 , R2, R4, R5 is independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, optionally substituted Ci-Ce alkyl acyl, optionally substituted Ci-Ce alkyl aminocarbonyl, optionally substituted carbony
  • R3 is selected from the group comprising
  • the ring A is a thiophene ring represented by formula: wherein: each R6, R7 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups.
  • the ring A is a thiophene ring represented by formula: wherein: each R6, R7 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group. Even more preferably, the ring A is a thiophene ring represented by formula: Even more preferably, the ring A is a thiophene ring represented by formula:
  • Halogen refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
  • C1-C10 alkyl refers to monovalent straight-chained and branched alkyl groups having 1 to 10 carbon atoms. This term is exemplified by groups such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec.
  • Ci-Cio-heteroalkyl includes both straight-chained and branched C1-C10 alkyl groups according to the definition above, having at least one or more heteroatoms selected from S, O and N.
  • Ci-Ce-haloalkyl includes both straight-chained and branched Ci-Ce alkyl groups according to the definition above, having at least one or more halogen selected from F, Cl, Br or I.
  • Carbonyl refers to the group -C(O)R where R includes hydrogen, “Ci-Ce- alkyl”, “aryl”, substituted aryl, “heteroaryl”, “Ci-Ce-alkyl aryl”, “Ci-Ce-alkyl heteroaryl”, or substituted amine (-NH-R’) wherein R’ is optionally substituted aryl.
  • Ci-Ce alkyl acyl refers to Ci-Ce alkyl groups having a Ci-Ce acyl substituent as defined herein.
  • Ci-Ce alkyl carbonyl refers to Ci-Ce alkyl groups having a “carbonyl” substituent as defined herein.
  • aminocarbonyl refers to the chemical moiety -C(0)NRR’ in which each of R and R’ is independently hydrogen, Ci-Ce alkyl, aryl, substituted aryl, heteroaryl, Ci-Ce alkyl aryl, or Ci-Ce alkyl heteroaryl.
  • Ci-Ce alkyl aminocarbonyl refers to Ci-Ce alkyl groups having an “aminocarbonyl” substituent as defined herein.
  • C3-C10 cycloalkyl refers to a saturated carbocyclic group of from 3 to 10 carbon atoms having a single ring (e.g., cyclohexyl) or multiple condensed rings (e.g., norborn yl).
  • Preferred cycloalkyl include cyclopentyl, cyclohexyl, norbornyl and the like.
  • Ci-Ce alkyl cycloalkyl refers to Ci-Ce-alkyl groups having a cycloalkyl substituent, including cyclohexylmethyl, cyclopentylpropyl, and the like.
  • C3-C10 heterocycloalkyl refers to Cs-Cio-cycloalkyl group according to the definition above, in which up to 3 carbon atoms are replaced by heteroatoms chosen from the group consisting of O, S, NR, R being defined as hydrogen or methyl.
  • Preferred heterocycloalkyl include cyclohexane in which 1 carbon atom is replaced by S(O)2, pyrrolidine, piperidine, piperazine, 1 -methylpiperazine, and the like.
  • optionally substituted C3-C10 heterocycloalkyl refers to a C3-C10 heterocycloalkyl as defined above that may be fused with 0 to 2 further groups selected from cycloalkyl, heterocycloalkyl, aryl or heteroaryl group. Preferred groups are benzene, toluene, phenol, aniline, anisole, and the like.
  • Ci-Ce-alkyl heterocycloalkyl refers to Ci-Ce-alkyl groups having a heterocycloalkyl substituent as defined herein.
  • C2-Cio-alkenyl refers to alkenyl groups preferably having from 2 to 10 carbon atoms and having at least 1 or 2 sites of alkenyl unsaturation.
  • Preferable alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl and octenyl, and the like.
  • alkenyl in the meaning of the present invention includes the cis and trans isomers.
  • Ci-C 6 alkoxy refers to group -O-R’ where R’ includes both straight-chained and branched "Ci-Ce alkyl” or " Ci-Ce haloalkyl” or " Ci-Ce heteroalkyl” or "aryl” or “heteroaryl” or "Ci-Ce-alkyl aryl” or “Ci-Ce-alkyl heteroaryl”.
  • Preferred alkoxy groups include by way of example, methoxy, ethoxy, propoxy, butoxy, phenoxy and the like.
  • Amino refers to -NRR’ in which each of R and R’ is independently hydrogen, Ci-Ce alkyl, substituted aryl, heteroaryl, Ci-Ce alkyl aryl, Ci-Ce alkyl heteroaryl, cycloalkyl, or heterocycloalkyl.
  • Ci-Ce-alkyl amino refers to Ci-Ce-alkyl groups having an amino substituent as defined herein.
  • C6-C10 aryl refers to an unsaturated aromatic carbocyclic group from 6 to 10 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl).
  • Preferred aryl groups include phenyl, naphthyl, phenantrenyl and the like.
  • C6-C10 aryl refers to a “C6-C10 aryl” as defined above that may be substituted with 0 to 3 further groups independently selected from halogen (Cl, Br, F, I), Ci-Ce-haloalkyl, and alkoxy as defined herein. Preferred groups are -Cl, -CF3 and -OCH3.
  • C6-C10 heteroaryl refers to a monocyclic heteroaromatic, or a bicyclic or a tricyclic fused- ring heteroaromatic group in which up to 3 carbon atoms are replaced by heteroatoms chosen from the group consisting of O, S, or N.
  • ether refers to -R-O-R’ in which each of R and R’ is independently Ci-Ce alkyl, “Ce-Cio aryl”, and Ci-Ce cycloalkyl.
  • the present invention also relates to an inhibitor of APT of formula (II): or a pharmaceutically acceptable salt thereof, wherein
  • Ring A is a thiophene ring or a furane ring
  • Each RAm is independently selected from the group comprising hydrogen, halogen, Ci- C10 alkyl, C2-C10 alkenyl, Ci-Ce alkoxy, Ci-Ce acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, Ce-Cio aryl, and/or Ce-Cio heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups; and m is an integer from 0 to 2;
  • Each R1 , R2, R4, R5 is independently selected from the group comprising hydrogen, optionally substituted Ci-Ce alkyl, optionally substituted Ce-Cio aryl; Preferably each R1 , R2, R4, R5 is hydrogen; and
  • R3 is selected from the group comprising
  • the ring A is a thiophene ring represented by formula:
  • the present invention also relates to an inhibitor of APT of formula (II) selected from the group comprising or a pharmaceutically acceptable salt thereof.
  • the inhibitor of APT of formula (II) is selected from the group comprising More preferably, the inhibitor of APT of formula (II) is
  • the present invention also relates to an inhibitor of APT of formula (II), as described herein, or a pharmaceutically acceptable salt thereof, for use as a medicament.
  • the present invention relates to an inhibitor of APT of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • the present invention also relates to a method of treating and/or preventing a viral and/or a bacterial infection, the method comprising administering an effective amount in a subject in need thereof of at least one inhibitor of APT of formula (II), or any pharmaceutically acceptable salt thereof as described herein.
  • the present invention also relates to an inhibitor of APT of formula , or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • the present invention also relates to a method of treating and/or preventing a viral and/or a bacterial infection, the method comprising administering an effective amount in a subject in need thereof an inhibitor of APT of formula , or any pharmaceutically acceptable salt thereof.
  • the present invention provides inhibitors of acyl protein thioesterase (APT) of formula (II) as described herein, and/or an inhibitor of APT of formula or any pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of viral infections mediated by or resulting from acyl protein thioesterases activity, and/or bacterial infections mediated by or resulting from acyl protein thioesterases activity.
  • APT acyl protein thioesterase
  • acyl protein thioesterases are enzymes that cleave off lipid modifications on proteins, located on the sulfur atom of cysteine residues linked via a thioester bond.
  • the APT such as acyl-protein thioesterase 1 (APT1 ) and acyl-protein thioesterase 2 (APT2) are involved in the depalmitoylation of proteins.
  • Viral infections mediated by or resulting from acyl protein thioesterases activity are infections of multiple human systems including infections of the upper and lower respiratory tract, infections of the skin, infections of the eye, infections of the gastrointestinal tract, infections of the mouth, infections of the urinary genital tract, infections of the intestinal tract, infections of the brain (e.g. meningitidis or encephalitis), and also systemic, multi organ infections such as haemorrhagic fevers.
  • viral infections or viral infections associated diseases mediated by or resulting from acyl protein thioesterases activity are selected from the group comprising COVID, SARS, flu, rheumatic diseases, encephalitis, haemorrhagic fevers, acquired immunodeficiency syndrome (AIDS), sarcoma and/or leukemia.
  • Bacterial infections or bacterial infections mediated by or resulting from acyl protein thioesterases activity are associated with infections of the upper and lower respiratory tract, infections of the skin, infections of the eye, infections of the gastro-intestinal tract, infections of the mouth, infections of the urinary tract, infections of the intestinal tract, infections of the uterus, and infections of the brain (e.g., meningitidis).
  • Bacterial infections associated diseases mediated by or resulting from acyl protein thioesterases activity are selected from the group comprising Typhoid fever, Salmonellosis, bacteria Gastroenteritis intestinal infection, bacteremia, Shigellosis, Bacteria pneumonia (e.g., legionnaires disease), endophthalmitis, bacteremia, septicemia, sepsis, endocarditis, salpingitis, skin infections, and/or meningitis.
  • the present invention provides inhibitors of acyl protein thioesterase (APT) as described herein, or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of a viral and/or bacterial infection selected from the group comprising, SARS, COVID, flu, rheumatic diseases, encephalitis, haemorrhagic fevers, acquired immunodeficiency syndrome (AIDS), sarcoma, leukemia, meningitidis, encephalitis, bacteraemia and sepsis.
  • APT acyl protein thioesterase
  • the present invention also provides inhibitors of acyl protein thioesterase (APT) of formula (II) as described herein, and/or an inhibitor of APT of formula or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of a microbial infection, wherein said microbial infection is a co-infection comprising a viral infection and a bacterial infection.
  • APT acyl protein thioesterase
  • viruses or viral families here represented are responsible of a variety of human diseases that comprise the infection of multiple human systems, including: the respiratory tract (upper and lower), the skin, the eye, the gastrointestinal tract, the mouth, the urinary genital and intestinal tract, the brain (e.g., meningitidis or encephalitis), and also systemic, multi organ infections such as haemorrhagic fevers.
  • said viral infection can be caused by a virus selected from the group comprising Coronaviruses (CoV) (such as severe acute respiratory syndrome coronavirus (SARS), Middle East respiratory syndrome (MERS), Mouse Hepatitis Virus (MHV), transmissible gastroenteritis virus (TGEV), Swine acute diarrhoea syndrome coronavirus (SADS-cov), Rousettus bat coronavirus HKU9, and any other bat CoVs), Influenza virus (such as A, B,C), Respiratory syncytial virus (hRSV), Newcastle disease virus (NDV), Measles virus, Sindbis virus, Semliki Forest virus (SFV), Rabies virus, Vesicular Stomatitis Virus (VSV), human immunodeficiency virus (HIV), murine leukemia viruses (MulV), Ebola virus, Marburg virus, Herpes simplex virus (HSV), Human cytomegalovirus (HCMV), Dengue virus (DENV), Zika virus (ZI
  • Coronaviruses
  • said Coronavirus (CoV) can be selected from SARS-CoV-2, SARS-CoV-1 , MHV, TGEV, SADS, MERS-CoV, HCoV-HKLH , HCoV-229E, HCoV-NL63 and/or HCoV- OC43.
  • said bacterial infection is caused by bacterial strains selected from the group comprising Bacillaceae, Vibrionaceae, Pectobacteriaceae Yersiniaceae, Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales and/or Pasteurellaceae.
  • bacterial families or strains are associated with infections of the respiratory tract (upper and lower), the skin, the eye, the gastro-intestinal tract, the mouth, the urinary and intestinal tract, uterus infections and other sexual transmitted diseases, brain infections (e.g., meningitidis), bacteraemia and sepsis.
  • a pathogen from family Bacillaceae is for example Bacillus anthracis
  • a pathogen from family Pectobacteriaceae is for example Erwinia spp (carotovora)
  • a pathogen from family Pseudomonadaceae is for example Pseudomonas spp (syringae)
  • a pathogen from family Enterobacteriaceae is for example Salmonella enterica or Shigella spp
  • a pathogen from family Legionellaceae is for example Legionella pneumophila
  • a pathogen from family Chlamydiaceae is for example Chlamydia trachomatis.
  • novel inhibitors of formula (II) as described herein have been synthesized (Example 1 1 , compounds C1 , C2, C3, C9, C12, C13, C16 and C17).
  • novel inhibitors of formula (II) as described herein demonstrate an EC50 that is decreased by about 3 to 8-fold compared to compound ML-349 (Example 12, figures 1 1 A and B).
  • compounds such as C5, C6, C7 that are not covered by formula (II) as described herein are not potent inhibitors of SARS-CoV2 replication in Calu-3 cells (Example 12, Figure 12).
  • inhibitors of formula (II) are effective without inducing cytotoxic effects in vitro (Example 14, figure 14) or in vivo (Example 16, Figure 16).
  • inhibitors of formula (II) as described herein such as C2, C3 or a compound of formula ML349, can inhibit the APT2-dependent anthrax toxin intoxication of host cells (Example 17, figure 17 and Example 18, figure 18). Results have also been demonstrated for different compounds of formula (II) such as C2, C9, C12, C13, C16 and C17 (Example 19, figures 19 and 20).
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one inhibitor of APT of formula (II) as described herein, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • pharmaceutically acceptable carrier means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe and possesses acceptable toxicities. Acceptable carriers include those that are acceptable for veterinary use as well as human pharmaceutical use.
  • pharmaceutically acceptable carrier as used in the specification and claims includes both one and more than one such carrier.
  • the present invention also relates to a pharmaceutical composition, comprising at least one inhibitor of APT of formula (II) as described herein, or pharmaceutically acceptable salts thereof, further comprising one or more additional therapeutic agents and a pharmaceutically acceptable carrier, diluent or excipient.
  • said one or more additional therapeutic agents are selected from the group comprising antibacterial agents and/or antiviral agents.
  • the at least one or more additional therapeutic agent is an antibacterial agent selected from the group comprising sulfonamides, penicillins, cephalosporins, aminoglycosides, chloramphenicol, tetracyclines, macrolides, lincosamides, streptogramins, glycopeptides, rifamycins, nitroimidiazoles, quinolones, trimethoprim, oxazolidinones, and/or lipopeptides and combinations thereof.
  • an antibacterial agent selected from the group comprising sulfonamides, penicillins, cephalosporins, aminoglycosides, chloramphenicol, tetracyclines, macrolides, lincosamides, streptogramins, glycopeptides, rifamycins, nitroimidiazoles, quinolones, trimethoprim, oxazolidinones, and/or lipopeptides and combinations thereof.
  • the at least one or more additional therapeutic agent is an antiviral agent selected from the group comprising Aribidol (umifenovir), Favilavir, APN01 , CCR5 antagonist leronlimab (PRO 140), Remdesivir (GS-5734), Galidesivir (BCX4430), Molnupiravir (MK-4482 I FJDD-2801 ), MK-7110 (CD24Fc).
  • Aribidol umifenovir
  • Favilavir Favilavir
  • APN01 APN01
  • CCR5 antagonist leronlimab PRO 140
  • Remdesivir GS-5734
  • Galidesivir BCX4430
  • Molnupiravir MK-4482 I FJDD-2801
  • MK-7110 CD24Fc
  • the compounds of the invention namely inhibitor of acyl protein thioesterase (APT) as described herein, or a pharmaceutically acceptable salt thereof, that can be used in the treatment and/or prevention of a microbial infection can be incorporated into a variety of formulations and medicaments for therapeutic administration. More particularly, one or more compound(s) as provided herein can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • API acyl protein thioesterase
  • administration of the compounds can be achieved in various ways, including oral, intranasal, intramuscular, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracranial and/or intratracheal administration.
  • the compound can be administered in a local rather than systemic manner, in a depot or sustained release formulation.
  • the compounds can be formulated with common excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for convenient oral administration or administered by the intramuscular or intravenous routes.
  • the compounds can be administered transdermally and can be formulated as sustained release dosage forms and the like.
  • the compounds can be administered alone, in combination with each other, or they can be used in combination with other known compounds.
  • Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (Mack Publishing Company (1985) Philadelphia, PA, 17th ed.), which is incorporated herein by reference. Moreover, for a brief review of methods for drug delivery, see, Langer, Science (1990) 249:1527-1533, which is incorporated herein by reference.
  • said pharmaceutical composition is administered by oral, inhalation, nebulization, intranasal, intrapulmonary, intradermal and/or intramuscular route of administration.
  • drug substances can be delivered to the respiratory system using various devices such as nebulizer or spray.
  • suitable unit doses for the compounds of the present invention can, for example, preferably contain between 0.1 mg to about 1000 mg, between 1 mg to about 500 mg, and between 1 mg to about 300 mg of the active compound. In another example, the unit dose is between 1 mg to about 100 mg.
  • Such unit doses can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total dosage for a 70 kg human adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration.
  • a preferred dosage is 0.01 to about 1.5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.
  • a typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient.
  • the timerelease effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release. It can be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art.
  • the present invention provides a kit comprising an inhibitor of APT of formula (II), as disclosed herein, or a pharmaceutically acceptable salt thereof, and information for use thereof.
  • the kit may further comprise one or more additional therapeutic agents, such as antibacterial agents and/or antiviral agents.
  • the present invention provides an inhibitor of acyl protein thioesterase (APT) of general formula (I): or a pharmaceutically acceptable salt thereof, wherein
  • R’ is independently selected from the group comprising optionally substituted thiophene ring, optionally substituted furane ring, optionally substituted C3-C10 cycloalkyl, or optionally substituted C6-C10 aryl;
  • Each R1 , R2, R3, R4, R5 is independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, optionally substituted Ci-Ce alkyl acyl, optionally substituted Ci-Ce alkyl aminocarbonyl, optionally substituted carbonyl, optionally substituted Ci-Ce alkyl carbonyl, optionally substituted C6-C10 aryl, ether, C2-C10 alkenyl, C6-C10 heteroaryl, C3- C10 cycloalkyl, C3-C10 heterocycloalkyl, Ci-Ce alkoxy, optionally substituted amino, and/or Ci-Ce alkyl amino; for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • R’ is independently selected from the group comprising optionally substituted thiophene ring, or optionally substituted furane ring.
  • R’ is a thiophene ring represented by formula: wherein: each R6, R7 are independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups.
  • R’ is a thiophene ring represented by formula: wherein: each R6, R7 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.
  • R’ is a thiophene ring represented by formula:
  • R’ is a thiophene ring represented by formula
  • the present invention relates to an inhibitor of acyl protein thioesterase (APT) of formula or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • APT acyl protein thioesterase
  • R’ is a furane ring represented by formula: wherein: each R8, R9, are independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups.
  • R’ is a furane ring represented by formula: wherein: each R8, R9 are independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, Ci-Ce alkoxy, Ci-Ce acyl, C3-C10 cycloalkyl, and/or optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.
  • the ring A is a furane ring represented by formula: wherein: each R8, R9 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, and/or optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.
  • the ring A is a furane ring represented by formula:
  • the present invention relates to an inhibitor of acyl protein thioesterase (APT) of formula (ML348), or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
  • APT acyl protein thioesterase
  • inhibitors of acyl protein thioesterases prevent intoxication of mammalian cells with Anthrax toxin by inhibiting the internalization and recycling of the toxin receptor capillary morphogenesis gene 2 (CMG2/ANTXR2) (Example 1 , Figure 1 ) and by blocking endocytosis and pore formation of the anthrax toxin (Example 2, Figure 2).
  • CMG2/ANTXR2 toxin receptor capillary morphogenesis gene 2
  • inhibitors of acyl protein thioesterase block SARS-CoV-2 cellular infection ( Figures 5 and 9) and replication ( Figures 6-7). It has also been demonstrated that inhibitors of acyl protein thioesterase block cellular infection by other type of virus such as HSV-2 ( Figure 8).
  • a dose response was determined for ML349 in Calu-3 cells and the EC50 is 8.6 pM ( Figure 10A).
  • RPE1 ATCC CRL-4000
  • VERO E6 ATCC CVCL-0574
  • CALU-3 ATCC HTB55
  • Verified siRNA for human APT2 were purchased from Qiagen.
  • APT2 target sequence 5’- CAGCTGCTTCTCAGTCATGAA-3’.
  • a pool of verified siRNA was used to target monkey APT1 and purchased from Qiagen.
  • APT1 target sequence 5’- TACCGACAGGACCCTGTGGAA-3’, 5’-GGTCACAGATATACGGTATNN-3’, 5’- CCGGTGTATGTGCGGCAATNN-3’.
  • a sequence targeting the viral glycoprotein VSV-G 5’-ATTGAACAAACGAAACAAGGA-3’ was used.
  • Transfections of 50 nM of siRNA were carried out using TRANSIT-X2 (MIRUS), and the cells were analysed at least 72 h after transfection.
  • MIRUS TRANSIT-X2
  • the antibodies were commercially available: rabbit anti-human CMG2 (Proteintech, RRID: AB_2056741 ); mouse anti-GAPDH (Acris Antibodies, 4A1 -MA0100, RRID_AB 1874646) ; mouse anti-LPXN (Sigma, SAB1400343), anti-Anthrax Protective Antigen (List Biological Laboratories, ref: 771 B); N-terminal rabbit anti-MEK2 (Santa Cruz, sc-524), rabbit anti-SPIKE (LIFESPAN, Cat#LS-C19510), HRP-conjugated secondary antibodies (Pierce); and for immunoprecipitation protein G beads were purchased from GE Healthcare, and Streptavidin beads from Sigma.
  • ML348, ML349 (Cayman Chemical company) and synthesised compounds (C1 , C2, C3, C9, C12, C13, C16, C17) were used at indicated times and concentration. All compounds were solubilised in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • Wild type PA (Anthrax toxin Protective Antigen) and LF (Anthrax toxin Lethal Factor) were produced in our laboratory by overexpression in E. coli as described (Feld et aL, 2012). Western Blotting
  • CMG2-APT2-Flaq Co-immunoprecipitation assays
  • RPE-1 cells cultured in 10 cm dishes (80% confluency) were transfected with plasmids encoding APT2-Flag for 24 h.
  • Transfected cells were pretreated with APT2 inhibitors at the indicated concentrations 4 h before toxin treatment (PA63 500 ng/ml and LF 50 ng/ml). Toxin treatment was carried out for the indicated time points in the presence of the drugs.
  • DMSO Dimethyl sulfoxide
  • VSV-CoV-2 Vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotypes (VSV-CoV-2) generated according to (Gasbarri et al, 2020) expressing a 19 amino acids C-terminal truncated spike protein (NCBI Reference sequence:NC_045512.2) were produced in HEK293F and titrated in Vero-E6.
  • Vero-E6 cells (13,000 cells per well) were seeded in a 96-well plate.
  • Compounds were serially diluted in DMEM and incubated with VSV-CoV-2 (MOI, 0.001 infectious units /cell) for 1 h at 37 °C. The mixture was added on cells for 1 h at 37 °C.
  • the monolayers were washed and overlaid with medium containing 2% FBS for 18 h.
  • the following day cells were fixed with paraformaldehyde 4%, stained with DAPI, and visualized using an ImageXpress Micro XL (Molecular Devices, San Jose, CA, USA) microplate reader and a 10x S Fluor objective.
  • the percentage of infected cells was estimated by counting the number of cells expressing GFP and the total number of cells (DAPI-positive cells) from four different fields per sample using MetaXpress software (Molecular Devices, San Jose, CA, USA).
  • Infected cells from 6 and 12 well plates and correspondent supernatants (when indicated - 150 pl from 6 well plates), were harvested and lysed in 220-330 pl of Maxwell® RSC Viral Total Nucleic Acid Purification Kit-lysis buffer from Promega, incubated at 80°C for 10 min, and used for Viral RNA extraction according to manufacturer’s instructions.
  • RNA concentration was measured and 500 ng or 1000 ng of total RNA was used for cDNA synthesis using iScript.
  • a 1 :5 dilution of cDNA was used to perform quantitative real-time PCR (Q-PCR) using Applied Biosystems SYBR Green Master Mix on 7900 HT Fast Q-PCR System (Applied Biosystems) with SDS 2.4 Software.
  • Infected cells (24 well plates) (cultured at 1 .5 x 10 A 5 cells per well, 24 h before infection) were harvested and lysed in 150 pl of lysis buffer (0.5% NP-40, 500 mM Tris-HCI pH7.4, 20 mM EDTA, 10 mM NaF, 30mM sodium pyrophosphate decahydrate, 2 mM benzamidine and protease inhibitor cocktail; Roche) for at least 30 min. Lysates were cleared by centrifugation (5000g, 3 min), and the protein content of the supernatant was determined.
  • lysis buffer 0.5% NP-40, 500 mM Tris-HCI pH7.4, 20 mM EDTA, 10 mM NaF, 30mM sodium pyrophosphate decahydrate, 2 mM benzamidine and protease inhibitor cocktail; Roche
  • Samples were resuspended in 50 pl Laemmli buffer, boiled for 5 min, and approximately 20-30 pg of protein lysate were used for Western blot analysis (as described) using antibodies against viral nucleocapsid N and host GAPDH. The percentage of infection was calculated as the densitometric ratio between N and GAPDH levels for each sample.
  • the number of viable cells in culture was measured by determining the reducing potential of cultured cells, thus metabolism, in Calu-3 cells.
  • the assay measures the reduction of a cell-permeable substrate, which is diffused to the medium and processed by an exogenously added reporter enzyme (NanoLuc® luciferase).
  • the reaction produces a luminescent signal that correlates with the number of viable cells.
  • Vero E6 cells were infected in 6-well plates [as mentioned previously]. Cells washed and scrapped in 1 ml PBS and recovered by mild centrifugation 400g 3 min. Supernatants were discard and cells were fixed in 4% PFA for at least 30 min, washed twice in PBS, and permeabilized for 5 min with 0.1 % Triton in PBS. The following blocking, antibody, and washing steps were all done with FACS buffer (2% FBS in PBS/EDTA) primarily at RT. Blocking was done for 15 min, primary antibody incubation with mouse anti-Spike antibody (LC-C19510) was performed at 1 :200 for 30 min at RT or at 1 :500 ON at 4 °C, and cells were washed twice.
  • FACS buffer 2% FBS in PBS/EDTA
  • Alexa-647-conjugated donkey anti-mouse was used as a secondary antibody at 1 :600 for 30 min at RT, after which cells were washed three times and kept cold before flow cytometry acquisition.
  • Analytical flow cytometry was performed using an LSRII or LSR Fortessa (BD; Becton Dickinson) instrument and results were analyzed using the FlowCore package in R.
  • Lactate dehydrogenase (LDH) cytotoxicity assays were performed, using In Situ Death Detection Kit (Roche, Indianapolis, IN), in Vero E6 or CALU-3 cells cultured in 12 well plates with equivalent culture conditions used for infection assays.
  • Wild-type HSV-2 viral suspensions were inoculated onto cells pre-treated with the indicated drugs for 1 h, at a multiplicity of infection (MOI) of 0.01 plaque-forming units (PFU) as described (Cagno et al, 2018). Viral inoculum was removed, and cells washed after 2 h, and infection was continued in the presence of the drugs for evaluation through a plaque-based assay.
  • MOI multiplicity of infection
  • PFU plaque-forming units
  • mice 1 1 -week-old male C57BL/6 mice were inoculated intraperitoneally with 400 to 600 pl of a 2 mg/ml working solution of C2 or ML349, resuspended in PEG300 50% (v/v) solution in PBS, to a final dose of 1 mg of compound per 25 g of body weight.
  • Control mice were inoculated in parallel with an equivalent volume (per body weight) of vehicle PEG300 50% (v/v) in PBS solution.
  • Drug and vehicle treatments were injected on days 1 , 3, 6, 8, and 10 of the experiment, whereas clinical parameters (body weight, blood pressure, and pulse) were monitored on days 1 , 2, 3, 6, 7, 8, 9, 10, and 13. After 13 days of treatment, mice were euthanatized, and different organs were harvested for histological analysis and weight measurements.
  • Example 1 Inhibition of APT2 activity increases surface expression of ANTXR2/CMG2
  • Example 2 Inhibition of APT2 activity prevents intoxication of mammalian cells with Anthrax toxin.
  • Mammalian cells were left untreated (control) or co-treated with ML349 (10 pM) or ML348 (10 pM) during 0, 20, 40 and 60 minutes. Cells were pre-treated with ML349 or ML348 during 2 h ( Figure 2A) or 4 h ( Figure 2B) prior to and during exposure to Anthrax toxin Protective Antigen (PA).
  • ML349 10 pM
  • ML348 10 pM
  • Treatment with ML349 blocks endocytosis of CMG2/ANTXR2, thus preventing pore formation of the anthrax toxin PA subunit.
  • ML349 thus prevents the protease subunit of anthrax toxin to reach the cytosol and in turn prevents the cleavage of a major host cell signaling regulator, the MAP kinase-kinase MEK.
  • Example 3 siRNA silencing of APT2 expression prevents intoxication of mammalian cells with Anthrax toxin.
  • Example 4 Inhibition of APT1 activity with ML348 diminishes SARS-CoV-2 Spike- pseudotyped particles (PPs) entry into host cell.
  • a cell-based assay was performed to determine the effects of different chemical inhibitors on the internalization of Vesicular Stomatitis Virus (VSV)-based particles pseudotyped with the spike fusion glycoprotein from SARS-CoV-2.
  • VSV Vesicular Stomatitis Virus
  • the effects of ML348 and ML349, inhibitors of APT1 and APT2 respectively, as well as Palmostatine, Bromopalmitate, Chlroroquine and Hydroxychloroquine were tested and are represented on Figure 4.
  • Vero E6 cells were infected with SARS-CoV-2-Spike- pseudotyped particles (VSV-S-CoV2) or VSV-G-pseudotyped particles(VSV-G) ( Figures 4A, B and C). Vero cells were either pre-treated (PRE-TREAT, Figure 4C) for 4 h washed, and infected with VSV-S- CoV2 or VSV-G, or infected in the presence of the same concentration of the chemical inhibitors (PRE+CO-TREAT). GFP-expressing cells were quantified by fluorescent microscopy.
  • the percentage of infection was calculated comparing the percentage of positive cells to the percentage in infected untreated controls (DMSO). Dose-response curve was calculated for ML348, ML349, and hydroxychloroquine. ( Figure 4C).
  • ML348 specifically inhibited the entry of PPs typed with SARS-CoV-2 spike protein to a similar extend as hydroxychloroquine (Figure 4A).
  • This inhibitory effect was dosedependent ( Figure 4C) and not observed upon treatment of target cells with unspecific inhibitors of palmitoylation and depalmitoylation such as Bromopalmitate and Palmostatine respectively, neither by treatment with the APT2 inhibitor ML349 ( Figure 4A and 4B).
  • Example 5 Inhibition of APT1 activity with ML348 blocks SARS-CoV-2 cellular infection.
  • the depalmitoylation-targeting drugs ML348, ML349 were tested during infection of Vero E6 cells with a clinical isolate of SARS-Cov-2. Infection was monitored using a standard plaque-based infection assay as described in materials and methods (Viral stock production and titration with plaque-based assays; SARS CoV-2 infections).
  • Vero E6 cells were pre-treated with ML348 or ML349 for 1 h washed, and infected with SARS-CoV-2, in the presence of drug. Infection was determined using a plaque assay and a dose-response curve was established for ML348 and ML349 ( Figure 5A). The EC50 for ML348 is 13.3 pM. Then, Vero E6 cells were infected with SARS-CoV-2 (MOI 0.1 ) for 24 h, in the presence of 12.5 pM ( Figure 5B). Drugs were added 1 h after viral inoculation, and replication was monitored by determining the % of high Spike-positive cells by flow cytometry. Results are mean ⁇ SEM and each dot represents one individual biological replicate.
  • Example 6 Inhibition of APT1 activity with ML348 inhibits SARS-CoV-2 intracellular replication.
  • ML348 occurs within a single cellular infection cycle (up to 8 to 12 h)
  • viral genomic RNA replication was monitored throughout time by probing for two viral genomic regions corresponding to the E and RdRp genes of SARS- CoV-2.
  • Vero E6 cells were treated with ML348 1 h after viral inoculation and throughout the time of infection.
  • Example 7 Inhibition of APT1 activity and silencing of APT1 expression blocks SARS- CoV-2 infection in Vero E6 cells.
  • Viral RNA replication in Vero E6 cells was also monitored after 24h of infection to follow multiple rounds of cellular infection.
  • the viral intracellular replication was monitored by determining the levels of the sub-genomic E RNA, a bona-fide marker of intracellular viral RNA replication.
  • E Quantification of viral RNA replication
  • SARS-CoV-2 MOI-O.1 for 24 h
  • Control treated without drug
  • Cells were inoculated for 1 h with SARS-CoV-2, washed and subsequently incubated in complete medium with 12.5 pM of ML348 or ML349 ( Figure 7 A and 7B).
  • Example 8 Inhibition of APT1 activity with ML348 blocks HSV-2 cellular infection.
  • ML348 or ML349 could limit infection by other type of viruses.
  • a plaque-based assays was performed using herpes simplex virus type 2 (HSV-2) according to materials and methods (HSV infection and inhibition assays). Vero E6 cells were pre-treated for 1 h in the presence of drug, washed, and infected with HSV-2 ( Figure 8 A). Infection was determined using a plaque assay. Dose-response was determined for ML348 and ML349. The EC50 for ML348 is 23.2 pM.
  • Example 9 Inhibition of APT2 activity blocks SARS-CoV-2 infection in Calu-3 lung- derived cells.
  • APT2 is often more abundant than APT1.
  • APT2 is highly enriched when compared to Vero E6 cells ( Figure 9A).
  • LYPLA1 AAT1
  • LYPLA2 AAT2
  • mRNA levels are measured in different cell lines ( Figure 9A). Values are TAG per Million (TPM) corresponding to 10*6 X ((reads mapped to transcript/transcript length)/ SUM (reads mapped to transcript/transcript length)). Results are the mean of independent experiments each from n > 2 biological replicates.
  • SARS-CoV-2 The replication of SARS-CoV-2 was monitored in Calu-3 cells at 24h post-inoculation and upon treatment with 12.5 pM of ML348 or ML349. This concentration is below the EC50 of ML348 for inhibition of viral replication in Vero E6.
  • Figure 9B shows the quantification of viral RNA replication (E, RdRp and E subgenomic) from Calu-3 cells and correspondent supernatants infected with SARS-CoV-2 (MOI-0.1 for 24 h), treated with ML348 or ML349.
  • the cells were inoculated for 1 h with SARS-CoV- 2, washed and subsequently incubated in complete medium with 12.5 pM of ML348 or ML349.
  • Example 10 Dose-response inhibition of SARS-CoV-2 replication in Calu-3 cells by ML349 and cytotoxicity evaluation of ML348 and ML349.
  • Calu-3 cells were infected with SARS-CoV-2. Infection was determined using Q-PCR by quantifying the mean levels of E and RdRp viral RNA (Figure 10A). Normalized results are mean ⁇ SEM. The dose-response was determined for ML349 and the EC50 is 8.6 pM.
  • a Lactate dehydrogenase activity assay was performed as described in the material and methods section. Vero E6 cells or Calu-3 cells were treated with increasing concentrations of ML348 for 48 h or ML349 for 24 h, respectively. The results of the cell viability are mean ⁇ SD of 2 biological replicates. Importantly, neither treatment of Vero E6 with ML348 for 48h nor treatment of Calu-3 with ML349 for 24h, triggered significant cellular toxicity at different ranges of concentrations ( Figure 10B and 10C).
  • Example 1 1 Synthesis of novel inhibitors of acyl protein thioesterase (APT) of general formula (II).
  • Compound SPC-EPFL-1 (C1 ) was prepared following General Procedure A using 1 -(4- fluorophenyl)piperazine (32 mg, 1 Eq, 0.18 mmol) to afford (5,5-dioxido-4H-thieno[3,2- c]thiochromen-2-yl)(4-(4-fluorophenyl)piperazin-1 -yl)methanone (42.6 mg, 54 %) as a white solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV].
  • Compound SPC-EPFL-2 (C2) was prepared following General Procedure A using 1 -(2- chlorophenyl)piperazine (35 mg, 1 Eq, 0.18 mmol) to give (4-(2-chlorophenyl)piperazin- 1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (41.4 mg, 51 %) as a white solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV].
  • Compound SPC-EPFL-3 (C3) was prepared following General Procedure A using 1 -(2,3- dichlorophenyl)piperazine (41 mg, 1 Eq, 0.18 mmol) to give (4-(2,3- dichlorophenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (30.4 mg, 35 %) as a solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV].
  • Compound SPC-EPFL-5 was prepared following General Procedure A using phenyl(piperazin-1 -yl)methanone (34 mg, 1 Eq, 0.18 mmol) to give (4-benzoylpiperazin- 1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (65.1 mg, 81 %) as a solid after purification by flash column chromatography [Silica-CS 12g, eluent: DCM/MeOH (0% MeOH for 3CV, 0-5% 15CV, 5% for 3CV, 5-10% for 3CV].
  • Compound SPC-EPFL-6 was prepared following General Procedure A using 2-(5- methoxy-1 H-indol-1 -yl)ethan-1 -amine (27 mg, 1 Eq, 0.14 mmol) to give N-(2-(5-methoxy- 1 H-indol-1 -yl)ethyl)-4H-thieno[3,2-c]thiochromene-2-carboxamide 5,5-dioxide (36.8 mg, 57 %) as a solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV].
  • Compound SPC-EPFL-9 (C9) was prepared following General Procedure A using 1 -o- tolyl-Piperazine (19 mg, 1 Eq, 0.1 mmol) to afford (5,5-dioxido-4H-thieno[3,2- c]thiochromen-2-yl)(4-(o-tolyl)piperazin-1 -yl)methanone (31 mg, 66 %) as a light yellow solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-50% for 15CV, 50% for 3CV].
  • Compound SPC-EPFL-13 (C13) was prepared following General Procedure A using 1 - (2,6-dimethylphenyl)piperazine (48 mg, 1 Eq, 0.25 mmol) to afford (4-(2,6- dimethylphenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (42 mg, 74 %) as a yellow solid after purification by flash column chromatography [Silica- CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-50% for 15CV, 50% for 3CV]. Yellow powder (42 mg, 0.09 mmol, 74%).
  • Compound SPC-EPFL-16 (C16) was prepared following General Procedure A using 1 - (2,6-dichlorophenyl)piperazine (58 mg, 1 Eq, 0.25 mmol) to afford (4-(2,6- dichlorophenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (56 mg, 91 %) as a Light yellow solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-60% for 15CV, 60% for 3CV].
  • Compound SPC-EPFL-17 (C17) was prepared following General Procedure A using 1 - (2,5-dichlorophenyl)piperazine (58 mg, 1 Eq, 0.25 mmol) to afford (4-(2,5- dichlorophenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (51 mg, 83 %) as a Light yellow solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-50% for 15CV, 50% for 3CV].
  • Example 12 Dose response inhibition of SARS-CoV-2 replication in Calu-3 cells by ML349 and novel inhibitors of acyl protein thioesterase (APT) according to formula (II).
  • APT acyl protein thioesterase
  • the improved inhibitory effect of the novel inhibitors of acyl protein thioesterase (APT) according to formula (II), such as C1 , C2 or C3 during SARS-CoV-2 replication in Calu-3 cells ( Figure 1 1 A) further demonstrate that ML349 and novel inhibitors of acyl protein thioesterase (APT) according to formula (II) are potent inhibitors of SARS-CoV-2 infection and that host cell depalmitoylation activity constitutes a safe strategy to inhibit viral infections.
  • inhibitors of acyl protein thioesterase (APT) of formula II wherein R3 is a substituted phenyl ring such as C1 , C2 or C3 have an improved inhibitory activity of SARS-CoV-2 replication compared to ML349.
  • molecules that are not covered by formula II wherein R3 is a substituted phenyl ring such as C5, C6 or C7 do not inhibit SARS-CoV-2 replication in Calu-3 cells ( Figure 12).
  • Example 13 Inhibition of APT2 activity with inhibitor C2 inhibits SARS-CoV-2 intracellular replication.
  • Cells were harvested at the time points 1 , 4, 6, 8 and 12 hours and host and viral RNA levels quantified by QPCR as described in materials and methods.
  • the data was normalized to the intracellular viral RNA levels harvested 1 h after inoculation before drug treatment and expressed as fold RNA replication of both viral genomic and subgenomic RNA. Normalized results are the mean ⁇ SEM of three independent biological replicates displayed within two equivalent graph plots with either linear or logarithmic scale for the fold RNA replication.
  • Example 14 Dose-response cytotoxicity evaluation of APT2 inhibition in Calu-3 cells by ML349 and novel inhibitors of acyl protein thioesterase (APT) according to formula (II).
  • a RealTime-GloTM MT Cell Viability Assay consisting of a nonlytic, homogeneous, bioluminescent method to measure cell viability in real time, was performed as described in material and method.
  • Calu-3 cells were treated with increasing concentration of ML349 and C2 or C3 over the course of 6 days. Culture medium was replaced every 2 days with freshly diluted compounds in order to ensure constant activity. The results of the cell viability represent the evolution of the number of viable cells in culture at the indicated time-points and are mean SEM of 4 biological replicates ( Figure 14A-C). Fitted curves for Control (no drug), non-toxic and cytotoxic compound concentrations are indicated.
  • APT2 activity with novel inhibitors of acyl protein thioesterase (APT) effectively and safely inhibits viral infections, such as SARS-CoV-2.
  • APT2 activity with novel inhibitors of acyl protein thioesterase (APT) effectively and safely inhibits viral infections, such as SARS-CoV-2.
  • Example 15 Inhibition of APT2 activity with compound C2 inhibits replication of SARS- CoV-2 variant Omicron BA.5.
  • Example 16 Toxicity evaluation of APT2 inhibition in vivo, in C57BL/6 mice by ML349 and compound C2.
  • Example 14 Following the experiment confirming that inhibition of APT2 with ML349, compound C2 and C3 did not induce significant cytotoxicity in vitro (Example 14), an experiment was carried out to evaluate the potential toxicity effect of inhibiting APT2 using ML349 or C2 in vivo in C57BL/6 mice. Mice were treated with 1 mg of ML349 or C2 per 25 g of body weight or an equivalent volume of drug delivery vehicle (control), inoculated intraperitoneally at alternate days through a period of 13 days. Toxicity was evaluated by monitoring the body weight, blood pressure, and pulse daily throughout treatment (Figure 16A-C).
  • Example 17 Evaluating APT2 inhibition mechanism by ML349, compound C2, or C3 during anthrax toxin cellular intoxication.
  • APT2 activity during anthrax intoxication can be monitored by probing for the formation of protein complexes between the anthrax receptor CMG2 and APT2 triggered by the binding of anthrax toxin protective antigen (PA) to CMG2 (Figure 17A). Formation of these complexes occurs upon de-acylation of CMG2 and is concomitant to the dissociation of CMG2 from the cytoskeleton components actin, vinculin, and talin, which ultimately promotes the internalization of the toxin and the receptor.
  • the levels of CMG2-APT2 complexes can be probed by western blot analysis of lysates from RPE-1 cells treated with PA ( Figure 17B).
  • ML349, C2, and C3 can reduce SARS-CoV-2 replication and inhibit the APT2- dependent anthrax toxin intoxication of host cells.
  • Example 18 Comparison of APT2 inhibition efficacy by ML349, compound C2, C3 during anthrax toxin cellular intoxication.
  • CMG2-APT2 complexes formed upon anthrax toxin protective antigen (PA) intoxication were also evaluated in cells pre and co-treated with only 2 pM of each compound to compare the potency of ML349 and compound C2, C3. At such concentrations C2 and C3, but not ML349, prevented the formation of APT2-CMG2 complexes, blocking cellular intoxication ( Figure 18). Therefore, as observed for the inhibition of SARS-CoV-2 replication, C2 and C3 inhibit intoxication of host cells by anthrax-toxin at lower concentrations than ML349.
  • PA anthrax toxin protective antigen
  • Example 19 Anthrax toxin cellular intoxication assays with compounds of formula (II). Novel compounds of formula (II) as described herein have been synthesized (Example 1 1 ). These compounds were tested for their capacity to inhibit the formation of CMG2- APT2 complexes in anthrax toxin cellular intoxication assays. Western blot analysis of a single parallel synchronized cellular intoxication experiment showed that compounds C9, C12, C13, C16, and C17 (used at 5 pM) inhibit the formation of CMG2-APT2 complexes beyond 50% (Figure 19).
  • Example 20 Inhibition of APT2 activity with compounds of formula (II) blocks SARS-CoV- 2 infection in Calu-3 lung derived cells
  • Figure 21 shows the levels of viral nucleocapsid protein N: cell lysates, were monitored by Western blot. The % of infection was quantified as the ratio between the levels of N and host GAPDH for three independent replicates for each compound with relation to Control cells (infected in the presence of drug carrier DMSO).
  • Figure 21 shows that 5 pM of compounds C9, C12, C13, C16, or C17 significantly inhibited SARS-CoV-2 replication beyond 50% and more potently than ML349. At 5 pM, C2 and C3 displayed the most potent inhibitory effect.

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Abstract

The present invention relates inhibitors of acyl protein thioesterases and method of preparation thereof. It also relates to the use of inhibitors of acyl protein thioesterases in the treatment and/or prevention of a microbial infection such as viral or bacterial infections or coinfection by virus and bacteria.

Description

INHIBITORS OF ACYL PROTEIN THIOESTERASES AGAINST MICROBIAL INFECTIONS
TECHNICAL FIELD
The present invention relates to inhibitors of acyl protein thioesterases (APT) and method of preparation thereof. It also relates to the use of inhibitors of APT in the treatment and/or prevention of a microbial infection such as viral or bacterial infections or coinfection by virus and bacteria.
BACKGROUND OF THE INVENTION
For infection to progress, pathogens such as viruses, bacteria or parasites must rely on interactions with membranes from target cells. Host-pathogen interactions frequently involve modifications of host or microbial proteins by a reversible post- translational lipidation called S-acylation, commonly referred to as S-palmitoylation.
S-acylation consists of the addition of an acyl chain, often palmitate, to cytosolic cysteines in target proteins via a thioester bond. This modification is reversible and therefore acts as a functional switch on target proteins (Zaballa & van der Goot, 2018). S-acylation and deacylation are crucial for membrane trafficking events and impact the cellular distribution, abundance, and function of proteins. During infection, S-acylation modifies numerous viral proteins from clinically relevant viruses (Veit, 2012), as well as multiple host receptors fundamental for internalization, and therefore virulence, of numerous pathogens and pathogenic molecules.
Pathogens are highly opportunistic organisms. Viruses often exploit S-acylation of viral proteins or host products to promote pathogenesis. [3-Coronaviruses (CoVs) have enormous zoonotic potential being responsible for various outbreaks of severe acute respiratory syndrome (SARS) in recent years including the pandemic Coronavirus disease-2019 (COVID-19). CoVs depend on S-acylation to produce fully infectious viruses (Mesquita et al, 2021 ). SARS-CoV-2, the etiological agent of COVID 19, contains several essential pathogenic molecules that are modified by S-acylation.
Vaccines, antibodies and pathogen-directed antibiotics or antivirals are effective therapeutic strategies that work by targeting the specific infectious agent(s) they were designed for. Host-directed anti-microbial drugs, in the other hand, have the potential to broadly target infectious agents with a lower risk of rapid resistance development (Fernandez-Oliva et al, 2019; Kaufmann et al, 2018).
Large spectrum therapeutic strategies are also invaluable for susceptible patients that cannot develop adequate immunity or are not eligible for vaccination such as immunocompromised individuals.
The emergence of drug-resistance and of new pathogens, such as SARS CoV-2, highlights the importance of alternative approaches to be used as first line wide-ranging interventions before specific therapies may be elaborated or immunity emerges.
Thus, there is a continuous and urgent necessity for the development of new drugs and molecular targets to strengthen the global public health preparedness against viral, bacterial, and other infection outbreaks.
SUMMARY OF THE INVENTION
The present invention provides an inhibitor of acyl protein thioesterase (APT) of
Figure imgf000003_0001
or a pharmaceutically acceptable salt thereof, wherein Ring A is a thiophene ring or a furane ring;
Each RAm is independently selected from the group comprising hydrogen, halogen, Ci- C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups; and m is an integer from 0 to 2;
Each R1 , R2, R4, R5 is independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, optionally substituted Ci-Ce alkyl acyl, optionally substituted Ci-Ce alkyl aminocarbonyl, optionally substituted carbonyl, optionally substituted Ci-Ce alkyl carbonyl, optionally substituted Ce-C aryl, ether, C2-C10 alkenyl, C6-C10 heteroaryl, C3- C10 cycloalkyl, C3-C10 heterocycloalkyl, Ci-Ce alkoxy, optionally substituted amino, and/or Ci-Ce alkyl amino; and
R3 is selected from the group comprising
Figure imgf000004_0001
The invention also provides said inhibitors of APT of formula (II), or pharmaceutically acceptable salts thereof, for use as a medicament.
Further, the invention provides said inhibitors of APT of formula (II), or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
The invention also relates to a pharmaceutical composition comprising at least one of said inhibitors of APT, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
The invention also provides a kit comprising said inhibitors of APT, or pharmaceutically acceptable salts thereof, and information for use thereof.
The invention further provides an inhibitor of APT of formula
Figure imgf000005_0001
(ML349), or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
The invention further provides an inhibitor of APT of formula
Figure imgf000005_0002
(ML348), or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : Labelling of surface proteins with NHS-biotin in mammalian cells untreated or treated with ML349 (10 pM) for the indicated times. Analysis of surface proteins ANTXR2/CMG2 by western blot. The quantification of surface ANTXR2/CMG2 is expressed as % of T=0. Results are mean ±SD.
Figure 2 (A) and (B): Synchronized anthrax intoxication assays in mammalian cells untreated (control) or co-treated with ML349 or ML348 (10 pM) for the indicated times. Anthrax toxin Protective Antigen (PA); (C): The dynamics of toxin pore formation (PA Pore); (D): MEK2 degradation. Quantification for each time point for cells pre-treated for 2 h and expressed as % of t=0 for each sample. All results are mean ±SEM.
Figure 3 (A): Synchronized anthrax intoxication assays in mammalian cells transfected with control siRNA or siRNA oligos targeting the expression of APT2 (siAPT2) for 72 h; (B) The dynamics of toxin pore formation (PA Pore) were quantified for each time point and expressed as % of t=0 for each sample. All results are mean ±SEM. Figure 4 (A), (B) and (C): Microscopy-based quantification assay of Vero E6 cells infected with SARS-CoV-2-Spike-pseudotyped particles (VSV-S-CoV2) (A, C) or VSV-G- pseudotyped particles (VSVG) (B). (C): Dose response curve calculated for ML348, ML349, and hydroxychloroquine. Results are mean ±SEM (A,B), or SD (C).
Figure 5 (A): Infection with SARS-CoV-2 determined using a plaque assay of Vero E6 cells pre-treated with ML348 or ML349 and infected in the presence of increasing concentrations of each drug. Dose-response curves were obtained using non-linear regression. (B): Vero E6 cells infected with SARS-CoV-2 (MOI 0.1 ) in the presence of 12.5 pM of ML348 or ML349. Replication was monitored by determining the % of high Spike-positive cells by flow cytometry. All results are mean ±SEM, and each data point (in B) represents an independent experiment.
Figure 6 (A, B): Vero E6 cells inoculated for 1 h with SARS-CoV-2, washed, and subsequently incubated in complete medium with 12.5 pM of ML348. Cells were harvested at the indicated time points and infection was determined by monitoring the levels of intracellular E and RNA-dependent RNA-polymerase-RdRp RNA transcripts by Q-PCR. T=0 was harvested 1 h after inoculation before drug treatment. All results are mean ±SEM.
Figure 7 (A, B): Quantification of viral RNA replication (E, RdRp and E subgenomic) from Vero E6 cells (A) and correspondent supernatants (B) infected with SARS-CoV-2 (MOI- 0.1 for 24 h). Cells were inoculated for 1 h with SARS-CoV-2, washed and subsequently incubated in complete medium with 12.5 pM of ML348 or ML349. (C): Cells were previously transfected with control or siRNA oligos targeting the expression of APT1 (siAPT) for 72 h. (D): Quantification of APT1 mRNA levels from the samples used in C. All results (A, B, C) are mean ±SEM and p values comparing RNA levels in relation to control conditions were obtained by (A, B, C) one-way ANOVA with Dunnet’s multiple comparison or (D) Paired two-tailed student’s t-test (*p < 0.05, ***p < 0.001 , ****p < 0.0001 ) Figure 8 (A): Vero E6 cells were pre-treated with drug for 1 h washed, and infected with HSV-2, in the presence of drug. Infection was determined using a plaque assay. Results are mean ±SEM, and dose-response curves were obtained using non-linear regression.
Figure 9 (A): LYPLA1 (APT1 ) and LYPLA2 (APT2) mRNA levels in different cell lines. Values are TAG per Million (TPM) corresponding to 10*6 X (reads mapped to transcript/transcript length)/ SUM (reads mapped to transcript/transcript length). (B and C): Quantification of viral RNA replication (E, RdRp and E subgenomic) from Calu-3 cells and correspondent supernatants infected with SARS-CoV-2 (MOI-0.1 for 24 h), treated as indicated. (D): Quantification of APT2 mRNA levels from the samples used in C. For all, each data point represents an individual experiment and results are mean (A) or mean ±SEM (B, C, D). All p values comparing RNA levels in relation to control conditions were obtained by (B, C) one-way ANOVA with Dunnet’s multiple comparison or (D) Paired two- tailed student’s t-test (*p < 0.05, **p < 0.01 , ****p < 0.0001 )
Figure 10: dose response inhibition of SARS-CoV-2 replication (infection at MQI=0.05- 01 ) in Calu-3 cells by ML349 and cytotoxicity evaluation of ML348 and ML349 in Vero E6 and CALU-3 cells, respectively. All results are mean ±SEM and the dose inhibition curve (A) was fitted using a nonlinear regression.
Figure 1 1 (A): dose-response inhibition of SARS-CoV-2 replication (infection at MQI=0.05-01 ) in human lung-derived Calu-3 cells by ML349 and compounds C1 , C2 and C3, 24 h post viral inoculation, quantified by QPCR of total viral RNA (E, RdRp and E sub-genomic). Drugs were added 1 h a post-viral inoculation (B): levels of housekeeping (HK) transcripts during infection, indicative of cell viability. For each individual concentration results are mean ±SEM of n >3. Dose inhibition curves were fitted using a nonlinear regression and EC50 ±SD concentrations are indicated
Figure 12 (A): dose response inhibitions of SARS-CoV-2 replication (infection at MOI «0.05-01 ) in human lung-derived Calu-3 cells by ML349-derived compounds C5, C6, and C7, 24 h post-viral inoculation, quantified by QPCR of total viral RNA (E, RdRp and E sub-genomic). (B): levels of housekeeping (HK) transcripts during infection, indicative of cell viability. For each concentration, results are mean ±SEM of n >3. Dose inhibition curves were fitted using nonlinear regression, and EC50 ±SD concentrations are indicated.
Figure 13 shows replication of SARS-CoV-2 inoculated in human lung-derived Calu-3 cells (MOI«0.5, 1 h), incubated with infection media supplemented with 5 pM of compound C2 and 0.5 pg/ml of P2G3 anti-Spike antibody, from 1 h post viral inoculation. Cells were harvested at the indicated time points and infection was determined by monitoring the levels of intracellular (A): genomic (E and RdRp) and (B): sub-genomic E (E-subgen) RNA transcripts by QPCR. Values were normalised to 1 for the timepoint 1 h post viral inoculation (before drug treatment) and results are mean ±SEM of n>3. Data is presented as equivalent side-by-side plot graphs with log or linear scale representation of viral RNA levels - Fold-infection.
Figure 14 (A, B and C): evaluation of levels of viable Calu-3 cells in the presence of the cell permeable RealTime-GloTM-Cell Viability reagent and incubated with the indicated concentrations of ML349, C2 and C3 for 6 days. Culture medium was replaced every 2 days using fresh ML349, C2 and C3 concentrations. Luminescence, corresponding to the levels of viable cells, was monitored at the indicated time points and the results are mean ±SEM of n=4. Fitted curves indicate Control (full-line), non-toxic (dotted-lines) and cytotoxic (>25 pM dashed-lines) concentrations. (D): dose-dependent cell viability at day 6 after drug treatment monitored as the % of viable cells normalised to Control (no drug). A significant decrease in cell viability was verified for all compounds when used at 25 to 100 pM (p<0.05 obtained by Two-way ANOVA).
Figure 15(A): dose-response inhibition of replication of SARS-CoV-2 variant Omicron BA.5 (infection at MOI =0.05-01 ) in human lung-derived Calu-3 cells by compound 2 (C2), 24 h post-viral inoculation, quantified by QPCR of total viral RNA (E, RdRp and E sub- genomic). (B): levels of housekeeping (HK) transcripts during infection, indicative of cell viability. For each concentration, results are mean ±SEM of n >3. The dose inhibition curve was fitted using nonlinear regression, and EC50 ±SD concentrations are indicated.
Figure 16: toxicity evaluation of inhibiting APT2 with ML349 and C2 in vivo in C57BL/6 mice. (A): Body weight (g); (B): blood pressure (Hgmm); and (C): pulse (beats per minute - bpm) were monitored for the indicated time points for every mouse mock- treated or treated with 1 mg of ML349 or C2 per 25 g of body weight for several days through 13 days. Mock-treated control mice were inoculated in parallel with equivalent volumes of vehicle solution used to resuspend ML349 and C2. At 13 days of treatment, mice were sacrificed, and the relative spleen weight (as % of body weight) was determined, (D): Results are mean ±SEM of n=8 mice per condition for A, B, and C and n=5 for D. In D, each data point represents an individual mouse.
Figure 17(A): Schematic representation of the APT2-dependent mechanism of synchronized anthrax toxin entry into host target cells. In brief, in 1 , the toxin binds to the S-acylated anthrax receptor (CMG2) at the cell surface; this leads to 2, the recruitment and binding of APT2 to CMG2, which, in turn, promotes, 3, the complete de-acylation of the receptor; and 4, its dissociation from the host cytoskeleton components (actin, vinculin, and talin) and binding to the host GTPase RhoA. These events enable 5, the endocytosis of the toxin and access to its host target. (B): western blot analysis of CMG2- immunoprecipitation (IP) fractions to monitor the co-immunoprecipitation (co-IP) of APT2- CMG2 complexes from cell lysates of control (untreated) or anthrax toxin-treated cells. Mammalian RPE-1 cells ectopically expressing FLAG-APT2, for 24 h, were left untreated or treated with anthrax toxin (protective antigen-PA subunit) in the presence of APT2 inhibitors, ML349 or compounds C2, C3, or control drug carrier DMSO. Drugs were added at a final concentration of 5 pM, 4 h before and during toxin treatment. B and respective quantification in C show that inhibition of APT2 activity with ML349 and compound of formula C2 and C3 reduces APT2-CMG2 complexes formed upon PA toxin treatment. (C): quantification of APT2-FLAG levels in equivalent CMG2 IP fractions. Results are mean ±SEM of n=4, (****p<0.0001 obtained by one-way ANOVA).
Figure 18: Western blot analysis of APT2-CMG2 complexes present in CMG2- immunoprecipitation (IP) fractions from cell lysates of control (untreated) or anthrax toxin- treated cells. (A): RPE-1 cells ectopically expressing APT2-FLAG for 24 h, left untreated or treated with anthrax toxin (protective antigen-PA subunit) in the presence of APT2 inhibitors, ML349 or compound 02, C3, or control drug carrier DMSO. Drugs were added at a final concentration of 2 pM, 4 h before and during toxin treatment. (B): quantification of APT2-FLAG levels in equivalent CMG2 fractions displayed in A. Figure 19: Quantification of APT2-FLAG levels in equivalent CMG2 fractions from western blot analysis of APT2-CMG2 complexes present in CMG2-immunoprecipitation (IP) fractions from cell lysates of control (untreated) or PA-toxin-treated cells: RPE-1 cells ectopically expressing APT2-FLAG for 24 h, left untreated or treated with anthrax toxin (protective antigen-PA subunit) in the presence of APT2 inhibitors, ML349 or compound of formula (II) (C9, C12, C13, C16, and C17), or control drug carrier DMSO. Drugs were added at a final concentration of 5 pM, 4 h before and during toxin treatment. Data are normalized to 100% for WT, and results are mean or mean ±SEM, and each data point represents an individual experiment.
Figure 20: Quantification of western blot analysis of the dynamics of toxin pore formation (PA-Pore) in RPE-1 mammalian cells treated with anthrax toxin (protective antigen-PA subunit) in the presence of APT2 inhibitors (C2, C9, C12, C13, C16 at 5 pM) for the indicated times (0, 30 and 60 min). Toxin pore formation (PA Pore) was quantified for each time-point and normalized to equivalent levels of PA63. Data were expressed as % of t0=1 for each sample.
Figure 21 : Inhibition of SARS-CoV-2 replication in human lung-derived Calu-3 cells (MOI«0.05-0.1 , 1 h), in the presence of the APT2 inhibitors, ML349, C2, C9, C12, C13, C15, C16 and C17 (used at 5 pM), from 1 h post-viral inoculation. Culture supernatants and infected cells were harvested 24 h post-viral inoculation. The levels of viral nucleocapsid protein N: cell lysates, were monitored by Western blot. The % of infection was quantified as the ratio between the levels of N and host GAPDH for three independent replicates for each compound with relation to Control cells (infected in the presence of drug carrier DMSO). Results are mean ±SEM of n=3, and p values were obtained by one-way ANOVA analysis.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention relates to an inhibitor of acyl protein thioesterase (APT) of formula (II):
Figure imgf000011_0001
or a pharmaceutically acceptable salt thereof, wherein
Ring A is a thiophene ring or a furane ring;
Each RAm is independently selected from the group comprising hydrogen, halogen, Ci- C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups; and m is an integer from 0 to 2; Each R1 , R2, R4, R5 is independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, optionally substituted Ci-Ce alkyl acyl, optionally substituted Ci-Ce alkyl aminocarbonyl, optionally substituted carbonyl, optionally substituted Ci-Ce alkyl carbonyl, optionally substituted C6-C10 aryl, ether, C2-C10 alkenyl, C6-C10 heteroaryl, C3- C10 cycloalkyl, C3-C10 heterocycloalkyl, Ci-Ce alkoxy, optionally substituted amino, and/or Ci-Ce alkyl amino; and
R3 is selected from the group comprising
Figure imgf000011_0002
Preferably, the ring A is a thiophene ring represented by formula:
Figure imgf000012_0001
wherein: each R6, R7 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups.
More preferably, the ring A is a thiophene ring represented by formula:
Figure imgf000012_0002
wherein: each R6, R7 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group. Even more preferably, the ring A is a thiophene ring represented by formula:
Figure imgf000012_0003
Even more preferably, the ring A is a thiophene ring represented by formula:
Figure imgf000013_0001
The following definitions are supplied to facilitate the understanding of the present invention.
As used herein, the term “comprise” is generally used in the sense of include, permitting the presence of one or more features or components.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
The following paragraphs provide definitions of the various chemical moieties that make up the compounds according to the invention and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.
“Halogen” refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
“C1-C10 alkyl” refers to monovalent straight-chained and branched alkyl groups having 1 to 10 carbon atoms. This term is exemplified by groups such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec. butyl, tert, butyl, n-pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, 2-ethylhexyl, 1 ,1 ,3,3-tetramethylbutyl, n-heptyl, 2,4,4 trimethylpentyl, 2- ethylhexyl, octyl, nonyl, decyl, and the like.
“Ci-Cio-heteroalkyl” includes both straight-chained and branched C1-C10 alkyl groups according to the definition above, having at least one or more heteroatoms selected from S, O and N. “Ci-Ce-haloalkyl” includes both straight-chained and branched Ci-Ce alkyl groups according to the definition above, having at least one or more halogen selected from F, Cl, Br or I.
“Ci-Ce acyl” or “Carbonyl” refers to the group -C(O)R where R includes hydrogen, “Ci-Ce- alkyl”, “aryl”, substituted aryl, “heteroaryl”, “Ci-Ce-alkyl aryl”, “Ci-Ce-alkyl heteroaryl”, or substituted amine (-NH-R’) wherein R’ is optionally substituted aryl.
“Ci-Ce alkyl acyl” refers to Ci-Ce alkyl groups having a Ci-Ce acyl substituent as defined herein.
“Ci-Ce alkyl carbonyl” refers to Ci-Ce alkyl groups having a “carbonyl” substituent as defined herein.
“aminocarbonyl” refers to the chemical moiety -C(0)NRR’ in which each of R and R’ is independently hydrogen, Ci-Ce alkyl, aryl, substituted aryl, heteroaryl, Ci-Ce alkyl aryl, or Ci-Ce alkyl heteroaryl.
“Ci-Ce alkyl aminocarbonyl” refers to Ci-Ce alkyl groups having an “aminocarbonyl” substituent as defined herein.
“C3-C10 cycloalkyl” refers to a saturated carbocyclic group of from 3 to 10 carbon atoms having a single ring (e.g., cyclohexyl) or multiple condensed rings (e.g., norborn yl). Preferred cycloalkyl include cyclopentyl, cyclohexyl, norbornyl and the like.
“Ci-Ce alkyl cycloalkyl” refers to Ci-Ce-alkyl groups having a cycloalkyl substituent, including cyclohexylmethyl, cyclopentylpropyl, and the like.
“C3-C10 heterocycloalkyl” refers to Cs-Cio-cycloalkyl group according to the definition above, in which up to 3 carbon atoms are replaced by heteroatoms chosen from the group consisting of O, S, NR, R being defined as hydrogen or methyl. Preferred heterocycloalkyl include cyclohexane in which 1 carbon atom is replaced by S(O)2, pyrrolidine, piperidine, piperazine, 1 -methylpiperazine, and the like.
As used herein, “optionally substituted C3-C10 heterocycloalkyl” refers to a C3-C10 heterocycloalkyl as defined above that may be fused with 0 to 2 further groups selected from cycloalkyl, heterocycloalkyl, aryl or heteroaryl group. Preferred groups are benzene, toluene, phenol, aniline, anisole, and the like.
"Ci-Ce-alkyl heterocycloalkyl" refers to Ci-Ce-alkyl groups having a heterocycloalkyl substituent as defined herein.
“C2-Cio-alkenyl” refers to alkenyl groups preferably having from 2 to 10 carbon atoms and having at least 1 or 2 sites of alkenyl unsaturation. Preferable alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl and octenyl, and the like. The term "alkenyl" in the meaning of the present invention includes the cis and trans isomers.
“Ci-C6 alkoxy” refers to group -O-R’ where R’ includes both straight-chained and branched "Ci-Ce alkyl" or " Ci-Ce haloalkyl" or " Ci-Ce heteroalkyl" or "aryl" or "heteroaryl" or "Ci-Ce-alkyl aryl" or "Ci-Ce-alkyl heteroaryl". Preferred alkoxy groups include by way of example, methoxy, ethoxy, propoxy, butoxy, phenoxy and the like.
“Amino” refers to -NRR’ in which each of R and R’ is independently hydrogen, Ci-Ce alkyl, substituted aryl, heteroaryl, Ci-Ce alkyl aryl, Ci-Ce alkyl heteroaryl, cycloalkyl, or heterocycloalkyl.
"Ci-Ce-alkyl amino” refers to Ci-Ce-alkyl groups having an amino substituent as defined herein.
“C6-C10 aryl” refers to an unsaturated aromatic carbocyclic group from 6 to 10 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl). Preferred aryl groups include phenyl, naphthyl, phenantrenyl and the like.
As used herein, optionally substituted "C6-C10 aryl" refers to a “C6-C10 aryl” as defined above that may be substituted with 0 to 3 further groups independently selected from halogen (Cl, Br, F, I), Ci-Ce-haloalkyl, and alkoxy as defined herein. Preferred groups are -Cl, -CF3 and -OCH3.
“C6-C10 heteroaryl” refers to a monocyclic heteroaromatic, or a bicyclic or a tricyclic fused- ring heteroaromatic group in which up to 3 carbon atoms are replaced by heteroatoms chosen from the group consisting of O, S, or N. “ether” refers to -R-O-R’ in which each of R and R’ is independently Ci-Ce alkyl, “Ce-Cio aryl”, and Ci-Ce cycloalkyl.
The present invention also relates to an inhibitor of APT of formula (II):
Figure imgf000016_0001
or a pharmaceutically acceptable salt thereof, wherein
Ring A is a thiophene ring or a furane ring;
Each RAm is independently selected from the group comprising hydrogen, halogen, Ci- C10 alkyl, C2-C10 alkenyl, Ci-Ce alkoxy, Ci-Ce acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, Ce-Cio aryl, and/or Ce-Cio heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups; and m is an integer from 0 to 2;
Each R1 , R2, R4, R5 is independently selected from the group comprising hydrogen, optionally substituted Ci-Ce alkyl, optionally substituted Ce-Cio aryl; Preferably each R1 , R2, R4, R5 is hydrogen; and
R3 is selected from the group comprising
Figure imgf000017_0001
5 Preferably, the ring A is a thiophene ring represented by formula:
Figure imgf000017_0002
The present invention also relates to an inhibitor of APT of formula (II) selected from the group comprising
Figure imgf000017_0003
Figure imgf000018_0001
or a pharmaceutically acceptable salt thereof.
Preferably, the inhibitor of APT of formula (II) is selected from the group comprising
Figure imgf000018_0002
More preferably, the inhibitor of APT of formula (II) is
Figure imgf000019_0001
In another aspect, the present invention also relates to an inhibitor of APT of formula (II), as described herein, or a pharmaceutically acceptable salt thereof, for use as a medicament.
In a further aspect, the present invention relates to an inhibitor of APT of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
The present invention also relates to a method of treating and/or preventing a viral and/or a bacterial infection, the method comprising administering an effective amount in a subject in need thereof of at least one inhibitor of APT of formula (II), or any pharmaceutically acceptable salt thereof as described herein.
In another aspect, the present invention also relates to an inhibitor of APT of formula
Figure imgf000019_0002
, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a viral and/or a bacterial infection. The present invention also relates to a method of treating and/or preventing a viral and/or a bacterial infection, the method comprising administering an effective amount in a subject in need thereof an inhibitor of APT of formula
Figure imgf000020_0001
, or any pharmaceutically acceptable salt thereof.
Further, the present invention provides inhibitors of acyl protein thioesterase (APT) of formula (II) as described herein, and/or an inhibitor of APT of formula
Figure imgf000020_0002
or any pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of viral infections mediated by or resulting from acyl protein thioesterases activity, and/or bacterial infections mediated by or resulting from acyl protein thioesterases activity.
As used herein, acyl protein thioesterases (APT) are enzymes that cleave off lipid modifications on proteins, located on the sulfur atom of cysteine residues linked via a thioester bond. In particular, the APT, such as acyl-protein thioesterase 1 (APT1 ) and acyl-protein thioesterase 2 (APT2) are involved in the depalmitoylation of proteins.
Viral infections mediated by or resulting from acyl protein thioesterases activity are infections of multiple human systems including infections of the upper and lower respiratory tract, infections of the skin, infections of the eye, infections of the gastrointestinal tract, infections of the mouth, infections of the urinary genital tract, infections of the intestinal tract, infections of the brain (e.g. meningitidis or encephalitis), and also systemic, multi organ infections such as haemorrhagic fevers.
For example, viral infections or viral infections associated diseases mediated by or resulting from acyl protein thioesterases activity are selected from the group comprising COVID, SARS, flu, rheumatic diseases, encephalitis, haemorrhagic fevers, acquired immunodeficiency syndrome (AIDS), sarcoma and/or leukemia.
Bacterial infections or bacterial infections mediated by or resulting from acyl protein thioesterases activity are associated with infections of the upper and lower respiratory tract, infections of the skin, infections of the eye, infections of the gastro-intestinal tract, infections of the mouth, infections of the urinary tract, infections of the intestinal tract, infections of the uterus, and infections of the brain (e.g., meningitidis).
Bacterial infections associated diseases mediated by or resulting from acyl protein thioesterases activity are selected from the group comprising Typhoid fever, Salmonellosis, bacteria Gastroenteritis intestinal infection, bacteremia, Shigellosis, Bacteria pneumonia (e.g., legionnaires disease), endophthalmitis, bacteremia, septicemia, sepsis, endocarditis, salpingitis, skin infections, and/or meningitis.
Thus, the present invention provides inhibitors of acyl protein thioesterase (APT) as described herein, or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of a viral and/or bacterial infection selected from the group comprising, SARS, COVID, flu, rheumatic diseases, encephalitis, haemorrhagic fevers, acquired immunodeficiency syndrome (AIDS), sarcoma, leukemia, meningitidis, encephalitis, bacteraemia and sepsis.
The present invention also provides inhibitors of acyl protein thioesterase (APT) of formula (II) as described herein, and/or an inhibitor of APT of formula
Figure imgf000022_0001
or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of a microbial infection, wherein said microbial infection is a co-infection comprising a viral infection and a bacterial infection.
Together, the viruses or viral families here represented (e.g. Orthomyxoviridae, Paramyxoviridae, Togaviridae, Rhabdoviridae, Filoviridae, Pneumoviridae Coronaviridae, Retroviridae, Herpesviridae, Flaviviridae, Hepeviridae, Bunyaviridae) are responsible of a variety of human diseases that comprise the infection of multiple human systems, including: the respiratory tract (upper and lower), the skin, the eye, the gastrointestinal tract, the mouth, the urinary genital and intestinal tract, the brain (e.g., meningitidis or encephalitis), and also systemic, multi organ infections such as haemorrhagic fevers.
In the present invention, preferably said viral infection can be caused by a virus selected from the group comprising Coronaviruses (CoV) (such as severe acute respiratory syndrome coronavirus (SARS), Middle East respiratory syndrome (MERS), Mouse Hepatitis Virus (MHV), transmissible gastroenteritis virus (TGEV), Swine acute diarrhoea syndrome coronavirus (SADS-cov), Rousettus bat coronavirus HKU9, and any other bat CoVs), Influenza virus (such as A, B,C), Respiratory syncytial virus (hRSV), Newcastle disease virus (NDV), Measles virus, Sindbis virus, Semliki Forest virus (SFV), Rabies virus, Vesicular Stomatitis Virus (VSV), human immunodeficiency virus (HIV), murine leukemia viruses (MulV), Ebola virus, Marburg virus, Herpes simplex virus (HSV), Human cytomegalovirus (HCMV), Dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus, Lassa virus, Nipah virus, Hepatitis E virus, Simian immunodeficiency virus, Lymphocytic choriomeningitis virus, and/or Puumala virus. For example, said Coronavirus (CoV) can be selected from SARS-CoV-2, SARS-CoV-1 , MHV, TGEV, SADS, MERS-CoV, HCoV-HKLH , HCoV-229E, HCoV-NL63 and/or HCoV- OC43.
In the present invention, preferably said bacterial infection is caused by bacterial strains selected from the group comprising Bacillaceae, Vibrionaceae, Pectobacteriaceae Yersiniaceae, Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales and/or Pasteurellaceae.
These bacterial families or strains are associated with infections of the respiratory tract (upper and lower), the skin, the eye, the gastro-intestinal tract, the mouth, the urinary and intestinal tract, uterus infections and other sexual transmitted diseases, brain infections (e.g., meningitidis), bacteraemia and sepsis.
A pathogen from family Bacillaceae is for example Bacillus anthracis, a pathogen from family Pectobacteriaceae is for example Erwinia spp (carotovora), a pathogen from family Pseudomonadaceae is for example Pseudomonas spp (syringae), a pathogen from family Enterobacteriaceae is for example Salmonella enterica or Shigella spp, a pathogen from family Legionellaceae is for example Legionella pneumophila, a pathogen from family Chlamydiaceae is for example Chlamydia trachomatis.
In the present invention, novel inhibitors of formula (II) as described herein, have been synthesized (Example 1 1 , compounds C1 , C2, C3, C9, C12, C13, C16 and C17).
The inhibition of SARS-CoV-2 intracellular replication has been observed in Calu-3 cells at EC50 concentrations below 2.5 pM without inducing cellular cytotoxicity. Advantageously, novel inhibitors of formula (II) as described herein, demonstrate an EC50 that is decreased by about 3 to 8-fold compared to compound ML-349 (Example 12, figures 1 1 A and B). In contrast, compounds such as C5, C6, C7 that are not covered by formula (II) as described herein, are not potent inhibitors of SARS-CoV2 replication in Calu-3 cells (Example 12, Figure 12). Furthermore, the inhibition of SARS-CoV-2 infection with an inhibitor of formula (II), such as C2 has been observed as early as 4 h after viral entry into cells (Figure 13A and 13B), thus confirming the inhibitory effect of the intracellular viral RNA replication (Example 13).
In addition, inhibitors of formula (II) are effective without inducing cytotoxic effects in vitro (Example 14, figure 14) or in vivo (Example 16, Figure 16).
Furthermore, it has been demonstrated that inhibition of APT2 activity with an inhibitor of general formula (II), such as C2 or a compound of formula ML349, inhibits replication of SARS-CoV-2 variant Omicron BA.5 (Example 15, Figure 15). Results have been demonstrated for different compounds of formula (II) such as C2, C3, C9, C12, C13, C16 and C17 (Example 20, figure 21 ).
Besides, it has been demonstrated that inhibitors of formula (II) as described herein such as C2, C3 or a compound of formula ML349, can inhibit the APT2-dependent anthrax toxin intoxication of host cells (Example 17, figure 17 and Example 18, figure 18). Results have also been demonstrated for different compounds of formula (II) such as C2, C9, C12, C13, C16 and C17 (Example 19, figures 19 and 20).
In another aspect, the present invention provides a pharmaceutical composition comprising at least one inhibitor of APT of formula (II) as described herein, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
As to the appropriate carriers, reference may be made to the standard literature describing these, e.g. to chapter 25.2 of Vol. 5 of “Comprehensive Medicinal Chemistry”, Pergamon Press 1990, and to “Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete”, by H.P. Fiedler, Editio Cantor, 2002. The term “pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe and possesses acceptable toxicities. Acceptable carriers include those that are acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier” as used in the specification and claims includes both one and more than one such carrier. The present invention also relates to a pharmaceutical composition, comprising at least one inhibitor of APT of formula (II) as described herein, or pharmaceutically acceptable salts thereof, further comprising one or more additional therapeutic agents and a pharmaceutically acceptable carrier, diluent or excipient.
Preferably, said one or more additional therapeutic agents are selected from the group comprising antibacterial agents and/or antiviral agents.
For example, the at least one or more additional therapeutic agent is an antibacterial agent selected from the group comprising sulfonamides, penicillins, cephalosporins, aminoglycosides, chloramphenicol, tetracyclines, macrolides, lincosamides, streptogramins, glycopeptides, rifamycins, nitroimidiazoles, quinolones, trimethoprim, oxazolidinones, and/or lipopeptides and combinations thereof.
For example, the at least one or more additional therapeutic agent is an antiviral agent selected from the group comprising Aribidol (umifenovir), Favilavir, APN01 , CCR5 antagonist leronlimab (PRO 140), Remdesivir (GS-5734), Galidesivir (BCX4430), Molnupiravir (MK-4482 I FJDD-2801 ), MK-7110 (CD24Fc).
The compounds of the invention, namely inhibitor of acyl protein thioesterase (APT) as described herein, or a pharmaceutically acceptable salt thereof, that can be used in the treatment and/or prevention of a microbial infection can be incorporated into a variety of formulations and medicaments for therapeutic administration. More particularly, one or more compound(s) as provided herein can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, intranasal, intramuscular, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracranial and/or intratracheal administration. Moreover, the compound can be administered in a local rather than systemic manner, in a depot or sustained release formulation. The compounds can be formulated with common excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for convenient oral administration or administered by the intramuscular or intravenous routes. The compounds can be administered transdermally and can be formulated as sustained release dosage forms and the like. The compounds can be administered alone, in combination with each other, or they can be used in combination with other known compounds. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (Mack Publishing Company (1985) Philadelphia, PA, 17th ed.), which is incorporated herein by reference. Moreover, for a brief review of methods for drug delivery, see, Langer, Science (1990) 249:1527-1533, which is incorporated herein by reference.
Preferably, said pharmaceutical composition is administered by oral, inhalation, nebulization, intranasal, intrapulmonary, intradermal and/or intramuscular route of administration.
In particular, drug substances can be delivered to the respiratory system using various devices such as nebulizer or spray.
The amount of a compound as provided herein that can be combined with a carrier material to produce a single dosage form will vary depending upon the disease treated, the subject in need thereof, and the particular mode of administration. However, as a general guide, suitable unit doses for the compounds of the present invention can, for example, preferably contain between 0.1 mg to about 1000 mg, between 1 mg to about 500 mg, and between 1 mg to about 300 mg of the active compound. In another example, the unit dose is between 1 mg to about 100 mg. Such unit doses can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total dosage for a 70 kg human adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration. A preferred dosage is 0.01 to about 1.5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area. A typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The timerelease effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release. It can be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art.
In another aspect, the present invention provides a kit comprising an inhibitor of APT of formula (II), as disclosed herein, or a pharmaceutically acceptable salt thereof, and information for use thereof. The kit may further comprise one or more additional therapeutic agents, such as antibacterial agents and/or antiviral agents.
In another aspect, the present invention provides an inhibitor of acyl protein thioesterase (APT) of general formula (I):
Figure imgf000027_0001
or a pharmaceutically acceptable salt thereof, wherein
R’ is independently selected from the group comprising optionally substituted thiophene ring, optionally substituted furane ring, optionally substituted C3-C10 cycloalkyl, or optionally substituted C6-C10 aryl; and
Each R1 , R2, R3, R4, R5 is independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, optionally substituted Ci-Ce alkyl acyl, optionally substituted Ci-Ce alkyl aminocarbonyl, optionally substituted carbonyl, optionally substituted Ci-Ce alkyl carbonyl, optionally substituted C6-C10 aryl, ether, C2-C10 alkenyl, C6-C10 heteroaryl, C3- C10 cycloalkyl, C3-C10 heterocycloalkyl, Ci-Ce alkoxy, optionally substituted amino, and/or Ci-Ce alkyl amino; for use in the treatment and/or prevention of a viral and/or a bacterial infection.
Preferably, R’ is independently selected from the group comprising optionally substituted thiophene ring, or optionally substituted furane ring.
In one embodiment, R’ is a thiophene ring represented by formula:
Figure imgf000028_0001
wherein: each R6, R7 are independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups.
Preferably, R’ is a thiophene ring represented by formula:
Figure imgf000028_0002
wherein: each R6, R7 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.
More preferably, R’ is a thiophene ring represented by formula:
Figure imgf000029_0001
Even more preferably, R’ is a thiophene ring represented by formula
Figure imgf000029_0002
Thus, the present invention relates to an inhibitor of acyl protein thioesterase (APT) of formula
Figure imgf000029_0003
or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
In another embodiment, R’ is a furane ring represented by formula:
Figure imgf000029_0004
wherein: each R8, R9, are independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups.
Preferably, R’ is a furane ring represented by formula:
Figure imgf000030_0001
wherein: each R8, R9 are independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, Ci-Ce alkoxy, Ci-Ce acyl, C3-C10 cycloalkyl, and/or optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.
More preferably, the ring A is a furane ring represented by formula:
Figure imgf000030_0002
wherein: each R8, R9 are independently selected from the group comprising hydrogen, C3-C10 cycloalkyl, and/or optionally substituted C3-C10 heterocycloalkyl, wherein said cycloalkyl, or optionally substituted heterocycloalkyl, may be fused with ring A and with 1 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.
Even more preferably, the ring A is a furane ring represented by formula:
Figure imgf000031_0001
Thus, the present invention relates to an inhibitor of acyl protein thioesterase (APT) of formula
Figure imgf000031_0002
(ML348), or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
As shown in the examples, inhibitors of acyl protein thioesterases prevent intoxication of mammalian cells with Anthrax toxin by inhibiting the internalization and recycling of the toxin receptor capillary morphogenesis gene 2 (CMG2/ANTXR2) (Example 1 , Figure 1 ) and by blocking endocytosis and pore formation of the anthrax toxin (Example 2, Figure 2).
Furthermore, inhibitors of acyl protein thioesterase (APT1 and 2) block SARS-CoV-2 cellular infection (Figures 5 and 9) and replication (Figures 6-7). It has also been demonstrated that inhibitors of acyl protein thioesterase block cellular infection by other type of virus such as HSV-2 (Figure 8). A dose response was determined for ML349 in Calu-3 cells and the EC50 is 8.6 pM (Figure 10A).
Besides, any viral infections, bacterial infections, pharmaceutical composition, formulations, and combinations that have been described herein are incorporated in this aspect of the invention. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
EXAMPLES
Materials and methods
Cell lines
RPE1 (ATCC CRL-4000), VERO E6 (ATCC CVCL-0574) and CALU-3 (ATCC HTB55) cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, penicillin, and streptomycin. siRNA
Verified siRNA for human APT2 were purchased from Qiagen. APT2 target sequence: 5’- CAGCTGCTTCTCAGTCATGAA-3’. A pool of verified siRNA was used to target monkey APT1 and purchased from Qiagen. APT1 target sequence: 5’- TACCGACAGGACCCTGTGGAA-3’, 5’-GGTCACAGATATACGGTATNN-3’, 5’- CCGGTGTATGTGCGGCAATNN-3’. As a control siRNA, a sequence targeting the viral glycoprotein VSV-G (5’-ATTGAACAAACGAAACAAGGA-3’) was used. Transfections of 50 nM of siRNA were carried out using TRANSIT-X2 (MIRUS), and the cells were analysed at least 72 h after transfection.
Antibodies and Reagents
The antibodies were commercially available: rabbit anti-human CMG2 (Proteintech, RRID: AB_2056741 ); mouse anti-GAPDH (Acris Antibodies, 4A1 -MA0100, RRID_AB 1874646) ; mouse anti-LPXN (Sigma, SAB1400343), anti-Anthrax Protective Antigen (List Biological Laboratories, ref: 771 B); N-terminal rabbit anti-MEK2 (Santa Cruz, sc-524), rabbit anti-SPIKE (LIFESPAN, Cat#LS-C19510), HRP-conjugated secondary antibodies (Pierce); and for immunoprecipitation protein G beads were purchased from GE Healthcare, and Streptavidin beads from Sigma.
ML348, ML349 (Cayman Chemical company) and synthesised compounds (C1 , C2, C3, C9, C12, C13, C16, C17) were used at indicated times and concentration. All compounds were solubilised in dimethyl sulfoxide (DMSO).
Wild type PA (Anthrax toxin Protective Antigen) and LF (Anthrax toxin Lethal Factor) were produced in our laboratory by overexpression in E. coli as described (Feld et aL, 2012). Western Blotting
Cells were washed three times in 1 x PBS at 4°C and lysed in Buffer (1 x PBS, 1 % Triton X-100, and protease inhibitor cocktail; Roche) for 30 min on ice. Lysates were then spun down at 5,000 rpm on a table-top centrifuge, and the protein content of supernatant were determined, the samples were boiled in Laemmli buffer for 5 min before separation via SDS-PAGE and western blotting against the different anti-bodies used in this study. Western blots were developed using the ECL protocol and imaged on a Fusion Solo from Vilber Lourmat. Densitometric analysis was performed using the software Bio-1 D from the manufacturer.
Surface biotinylation
Surface biotinylation was performed on RPE1 cells with endogenous CMG2. After toxin treatment (PA63 500ng/ml and LF 50ng/ml), cells were allowed to cool down shaking at 4°C for 15 min to arrest endocytosis. Cells were then washed three times with cold PBS and treated with EZ-Link Sulfo-NHS-SS-Biotin No weight (Thermo Scientific) for 30min shaking at 4°C. Cells were then washed 3 times for 5 min with 100mM NH4CI and lysed in lysis Buffer for 1 h at 4°C. Lysate were then centrifuged for 5 minutes at 5000rpm and the supernatant incubated with streptavidin agarose beads (Sigma) overnight on a wheel at 4°C. Western blots were performed.
Oligonucleotides, Plasmids and Sequences
All Q-PCR primers are described in detail.
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000034_0002
CMG2-APT2-Flaq Co-immunoprecipitation assays For immunoprecipitations, RPE-1 cells cultured in 10 cm dishes (80% confluency) were transfected with plasmids encoding APT2-Flag for 24 h. Transfected cells were pretreated with APT2 inhibitors at the indicated concentrations 4 h before toxin treatment (PA63 500 ng/ml and LF 50 ng/ml). Toxin treatment was carried out for the indicated time points in the presence of the drugs. Cells were washed and lysed for 30 min at 4°C in PBS-1 % triton-X, with protease inhibitors cocktail (Roche), centrifuged for 3 min at 2000 g, and supernatants further precleared with protein G-agarose conjugated beads for 30 min. Cleared supernatants were incubated for 16 h at 4°C with CMG2 antibodies and beads. Beads were washed three times in lysis buffer, resuspended in 60 pl Laemmli buffer, and boiled, and the CMG2 immunoprecipitated fractions were processed for Western blot analysis as described.
Viral Stock production and titration with plaque-based assays
All viral stocks were produced and isolated from supernatants of Vero E6 cells, cultured in T75 culture flasks to a confluency of 80-90%, and infected with an original passage 2 (P2) SARS-CoV-2 virus (lineage B.1 ), for 48 or 72 h, at MOI«0.05, in 10 ml DMEM supplemented with 2.5% FCS. Stocks were obtained from the following strain: hCoV- 19/Switzerland/GE-SNRCI-29943121/2020, (GISAID ID: EPI _ISL_ 414019). For Omicron stocks, cells were infected with BA.5 isolates (ID: EPI_ISL_12268493.2 to produce P2 working stocks. Passage 3 SARS-CoV-2 B.1 or passage 2 Omicron BA.5 supernatants were harvested, clear of cell debris by centrifugation (500 g 10 min) and filtration (0.45 pm), aliquoted and stored at -80 C. Viral titters were quantified by determining the number of individual plaque forming units after 48 h of infection in confluent Vero E6 cells. In brief, viral stocks were serially diluted (10-fold) in serum-free medium and (400 pl) inoculated in triplicate 48 wells, confluent Vero E6 cells (2.5 x 105) cells per well. After 1 h, inoculums were discarded, and cells overlaid with a mixture of 0.4% of Avicel-3515 (Dupont) (from 2% stock) in DMEM supplemented with 5% FCS and pen/strep for additional 48 h. Overlays were discarded, and cells fixed in 4% PFA for 30 min at RT. Fixed cells were washed in PBS and stained with 0.1 % crystal violet solution (in 20% ethanol/water) for 15 min. Staining solution was discard and wells washed twice in water. Plates were allowed to dry and analyzed for quantification of the cytopathic effect as number of individual Plaque forming units (PFU) per ml (Avg PFU*1/Volume*1 /dilution factor). Such un-concentrated viral stocks yielded between 0.5 to 3x106 PFU/ml.
SARS CoV-2 infections
All infections for experimental analysis were done using passage 3 SARS-CoV-2 (B.1 ) stocks. BA.5 passage 2 was used for experiments using the Omicron variant. Vero E6 cells or CALU-3 cells seeded to a confluency of 90 to 100%, were, washed twice in warm serum-free medium and inoculated with the indicated MOI of SARS-CoV-2, diluted in serum free medium (1 ml for 6-well plates and 500 pl for 12-well plates). 1 hour after inoculation cells were washed with complete medium and infection allowed to proceed for the indicated time points in DMEM supplemented with 2.5% FCS, penicillin and streptomycin. For plaque-based assays culture medium was supplemented with Avicel overlay as described.
Drug treatments
All drug treatments were done as indicated in complete culture and/or infection medium. Equivalent volume of Dimethyl sulfoxide (DMSO), used as a solvent, was added for control samples.
VSV-CoV-2 production and Inhibition Assays
Vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotypes (VSV-CoV-2) generated according to (Gasbarri et al, 2020) expressing a 19 amino acids C-terminal truncated spike protein (NCBI Reference sequence:NC_045512.2) were produced in HEK293F and titrated in Vero-E6. For infection, Vero-E6 cells (13,000 cells per well) were seeded in a 96-well plate. Compounds were serially diluted in DMEM and incubated with VSV-CoV-2 (MOI, 0.001 infectious units /cell) for 1 h at 37 °C. The mixture was added on cells for 1 h at 37 °C. The monolayers were washed and overlaid with medium containing 2% FBS for 18 h. The following day cells were fixed with paraformaldehyde 4%, stained with DAPI, and visualized using an ImageXpress Micro XL (Molecular Devices, San Jose, CA, USA) microplate reader and a 10x S Fluor objective. The percentage of infected cells was estimated by counting the number of cells expressing GFP and the total number of cells (DAPI-positive cells) from four different fields per sample using MetaXpress software (Molecular Devices, San Jose, CA, USA).
Analysis of intracellular viral RNA-Q-PCR
Infected cells (from 6 and 12 well plates) and correspondent supernatants (when indicated - 150 pl from 6 well plates), were harvested and lysed in 220-330 pl of Maxwell® RSC Viral Total Nucleic Acid Purification Kit-lysis buffer from Promega, incubated at 80°C for 10 min, and used for Viral RNA extraction according to manufacturer’s instructions. RNA concentration was measured and 500 ng or 1000 ng of total RNA was used for cDNA synthesis using iScript. A 1 :5 dilution of cDNA was used to perform quantitative real-time PCR (Q-PCR) using Applied Biosystems SYBR Green Master Mix on 7900 HT Fast Q-PCR System (Applied Biosystems) with SDS 2.4 Software. Primers used are described in Supplementary table 1 . All data (always in triplicate) were normalized to Ct values from three housekeeping (HK) genes ALAS-1 , Guss and TBP, except for supernatants from replication experiments at 24 h, which Ct data were normalized using Ct values from correspondent cellular HK genes. Results were expressed as 2A(- AACt)*100%.
Analysis of intracellular viral Nucleocapsid-N levels by Western blot
Infected cells (24 well plates) (cultured at 1 .5 x 10A5 cells per well, 24 h before infection) were harvested and lysed in 150 pl of lysis buffer (0.5% NP-40, 500 mM Tris-HCI pH7.4, 20 mM EDTA, 10 mM NaF, 30mM sodium pyrophosphate decahydrate, 2 mM benzamidine and protease inhibitor cocktail; Roche) for at least 30 min. Lysates were cleared by centrifugation (5000g, 3 min), and the protein content of the supernatant was determined. Samples were resuspended in 50 pl Laemmli buffer, boiled for 5 min, and approximately 20-30 pg of protein lysate were used for Western blot analysis (as described) using antibodies against viral nucleocapsid N and host GAPDH. The percentage of infection was calculated as the densitometric ratio between N and GAPDH levels for each sample.
Realtime-Glo™ mt cell viability assay
The number of viable cells in culture was measured by determining the reducing potential of cultured cells, thus metabolism, in Calu-3 cells. The assay measures the reduction of a cell-permeable substrate, which is diffused to the medium and processed by an exogenously added reporter enzyme (NanoLuc® luciferase). The reaction produces a luminescent signal that correlates with the number of viable cells. The luminescence signal was monitored every day for 6 days in Calu-3 cells cultured in 96-well plates (50000 cells/well), 24 h prior addition of the substrate and increasing concentrations of ML349, C2, and C3. Two wells per compound per concentration were average for 4 independent biological replicates. Results are mean ±SEM of n=4. Dose-dependent viability curves, and cytotoxic concentration, were determined on day 6 of treatment using nonlinear regression and ANOVA analysis (Prism).
Analytical flow cytometry of SARS-CoV-2 infected cells
Vero E6 cells were infected in 6-well plates [as mentioned previously]. Cells washed and scrapped in 1 ml PBS and recovered by mild centrifugation 400g 3 min. Supernatants were discard and cells were fixed in 4% PFA for at least 30 min, washed twice in PBS, and permeabilized for 5 min with 0.1 % Triton in PBS. The following blocking, antibody, and washing steps were all done with FACS buffer (2% FBS in PBS/EDTA) primarily at RT. Blocking was done for 15 min, primary antibody incubation with mouse anti-Spike antibody (LC-C19510) was performed at 1 :200 for 30 min at RT or at 1 :500 ON at 4 °C, and cells were washed twice. Alexa-647-conjugated donkey anti-mouse was used as a secondary antibody at 1 :600 for 30 min at RT, after which cells were washed three times and kept cold before flow cytometry acquisition. Analytical flow cytometry was performed using an LSRII or LSR Fortessa (BD; Becton Dickinson) instrument and results were analyzed using the FlowCore package in R.
LDH cytotoxicity assay
Lactate dehydrogenase (LDH) cytotoxicity assays were performed, using In Situ Death Detection Kit (Roche, Indianapolis, IN), in Vero E6 or CALU-3 cells cultured in 12 well plates with equivalent culture conditions used for infection assays.
HSV infection and inhibition assays
Wild-type HSV-2 viral suspensions were inoculated onto cells pre-treated with the indicated drugs for 1 h, at a multiplicity of infection (MOI) of 0.01 plaque-forming units (PFU) as described (Cagno et al, 2018). Viral inoculum was removed, and cells washed after 2 h, and infection was continued in the presence of the drugs for evaluation through a plaque-based assay.
Evaluation of Toxicity in vivo in C57BL/6 mice
1 1 -week-old male C57BL/6 mice were inoculated intraperitoneally with 400 to 600 pl of a 2 mg/ml working solution of C2 or ML349, resuspended in PEG300 50% (v/v) solution in PBS, to a final dose of 1 mg of compound per 25 g of body weight. Control mice were inoculated in parallel with an equivalent volume (per body weight) of vehicle PEG300 50% (v/v) in PBS solution. Drug and vehicle treatments were injected on days 1 , 3, 6, 8, and 10 of the experiment, whereas clinical parameters (body weight, blood pressure, and pulse) were monitored on days 1 , 2, 3, 6, 7, 8, 9, 10, and 13. After 13 days of treatment, mice were euthanatized, and different organs were harvested for histological analysis and weight measurements.
Quantification and statistical analysis Unless otherwise indicated, all data were repeated at least three times independently. Unless otherwise stated, each data point corresponds to one independent experiment with consistent results. Statistical analysis was carried out using Prism software. Data representations and statistical details can be found in the description of the figures. For ANOVA analysis, p values were obtained by post hoc tests to compare every mean and pair of means (Tukey’s & Sidak’s) or to compare every mean to a control sample (Dunnet’s). Half-maximal effective concentrations (EC50) were obtained using dose inhibition curves fitted using nonlinear regression with a variable slope (Prism).
Targeting depalmitoylation inhibits intoxication by bacterial toxins
Example 1 : Inhibition of APT2 activity increases surface expression of ANTXR2/CMG2 Experiments were performed by surface biotinylation as described in materials and methods. Labelling of surface proteins with NHS-biotin was carried out in mammalian cells left untreated or treated with ML349 (10 pM) for the indicated times. The surface proteins were pulled down with streptavidin beads. The corresponding cell extracts were analyzed by western blot using antibodies and reagents as described in materials and methods. Leupaxin (LPXN) and glyceraldehyde-3-phosphate deshydrogenase (GAPDH) are blotted as positive and negative controls respectively. The quantification of surface ANTXR2/CMG2 is expressed as % of t=0 and results are mean ±SD (n>3) (Figure 1 ).
In these assays, pre-incubation of cells with the ML349 gradually blocks the internalization of the toxin receptor capillary morphogenesis gene 2 (CMG2/ANTXR2), causing its accumulation in the plasma membrane (Figure 1 ).
In this example, the inhibition of the thioesterase APT2 with ML349 prevents the endocytosis of the anthrax toxin receptor capillary morphogenesis gene 2. These results suggest that the inhibition of depalmitoylation through specific drugs that affect acyl protein thioesterases (APTs) could block the entry of pathogenic toxins into the host cells.
Example 2: Inhibition of APT2 activity prevents intoxication of mammalian cells with Anthrax toxin.
Experiments were performed by synchronized anthrax intoxication assays in mammalian cells. The results are represented on Western blots (Figure 2A and B).
Mammalian cells were left untreated (control) or co-treated with ML349 (10 pM) or ML348 (10 pM) during 0, 20, 40 and 60 minutes. Cells were pre-treated with ML349 or ML348 during 2 h (Figure 2A) or 4 h (Figure 2B) prior to and during exposure to Anthrax toxin Protective Antigen (PA).
The dynamics of toxin pore formation (PA Pore) (Figure 2C) and MEK2 degradation (Figure 2D) were quantified for each time point for cells pre-treated for 2 h and expressed as % of t=0 for each sample. Results are mean ±SD (n>3).
Treatment with ML349, blocks endocytosis of CMG2/ANTXR2, thus preventing pore formation of the anthrax toxin PA subunit. ML349 thus prevents the protease subunit of anthrax toxin to reach the cytosol and in turn prevents the cleavage of a major host cell signaling regulator, the MAP kinase-kinase MEK.
Example 3: siRNA silencing of APT2 expression prevents intoxication of mammalian cells with Anthrax toxin.
Experiments were performed using siRNA silencing APT2 as described in materials and methods in paragraph siRNA.
Synchronized anthrax intoxication assays were performed in mammalian cells transfected with control or siRNA oligos targeting the expression of APT2 (siAPT2) as described in materials and methods (siRNA). Mammalian cells were left in culture for 72 h after transfection and then intoxicated with Anthrax toxin Protective Antigen (PA). Results are represented on Western blot (Figure 3A). The dynamics of toxin pore formation (PA Pore) measured by western blot in Figure 3A were quantified for each time point 0, 20, 40 and 60 minutes, and values expressed as % of t=0 for each sample (Figure 3B). Results are mean ±SD (n>3).
The results show that silencing APT2 expression (Figure 3 A) recapitulates the effects of inhibiting its enzymatic activity with ML349 depicted in Figure 2A and 2B. Thus, inhibition of depalmitoylation can function as an effective strategy to block host cellular intoxication with bacterial toxins.
Targeting depalmitoylation inhibits Viral infections
Example 4: Inhibition of APT1 activity with ML348 diminishes SARS-CoV-2 Spike- pseudotyped particles (PPs) entry into host cell. A cell-based assay was performed to determine the effects of different chemical inhibitors on the internalization of Vesicular Stomatitis Virus (VSV)-based particles pseudotyped with the spike fusion glycoprotein from SARS-CoV-2. The effects of ML348 and ML349, inhibitors of APT1 and APT2 respectively, as well as Palmostatine, Bromopalmitate, Chlroroquine and Hydroxychloroquine were tested and are represented on Figure 4.
Experiments were realized as described in materials and methods (VSV-CoV-2 production and Inhibition Assays). Vero E6 cells were infected with SARS-CoV-2-Spike- pseudotyped particles (VSV-S-CoV2) or VSV-G-pseudotyped particles(VSV-G) (Figures 4A, B and C). Vero cells were either pre-treated (PRE-TREAT, Figure 4C) for 4 h washed, and infected with VSV-S- CoV2 or VSV-G, or infected in the presence of the same concentration of the chemical inhibitors (PRE+CO-TREAT). GFP-expressing cells were quantified by fluorescent microscopy. The percentage of infection was calculated comparing the percentage of positive cells to the percentage in infected untreated controls (DMSO). Dose-response curve was calculated for ML348, ML349, and hydroxychloroquine. (Figure 4C). The 50% effective concentrations (EC50) were determined using Prism software (GraphPad Software, San Diego, CA): EC50 (ML348=19pM, EC50 (hydroxychloroquine)=1 1 pM).
ML348 specifically inhibited the entry of PPs typed with SARS-CoV-2 spike protein to a similar extend as hydroxychloroquine (Figure 4A). This inhibitory effect was dosedependent (Figure 4C) and not observed upon treatment of target cells with unspecific inhibitors of palmitoylation and depalmitoylation such as Bromopalmitate and Palmostatine respectively, neither by treatment with the APT2 inhibitor ML349 (Figure 4A and 4B).
Example 5: Inhibition of APT1 activity with ML348 blocks SARS-CoV-2 cellular infection. The depalmitoylation-targeting drugs ML348, ML349 were tested during infection of Vero E6 cells with a clinical isolate of SARS-Cov-2. Infection was monitored using a standard plaque-based infection assay as described in materials and methods (Viral stock production and titration with plaque-based assays; SARS CoV-2 infections).
Vero E6 cells were pre-treated with ML348 or ML349 for 1 h washed, and infected with SARS-CoV-2, in the presence of drug. Infection was determined using a plaque assay and a dose-response curve was established for ML348 and ML349 (Figure 5A). The EC50 for ML348 is 13.3 pM. Then, Vero E6 cells were infected with SARS-CoV-2 (MOI 0.1 ) for 24 h, in the presence of 12.5 pM (Figure 5B). Drugs were added 1 h after viral inoculation, and replication was monitored by determining the % of high Spike-positive cells by flow cytometry. Results are mean ±SEM and each dot represents one individual biological replicate.
It has been found that SARS-CoV-2 cellular infection was inhibited when carried out in the presence of increasing micromolar doses of ML348 (EC50~13.3 pM) reaching full inhibition (Figure 5A). Complementary infection assays using a ML348 concentration at 12.5 pM below the determined EC50, demonstrated that treatment of infected cells with ML348, after viral inoculation and throughout the remaining time of infection, greatly reduced the number of Spike-positive infected cells, confirming the inhibitory effect of ML348 (Figure 5B).
Example 6: Inhibition of APT1 activity with ML348 inhibits SARS-CoV-2 intracellular replication.
To understand whether the effect of ML348 occurs within a single cellular infection cycle (up to 8 to 12 h), viral genomic RNA replication was monitored throughout time by probing for two viral genomic regions corresponding to the E and RdRp genes of SARS- CoV-2. Vero E6 cells were treated with ML348 1 h after viral inoculation and throughout the time of infection.
Vero E6 cells were inoculated for 1 h with SARS-CoV-2, washed and subsequently incubated in complete medium with 12.5 pM of ML348. Cells were harvested at the time points 0, 4, 8 and 12 hours (Figure 6A and 6B). Infection was determined by monitoring the levels of intracellular E and RNA-dependent RNA polymerase-RdRp transcripts by Q- PCR as described in materials and methods. t=0 was harvested 1 h after inoculation before drug treatment. Normalized results, with relation to control (w.r.t.) are the mean ± SD of three independent biological replicates.
ML348 blocked SARS-CoV-2 intracellular viral RNA replication as early as 8h after viral inoculation (Figure 6A and 6B). Example 7: Inhibition of APT1 activity and silencing of APT1 expression blocks SARS- CoV-2 infection in Vero E6 cells.
Viral RNA replication in Vero E6 cells was also monitored after 24h of infection to follow multiple rounds of cellular infection. In these experiments, the viral intracellular replication was monitored by determining the levels of the sub-genomic E RNA, a bona-fide marker of intracellular viral RNA replication.
Quantification of viral RNA replication (E, RdRp and E subgenomic) was analyzed from Vero E6 cells or correspondent supernatants of Vero E6 cells infected with SARS-CoV-2 (MOI-O.1 for 24 h), treated without drug (Control), with ML348 or ML349. Cells were inoculated for 1 h with SARS-CoV-2, washed and subsequently incubated in complete medium with 12.5 pM of ML348 or ML349 (Figure 7 A and 7B).
As already observed in cells treated with ML348 after viral inoculation (see Example 6), the viral RNA replication was significantly reduced by ML348 both in cells and in culture supernatants containing released virions (Figures 7A and 7B).
In a further experiment, cells were previously transfected with control or siRNA oligos targeting the expression of APT1 (siAPT) for 72h as described in materials and methods (siRNA; Figure 7C).
The quantification of APT1 mRNA levels from the samples used in Figure 7C is represented on Figure 7D. The depletion is approximately 60%. All results were normalized to the mean of control samples and are mean ± SEM of n >3. p values were obtained by ANOVA analysis and *p<0.05, ***p<0.001 , ****p<0.0001 .
The effects caused by inhibition of the enzymatic activity of APT1 by ML348 (Figures 7A and 7B) can be recapitulated by carrying SARS-CoV-2 infections in Vero E6 cells siRNA- treated for monkey APT1 (Figure 7C). These experiments confirmed the specificity of ML348 inhibition during SARS-CoV-2 infection.
Example 8: Inhibition of APT1 activity with ML348 blocks HSV-2 cellular infection.
In this experiment, it was evaluated whether ML348 or ML349 could limit infection by other type of viruses. A plaque-based assays was performed using herpes simplex virus type 2 (HSV-2) according to materials and methods (HSV infection and inhibition assays). Vero E6 cells were pre-treated for 1 h in the presence of drug, washed, and infected with HSV-2 (Figure 8 A). Infection was determined using a plaque assay. Dose-response was determined for ML348 and ML349. The EC50 for ML348 is 23.2 pM.
ML348 treatment reduces infection of HSV-2 in a dose-dependent manner (Figure 8). These data highlight the larger anti-viral scope of targeting depalmitoylation, using a specific inhibitor of APT1 activity.
Example 9: Inhibition of APT2 activity blocks SARS-CoV-2 infection in Calu-3 lung- derived cells.
The mRNA expression of APTs varies between cell lines. APT2 is often more abundant than APT1. In human airway epithelial cells, such as the lung-derived cell line, CALU-3, APT2 is highly enriched when compared to Vero E6 cells (Figure 9A).
Given the tropism of SARS-CoV-2 to lung cells, the effect of inhibiting depalmitoylation on viral replication was evaluated in Calu-3 cells.
The LYPLA1 (APT1 ) and LYPLA2 (APT2) mRNA levels are measured in different cell lines (Figure 9A). Values are TAG per Million (TPM) corresponding to 10*6 X ((reads mapped to transcript/transcript length)/ SUM (reads mapped to transcript/transcript length)). Results are the mean of independent experiments each from n > 2 biological replicates.
The replication of SARS-CoV-2 was monitored in Calu-3 cells at 24h post-inoculation and upon treatment with 12.5 pM of ML348 or ML349. This concentration is below the EC50 of ML348 for inhibition of viral replication in Vero E6.
Figure 9B shows the quantification of viral RNA replication (E, RdRp and E subgenomic) from Calu-3 cells and correspondent supernatants infected with SARS-CoV-2 (MOI-0.1 for 24 h), treated with ML348 or ML349. The cells were inoculated for 1 h with SARS-CoV- 2, washed and subsequently incubated in complete medium with 12.5 pM of ML348 or ML349.
In another experiment, the cells were previously transfected with control or siRNA oligos targeting the expression of APT2 (siAPT2) for 72h (Figure 9C). Figure 9D shows the quantification of APT2 mRNA levels from the samples used in Figure 9C. All results were normalized to the mean of control samples and are mean ± SEM of n >3. p values were obtained by ANOVA analysis and *p<0.05, ***p<0.001 , ****p<0.0001 .
The treatment of cells with ML349 significantly diminishes viral intracellular replication and secretion to the culture supernatant (Figure 9B). Correspondently, partial depletion of APT2 in Calu-3 also diminished intracellular replication of SARS-CoV-2 after 24h of infection in Calu-3 cells (Figure 9C and 9D). Thus, these experiments in Calu-3 cells confirm that targeting depalmitoylation activity represents a valid strategy to inhibit SARS- CoV-2 infection.
Example 10: Dose-response inhibition of SARS-CoV-2 replication in Calu-3 cells by ML349 and cytotoxicity evaluation of ML348 and ML349.
In this experiment, the EC50 of ML349 inhibiting SARS-CoV-2 intracellular replication was determined in Calu-3. In addition, an analysis of the potential cytotoxic effects caused by the drug-treatment protocols was performed.
Calu-3 cells were infected with SARS-CoV-2. Infection was determined using Q-PCR by quantifying the mean levels of E and RdRp viral RNA (Figure 10A). Normalized results are mean ± SEM. The dose-response was determined for ML349 and the EC50 is 8.6 pM.
A Lactate dehydrogenase activity assay was performed as described in the material and methods section. Vero E6 cells or Calu-3 cells were treated with increasing concentrations of ML348 for 48 h or ML349 for 24 h, respectively. The results of the cell viability are mean ± SD of 2 biological replicates. Importantly, neither treatment of Vero E6 with ML348 for 48h nor treatment of Calu-3 with ML349 for 24h, triggered significant cellular toxicity at different ranges of concentrations (Figure 10B and 10C).
The inhibitory effect of ML349 during SARS-CoV-2 replication in Calu-3 cells (EC50 ~ 8.6 pM) (Figure 10A) and the one observed for ML348 in infection of Vero E6 cells (EC50~13.3 pM) demonstrate that both drugs ML348 and ML349 are potent inhibitors for SARS-CoV-2 infection. Altogether, targeting host cell depalmitoylation activity constitutes a safe strategy to inhibit viral infections such as SARS-CoV-2, or HSV, and bacterial-mediated intoxication.
Example 1 1 : Synthesis of novel inhibitors of acyl protein thioesterase (APT) of general formula (II).
Synthesis of carboxylic acid precursor
4-chloro-2H-thiochromene-3-carbaldehyde
Figure imgf000046_0001
Phosphorus oxychloride (647 pL, 1.14 Eq, 6.94 mmol) was added dropwise to ice-cold anhydrous DMF solution (12 mL, 0.48 molar), followed by dropwise addition of thiochroman-4-one (1.00 g, 6.09 mmol). The reaction mixture was stirred for 30 minutes at 0°C, then heated at 80°C for 2 hours, and quenched with 4mL of ice-cold aqueous sodium acetate (25%). The mixture was extracted with CH2CI2 (4 x 30mL), washed with brine, dried with Na2SO4 and concentrated. The crude product, 4-chloro-2H- thiochromene-3-carbaldehyde (1 .12 g, 5.34 mmol), was obtained as a brown oil.
1 H NMR (400MHz, Chloroform-d) 5 10.32 (s, 1 H), 7.95-7.88 (m, 1 H), 7.34 - 7.40 (m, 3H), 3.71 (s, 2H).
Ethyl 4H-thieno[3,2-c]thiochromene-2-carboxylate
Figure imgf000046_0002
69% yield
4-chloro-2H-thiochromene-3-carbaldehyde (1.28 g, , 6.08 mmol) is slowly added to a mixture of ice-cold ethyl 2-mercaptoacetate (1 .33 mL, 2 Eq, 12.2 mmol), sodium ethoxide (1.65 g, 4 Eq, 24.3 mmol) in ethanol (7.06 mL, 0.86 molar). The reaction mixture was stirred overnight at room temperature and then heated at 80°C for 2 hours. Solvents were removed and EtOAc was added to the residue. The organic layer was washed with brine, dried and purified by flash column chromatography (20% EtOAc, 80% Cyclohexane) to yield ethyl 4H-thieno[3,2-c]thiochromene-2-carboxylate (1 .33 g, 4.83 mmol, 80 %).
1 H NMR (400MHz, Chloroform-d) 5 7.57 (s, 1 H), 7.54 - 7.45 (m, 1 H), 7.39 - 7.33 (m, 1 H), 7.20 - 7.15 (m, 2H), 4.36 (q, J = 7.14 Hz, 2H), 3.94 (s, 2H), 1 .39 (t, J = 7.12 Hz, 3H).
Ethyl 4H-thieno[3,2-c]thiochromene-2-carboxylate 5,5-dioxide
Figure imgf000047_0001
mCPBA (3.75 g, 3.00 Eq, 21 .70 mmol) was added portionwise to an ice-cold solution of ethyl 4H-thieno[3,2-c]thiochromene-2-carboxylate (2.00 g, 1.00 Eq, 7.24 mmol) in CH2CI2 (80 mL). The reaction was warmed to room temperature and stirred until complete conversion (3 h, TLC). The reaction mixture was quenched by adding a mixture of sat. aq. Na2S2O3 (10 mL) and sat. aq. NaHCO3 (50 mL) and stirred for 1 h. The layers were separated and the aqueous layer was extracted with CH2CI2, dried over Na2SO4, filtered, and concentrated under vacuum. The compound was purified by flash column chromatography (CH2CI2 100% to CH2CI2:MeOH 95:5) to yield ethyl 4H-thieno[3,2- c]thiochromene-2-carboxylate 5,5-dioxide (710 mg, 7.24 mmol, 32%) as a yellow powder. 1 H NMR (400 MHz, DMSO-d6) 5 7.99 (ddd, J = 7.8, 1.3, 0.5 Hz, 1 H), 7.89 (dd, J = 7.8, 1 .2 Hz, 1 H), 7.81 (td, J = 7.6, 1 .3 Hz, 1 H), 7.70 (td, J = 7.6, 1 .2 Hz, 1 H), 4.96 (s, 1 H), 4.35 (q, J = 7.1 Hz, 1 H), 1 .33 (t, J = 7.1 Hz, 2H).
4H-thieno[3,2-c]thiochromene-2-carboxylic acid 5,5-dioxide
Figure imgf000047_0002
To a solution of ethyl 4H-thieno[3,2-c]thiochromene-2-carboxylate 5,5-dioxide (598 mg, 1 .00 Eq, 1 .94 mmol) in a mixture of THF/MeOH/H2O (2:1 :1 ) (30 mL) was added lithium hydroxide monohydrate (407 mg, 5.00 Eq, 9.70 mmol). The reaction mixture was stirred at room temperature for 16 h then partially concentrated under vacuum. Water was added and the solution was washed with EtOAc (2x) and acidified with aq. 6M HCL The aqueous layer was washed with EtOAc. Finally, the organic layers were combined, washed with brine, dried over Na2SO4 and concentrated under vacuum. 4H-thieno[3,2- c]thiochromene-2-carboxylic acid 5,5-dioxide (533 mg, 1 .94 mmol, 98%) was obtained as a white powder.
1 H NMR (400 MHz, DMSO-d6) 5 13.53 (s, 1 H), 7.98 (ddd, J = 7.8, 1 .3, 0.5 Hz, 1 H), 7.89 - 7.76 (m, 3H), 7.68 (td, J = 7.6, 1 .3 Hz, 1 H), 4.94 (s, 2H).
Synthesis of amide derivatives
General procedure for amide coupling
Figure imgf000048_0001
To a solution of 4H-thieno[3,2-c]thiochromene-2-carboxylic acid 5,5-dioxide (1.00 Eq) in anhydrous DMF (0.1 M) under N2 atmosphere, DIPEA (2.00 Eq), HBTU (3.00 Eq) and the secondary amine (2.00 Eq) were added. The mixture was stirred under N2 at room temperature for 16 h. Water was added to the solution and the aqueous layer was washed with EtOAc. The organic layer was washed with sat. aq. NH4CI, brine and, dried over Na2SO4. The solvent was evaporated and the crude residue was purified by column chromatography (cHex:EtOAc) to afford the final compound.
(4-(4-fluorophenyl)piperazin- 1 -yl)(4H-thieno[3,2-c]thiochromen-2-yl)methanone (SPC-
EPFL-1)
Figure imgf000048_0002
Compound SPC-EPFL-1 (C1 ) was prepared following General Procedure A using 1 -(4- fluorophenyl)piperazine (32 mg, 1 Eq, 0.18 mmol) to afford (5,5-dioxido-4H-thieno[3,2- c]thiochromen-2-yl)(4-(4-fluorophenyl)piperazin-1 -yl)methanone (42.6 mg, 54 %) as a white solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV]. 1 H NMR (400MHz, DMSO-d6) 5 8.01 - 7.92 (m, 1 H), 7.81 -7.75 (m, 2H), 7.68 - 7.60 (m, 1 H), 7.55 (s, 1 H), 7.15 - 7.05 (m, 2H), 7.02 - 6.95 (m, 2H), 4.90 (s, 2H), 3.81 - 3.75 (m, 4H), 3.18 (app t, J = 5.2 Hz, 4H).
(4-(2-chlorophenyl)piperazin- 1 -yl)(4H-thieno[3,2-c]thiochromen-2-yl)methanone (SPC- EPFL-2)
Figure imgf000049_0001
Compound SPC-EPFL-2 (C2) was prepared following General Procedure A using 1 -(2- chlorophenyl)piperazine (35 mg, 1 Eq, 0.18 mmol) to give (4-(2-chlorophenyl)piperazin- 1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (41.4 mg, 51 %) as a white solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV]. 1 H NMR (400MHz, DMSO-d6) 5 8.02 - 7.93 (m, 1 H), 7.83 - 7.76 (m, 2H), 7.70 - 7.60 (m, 1 H), 7.56 (s, 1 H), 7.45 (dd, J = 7.9, 1 .5 Hz, 1 H), 7.36 - 7.28 (m, 1 H), 7.19 (dd, J = 8.1 , 1 .5 Hz, 1 H), 7.09 (td, J = 7.6, 1 .5 Hz, 1 H), 4.89 (s, 2H), 3.89 - -3.81 (m, 4H), 3.06 (app t, J = 5Hz, 4H).
(4-(2,3-dichlorophenyl)piperazin-1-yl)(4H-thieno[3,2-c]thiochromen-2-yl)methanone (SPC-EPFL-3)
Figure imgf000049_0002
Compound SPC-EPFL-3 (C3) was prepared following General Procedure A using 1 -(2,3- dichlorophenyl)piperazine (41 mg, 1 Eq, 0.18 mmol) to give (4-(2,3- dichlorophenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (30.4 mg, 35 %) as a solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV].
1 H NMR (400MHz, DMSO-d6) 5 8.01 - 7.96 (m, 1 H), 7.83 - 7.74 (m, 2H), 7.70 - 7.60 (m, 1 H), 7.56 (s, 1 H), 7.37 - 7.31 (m, 2H), 7.19 - 7.1 1 (m, 1 H), 4.89 (s, 2H), 3.95 - 3.98 (m, 4H), 3.08 (app t, J = 4.91 Hz, 4H).
(4-(4H-thieno[3,2-c]thiochromene-2-carbonyl)piperazin- 1 -yl) (phenyl)methanone (SPC-
Figure imgf000050_0001
Compound SPC-EPFL-5 was prepared following General Procedure A using phenyl(piperazin-1 -yl)methanone (34 mg, 1 Eq, 0.18 mmol) to give (4-benzoylpiperazin- 1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (65.1 mg, 81 %) as a solid after purification by flash column chromatography [Silica-CS 12g, eluent: DCM/MeOH (0% MeOH for 3CV, 0-5% 15CV, 5% for 3CV, 5-10% for 3CV].
1 H NMR (400MHz, DMSO-d6) 5 7.97 (dd, J = 7.6 Hz, 1 H), 7.75-7.82 (m, 2H), 7.61 -7.67 (m, 1 H), 7.54 (s, 1 H), 7.40-7.50 (m, 5H), 4.88 (s, 2H), 3.35-3.90 (br m, 8H),
N-(2-(5-methoxy- 1 H-indol- 1 -yl)ethyl)-4H-thieno[3,2-c]thiochromene-2-carboxamide (SPC-EPFL-6)
Figure imgf000050_0002
Compound SPC-EPFL-6 was prepared following General Procedure A using 2-(5- methoxy-1 H-indol-1 -yl)ethan-1 -amine (27 mg, 1 Eq, 0.14 mmol) to give N-(2-(5-methoxy- 1 H-indol-1 -yl)ethyl)-4H-thieno[3,2-c]thiochromene-2-carboxamide 5,5-dioxide (36.8 mg, 57 %) as a solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-100% for 15CV, 100% for 3CV]. 1 H NMR (400MHz, DMSO-d6) 5 8.87 (t, J = 5.8 Hz, 1 H), 7.95 (d, J = 7.6 Hz, 1 H), 7.75 - 7.80 (m, 2H), 7.67 - 7.61 (m, 2H), 7.41 (d, J = 8.8 Hz, 1 H), 7.28 (d, J = 3.0 Hz, 1 H), 7.04 (d, J = 2.4 Hz, 1 H), 6.78 (dd, J = 8.8, 2.4 Hz, 1 H), 6.32 (d, J = 3.0 Hz, 1 H), 4.94 (s, 2H), 4.32 (t, J = 6.0 Hz, 2H), 3.59 (q, J = 6.0 Hz, 2H), 3.74 (s, 3H).
(4-(3-aminobicyclo[ 1.1.1 ]pentan- 1 -yl)piperazin- 1 -yl)(4H-thieno[3,2-c]thiochromen-2- yl)methanone (SPC-EPFL-7)
Figure imgf000051_0001
Tert-butyl(4-(4-(5,5-dioxido-4H-thieno[3,2-c]thiochromene-2-carbonyl)piperazin-1 - yl)bicyclo[1 .1 .1 ] pentan-2-yl)carbamate was prepared following General Procedure A using tert-butyl (4-(piperazin-1 -yl)bicyclo[1 .1 .1 ]pentan-2-yl)carbamate (48 mg, 1 Eq, 0.18 mmol). The compound was purified by flash column chromatography [Silica-CS 12g, eluent: DCM/MeOH (0% MeOH for 3CV, 0-5% 15CV, 5% for 3CV, 5-10% for 3CV], To a solution of tert-butyl(3-(4-(5,5-dioxido-4H-thieno[3,2-c]thiochromene-2- carbonyl)piperazin-1 -yl)bicyclo[1 .1 .1 ]pentan-1 -yl)carbamate (60 mg, 1 Eq, 0.1 1 mmol) in 1 ,4-dioxane (1 .1 mL, 0.1 molar) was added HCI in dioxane 4M (0.42 mL, 4 molar, 15 Eq, 1.7 mmol) dropwise at 0°C. The reaction mixture was stirred at room temperature overnight. SPC-EPFL-7 (9 mg, 20%) was obtained as a solid after purification by flash column chromatography.
1 H NMR (400MHz, DMSO-d6) 5 7.97 (dt, J = 7.7, 1 .0 Hz, 1 H), 7.76-7.82 (m, 2H), 7.61 - 7.72 (m, 1 H), 7.49 (s, 1 H), 4.88 (s, 2H), 3.62 - 3.68 (m, 4H), 2.41 (app t, J = 5.1 Hz, 4H), 1.65 (s, 6H).
(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl) (4-(o-tolyl)piperazin- 1 -yl)methanone
(SPC-EPFL-9)
Figure imgf000052_0001
Compound SPC-EPFL-9 (C9) was prepared following General Procedure A using 1 -o- tolyl-Piperazine (19 mg, 1 Eq, 0.1 mmol) to afford (5,5-dioxido-4H-thieno[3,2- c]thiochromen-2-yl)(4-(o-tolyl)piperazin-1 -yl)methanone (31 mg, 66 %) as a light yellow solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-30% 5CV, 30-50% for 15CV, 50% for 3CV]. 1 H NMR (400 MHz, CDCI3) 5 8.05 (d, J = 7.8 Hz, 1 H), 7.72 - 7.59 (m, 2H), 7.54 (td, J = 7.4, 1 .5 Hz, 1 H), 7.28 - 7.13 (m, 3H), 7.08 - 6.98 (m, 2H), 4.44 (s, 2H), 3.93 (app ft, J = 4.9 Hz, 4H), 2.98 (app t, J = 4.9 Hz, 4H), 2.35 (s, 3H).
(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)(4-(2-fluoro-4-
(trifluoromethyl)phenyl)piperazin- 1 -yl)methanone (SPC-EPFL- 12)
Figure imgf000052_0002
Compound SPC-EPFL-12 (C12) was prepared following General Procedure A using 1 - (2-fluoro-4-(trifluoromethyl)phenyl)piperazine (71 mg, 1 Eq, 0.28 mmol) to afford (5,5- dioxido-4H-thieno[3,2-c]thiochromen-2-yl)(4-(2-fluoro-4-
(trifluoromethyl)phenyl)piperazin-1 -yl)methanone (43 mg, 59 %) as a Pink powder after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (10% EtOAc for 3CV, 10-50% for 15CV, 50% for 3CV].
1 H NMR (400 MHz, CDCI3) 5 8.05 (dd, J = 7.8, 1.3 Hz, 1 H), 7.72 - 7.59 (m, 2H), 7.55 (td, J = 7.9, 7.2, 1 .4 Hz, 1 H), 7.40 - 7.27 (m, 2H), 7.23 (s, 1 H), 7.04 - 6.95 (m, 1 H), 4.45 (s, 2H), 4.07 - 3.75 (m, 4H), 3.23 (app t, J = 5.0 Hz, 4H).
(4-(2,6-dimethylphenyl)piperazin-1-yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2- yl)methanone (SPC-EPFL-13)
Figure imgf000053_0001
Compound SPC-EPFL-13 (C13) was prepared following General Procedure A using 1 - (2,6-dimethylphenyl)piperazine (48 mg, 1 Eq, 0.25 mmol) to afford (4-(2,6- dimethylphenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (42 mg, 74 %) as a yellow solid after purification by flash column chromatography [Silica- CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-50% for 15CV, 50% for 3CV]. Yellow powder (42 mg, 0.09 mmol, 74%). 1 H NMR (400 MHz, DMSO- d6) 5 7.97 (d, J = 7.8 Hz, 1 H), 7.85 - 7.76 (m, 2H), 7.66 (ddd, J = 8.3, 6.3, 2.4 Hz, 1 H), 7.55 (s, 1 H), 7.04 - 6.91 (m, 3H), 4.90 (s, 2H), 3.80 (app t, J = 4.9 Hz, 4H), 3.1 1 (app t, J = 4.9 Hz, 4H), 2.31 (s, 6H).
(4-(2,6-dichlorophenyl)piperazin-1-yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2- yl)methanone (SPC-EPFL-16)
Figure imgf000053_0002
Compound SPC-EPFL-16 (C16) was prepared following General Procedure A using 1 - (2,6-dichlorophenyl)piperazine (58 mg, 1 Eq, 0.25 mmol) to afford (4-(2,6- dichlorophenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (56 mg, 91 %) as a Light yellow solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-60% for 15CV, 60% for 3CV].
1 H NMR (400 MHz, DMSO- d6) 5 8.00 - 7.93 (m, 1 H), 7.85 - 7.75 (m, 2H), 7.65 (ddd, J = 7.8, 6.5, 2.2 Hz, 1 H), 7.55 (s, 1 H), 7.46 (d, J = 8.1 Hz, 2H), 7.20 (dd, J = 8.4, 7.7 Hz, 1 H), 4.89 (s, 2H), 3.82 (app t, J = 4.9 Hz, 4H), 3.27 - 3.20 (m, 4H). (4-(2,5-dichlorophenyl)piperazin-1-yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2- yl)methanone (SPC-EPFL-17)
Figure imgf000054_0001
Compound SPC-EPFL-17 (C17) was prepared following General Procedure A using 1 - (2,5-dichlorophenyl)piperazine (58 mg, 1 Eq, 0.25 mmol) to afford (4-(2,5- dichlorophenyl)piperazin-1 -yl)(5,5-dioxido-4H-thieno[3,2-c]thiochromen-2-yl)methanone (51 mg, 83 %) as a Light yellow solid after purification by flash column chromatography [Silica-CS 12g, eluent: Cyclohexane/EtOAc (0% EtOAc for 3CV, 0-50% for 15CV, 50% for 3CV].
1 H NMR (400 MHz, DMSO- d6) 5 7.97 (dt, J = 7.8, 1 .0 Hz, 1 H), 7.85 - 7.75 (m, 2H), 7.65 (ddd, J = 7.8, 6.2, 2.5 Hz, 1 H), 7.56 (s, 1 H), 7.48 (d, J = 8.5 Hz, 1 H), 7.21 (d, J = 2.4 Hz, 1 H), 7.15 (dd, J = 8.5, 2.4 Hz, 1 H), 4.89 (s, 2H), 3.86 (br s, 4H), 3.09 (app t, J = 5.0 Hz, 4H).
Example 12: Dose response inhibition of SARS-CoV-2 replication in Calu-3 cells by ML349 and novel inhibitors of acyl protein thioesterase (APT) according to formula (II).
In figure 1 1 , the dose dependent inhibition curves and correspondent EC50 of ML349 and novel inhibitors of acyl protein thioesterase (APT) according to formula (II) (SPC-EPFL1 - C1 , SPC-EPFL2-C2, and SPC-EPFL3-C3) in inhibiting SARS-CoV-2 intracellular replication was determined in Calu-3 by quantifying total viral RNA at 24 h post viral inoculation with SARS-CoV-2 at MOI=0.05 to 0.1. Analysis of the potential cytotoxic effects caused by the drug-treatment during infection was assessed by comparing the levels of housekeeping transcripts during infection.
Infection and cell viability were determined using qPCR to quantify the mean levels of E, RdRp and Esub genomic viral RNA (Figure 1 1 A) and host ALAS-1 and RPL27 transcripts (Figure 1 1 B), respectively. Normalized results are mean ± SEM and the dose-response curves are indicated for the 4 compounds. The observed EC50 concentrations correspond to ML349EC50=7,9 pM, C1 EC5O=6.7 pM, C2EC5O=2.3 pM and C3EC5O=O.96 pM. Advantageously, novel inhibitors of general formula (II) such as C2 or C3 demonstrate an EC50 that is decreased by about 3 to 8-fold compared to compound ML-349 (Example 12, figures 1 1 A and B).
The levels of housekeeping transcripts during infection remained constant through all tested concentrations of each studied compound. Therefore, treatment of SARS-CoV-2- infected Calu-3 cells with ML349, C1 , C2 or C3 for 24h, does not significantly induce cellular toxicity at different ranges of concentrations (Figure 1 1 B).
The improved inhibitory effect of the novel inhibitors of acyl protein thioesterase (APT) according to formula (II), such as C1 , C2 or C3 during SARS-CoV-2 replication in Calu-3 cells (Figure 1 1 A) further demonstrate that ML349 and novel inhibitors of acyl protein thioesterase (APT) according to formula (II) are potent inhibitors of SARS-CoV-2 infection and that host cell depalmitoylation activity constitutes a safe strategy to inhibit viral infections.
It has been demonstrated that inhibitors of acyl protein thioesterase (APT) of formula II wherein R3 is a substituted phenyl ring such as C1 , C2 or C3 have an improved inhibitory activity of SARS-CoV-2 replication compared to ML349. In contrast, molecules that are not covered by formula II wherein R3 is a substituted phenyl ring such as C5, C6 or C7 do not inhibit SARS-CoV-2 replication in Calu-3 cells (Figure 12).
Example 13: Inhibition of APT2 activity with inhibitor C2 inhibits SARS-CoV-2 intracellular replication.
It has been previously demonstrated that ML348 blocked SARS-CoV-2 intracellular viral RNA replication in Vero E6 as early as 8 h after viral inoculation (Figure 6A and 6B). To understand if targeting APT2 in Calu-3 cells was blocking infection in an equivalent manner (up to 8 to 12 h), viral genomic and sub-genomic RNA replication was monitored throughout time by probing for two viral genomic regions corresponding to the E and RdRp genes, and the E-sub genomic transcript of SARS-CoV-2. Calu-3 cells were first inoculated with SARS-CoV-2 at MOI=0.5 for 1 h, washed thoroughly in culture medium to removed extracellular virions and subsequently incubated in complete medium with 5 pM of C2 and 0.5 pg/ml of anti-Spike antibody (Fenwick et aL, 2022) to neutralized of virions released within the first round of viral cellular life cycle (approximately from 6 h onwards in Calu-3) (Cortese et aL, 2020), restricting the quantification of the viral RNA replication to the initial round of infection.
Cells were harvested at the time points 1 , 4, 6, 8 and 12 hours and host and viral RNA levels quantified by QPCR as described in materials and methods. The data was normalized to the intracellular viral RNA levels harvested 1 h after inoculation before drug treatment and expressed as fold RNA replication of both viral genomic and subgenomic RNA. Normalized results are the mean ± SEM of three independent biological replicates displayed within two equivalent graph plots with either linear or logarithmic scale for the fold RNA replication.
The results show that blocking APT2 activity with C2 strongly inhibits SARS-CoV-2 infection as early as 4 h after viral entry into cells (Figure 13A and 13B), thus confirming an inhibitory effect of the intracellular viral RNA replication.
Example 14: Dose-response cytotoxicity evaluation of APT2 inhibition in Calu-3 cells by ML349 and novel inhibitors of acyl protein thioesterase (APT) according to formula (II).
A RealTime-Glo™ MT Cell Viability Assay, consisting of a nonlytic, homogeneous, bioluminescent method to measure cell viability in real time, was performed as described in material and method.
Calu-3 cells were treated with increasing concentration of ML349 and C2 or C3 over the course of 6 days. Culture medium was replaced every 2 days with freshly diluted compounds in order to ensure constant activity. The results of the cell viability represent the evolution of the number of viable cells in culture at the indicated time-points and are mean SEM of 4 biological replicates (Figure 14A-C). Fitted curves for Control (no drug), non-toxic and cytotoxic compound concentrations are indicated.
After 6 days of drug-treatment ML349, C2 and C3 were observed to induced cellular toxicity at very high concentrations (from 25 pM onwards, Figure 14D), at least 10-fold more concentrated than the determined EC50 (C1 EC5O=6.7 pM, C2EC5O=2.3 pM and C3EC50~0.96 pM).
Thus, blocking APT2 activity with novel inhibitors of acyl protein thioesterase (APT) according to the present invention effectively and safely inhibits viral infections, such as SARS-CoV-2. Example 15: Inhibition of APT2 activity with compound C2 inhibits replication of SARS- CoV-2 variant Omicron BA.5.
An experiment was performed to evaluate if the compound C2 inhibits multiple SARS- CoV-2 strains. The dose-dependent effect of C2 was determined during the intracellular replication of SARS-CoV-2 variant of concern Omicron BA5 in Calu-3 cells by quantifying the total viral RNA at 24 h post-viral inoculation with SARS-CoV-2 BA5 at MOI =0.05 to 0.1. Infection and cell viability were measured using QPCR of E, RdRp, and E sub- genomic viral RNA (Figure 15A) and host ALAS-1 and RPL27 RNA (Figure 15B), respectively.
Normalized mean results show that C2 inhibits replication of SARS-CoV-2 BA5 strain with an EC50«2.031 pM (Figure 15A) without significantly affecting the levels of housekeeping transcripts during infection (Figure 15B). These data confirm that inhibition of APT2 activity with C2 potently blocks replication of different SARS-CoV-2 strains.
Example 16: Toxicity evaluation of APT2 inhibition in vivo, in C57BL/6 mice by ML349 and compound C2.
Following the experiment confirming that inhibition of APT2 with ML349, compound C2 and C3 did not induce significant cytotoxicity in vitro (Example 14), an experiment was carried out to evaluate the potential toxicity effect of inhibiting APT2 using ML349 or C2 in vivo in C57BL/6 mice. Mice were treated with 1 mg of ML349 or C2 per 25 g of body weight or an equivalent volume of drug delivery vehicle (control), inoculated intraperitoneally at alternate days through a period of 13 days. Toxicity was evaluated by monitoring the body weight, blood pressure, and pulse daily throughout treatment (Figure 16A-C). In addition, the relative spleen weight harvested after 13 days of treatment was also monitored (Figure 16D), and a histological analysis of multiple organs (nose, trachea, lung, brain, small and large intestine, liver, kidney) was also performed (data not shown). Inhibiting APT2 using ML349 or C2 did not significantly change body weight, blood pressure, and pulse throughout treatment, neither did it cause any detectable pathological signs in the spleen (relative weight) after 13 days of treatment (Figure 16). The histological analysis of nose, trachea, lung, brain, small and large intestine, liver and kidney also did not show any ML349- or C2- related change in tissue morphology (data not shown). In conclusion, treatment of mice with APT2 inhibitors, ML349 and C2, at 1 mg of compound per 25 g of body weight, on alternate days does not induce any apparent toxic effect.
Example 17: Evaluating APT2 inhibition mechanism by ML349, compound C2, or C3 during anthrax toxin cellular intoxication.
Internalization of the anthrax toxin and degradation of its host target MEK2 requires APT2 activity (Figure 1 -3).
APT2 activity during anthrax intoxication can be monitored by probing for the formation of protein complexes between the anthrax receptor CMG2 and APT2 triggered by the binding of anthrax toxin protective antigen (PA) to CMG2 (Figure 17A). Formation of these complexes occurs upon de-acylation of CMG2 and is concomitant to the dissociation of CMG2 from the cytoskeleton components actin, vinculin, and talin, which ultimately promotes the internalization of the toxin and the receptor. The levels of CMG2-APT2 complexes can be probed by western blot analysis of lysates from RPE-1 cells treated with PA (Figure 17B). Inhibition of APT2 activity with 5 pM ML349, 4 h before and during toxin treatment, prevents the formation of CMG2-APT2 complexes (Figure 17B). Equivalent treatment of RPE-1 cells with compound C2 and C3, also blocks the formation of APT2-CMG2 complexes upon PA intoxication.
Thus ML349, C2, and C3 can reduce SARS-CoV-2 replication and inhibit the APT2- dependent anthrax toxin intoxication of host cells.
Example 18: Comparison of APT2 inhibition efficacy by ML349, compound C2, C3 during anthrax toxin cellular intoxication.
The levels of CMG2-APT2 complexes formed upon anthrax toxin protective antigen (PA) intoxication were also evaluated in cells pre and co-treated with only 2 pM of each compound to compare the potency of ML349 and compound C2, C3. At such concentrations C2 and C3, but not ML349, prevented the formation of APT2-CMG2 complexes, blocking cellular intoxication (Figure 18). Therefore, as observed for the inhibition of SARS-CoV-2 replication, C2 and C3 inhibit intoxication of host cells by anthrax-toxin at lower concentrations than ML349.
Example 19: Anthrax toxin cellular intoxication assays with compounds of formula (II). Novel compounds of formula (II) as described herein have been synthesized (Example 1 1 ). These compounds were tested for their capacity to inhibit the formation of CMG2- APT2 complexes in anthrax toxin cellular intoxication assays. Western blot analysis of a single parallel synchronized cellular intoxication experiment showed that compounds C9, C12, C13, C16, and C17 (used at 5 pM) inhibit the formation of CMG2-APT2 complexes beyond 50% (Figure 19).
Experiments were also performed to evaluate the dynamics of the formation of the SDS- resistant toxin pore in the presence of the same compounds (Figure 20). In agreement with the observations from Figure 19, compounds C2, C9, C13, C16 and C17 reduced the formation of PA pores when mammalian cells were pre- and co-treated with 5 pM of each compound during PA treatment. Furthermore, the observed inhibition was equivalent to the inhibitory effect of C2 in the same assay.
Example 20: Inhibition of APT2 activity with compounds of formula (II) blocks SARS-CoV- 2 infection in Calu-3 lung derived cells
Experiments were conducted to compare the effect of inhibiting APT2 activity during SARS-CoV-2 replication in human lung-derived cells with ML349 and different compounds of formula (II). Calu-3 cells were inoculated with SARS-CoV-2 at MOI =0.05 to 0.1 for 1 h, washed, and co-treated with 5 pM of each APT2 inhibitor or an equivalent volume of drug vehicle. At 24 h post-inoculation, infected cells were harvested for quantitative analysis of the levels of the Nucleocapsid protein N. The intracellular levels of N were monitored by western blot as a readout for viral intracellular replication.
Figure 21 shows the levels of viral nucleocapsid protein N: cell lysates, were monitored by Western blot. The % of infection was quantified as the ratio between the levels of N and host GAPDH for three independent replicates for each compound with relation to Control cells (infected in the presence of drug carrier DMSO).
Figure 21 shows that 5 pM of compounds C9, C12, C13, C16, or C17 significantly inhibited SARS-CoV-2 replication beyond 50% and more potently than ML349. At 5 pM, C2 and C3 displayed the most potent inhibitory effect.
Altogether these data demonstrated that compounds of formula (II) as described herein have potent and non-toxic anti-viral and anti-toxin effects.

Claims

1 . An inhibitor of acyl protein thioesterase (APT) of formula (II):
Figure imgf000060_0001
or a pharmaceutically acceptable salt thereof, wherein
Ring A is a thiophene ring or a furane ring;
Each RAm is independently selected from the group comprising hydrogen, halogen, Ci-
C-io alkyl, C2-C10 alkenyl, C1-C6 alkoxy, C1-C6 acyl, C3-C10 cycloalkyl, optionally substituted C3-C10 heterocycloalkyl, C6-C10 aryl, and/or C6-C10 heteroaryl, wherein said cycloalkyl, optionally substituted heterocycloalkyl, aryl or heteroaryl groups may be fused with ring A and with 0 to 2 further cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups; and m is an integer from 0 to 2;
Each R1 , R2, R4, R5 is independently selected from the group comprising hydrogen, halogen, C1-C10 alkyl, optionally substituted Ci-Ce alkyl acyl, optionally substituted Ci-Ce alkyl aminocarbonyl, optionally substituted carbonyl, optionally substituted Ci-Ce alkyl carbonyl, optionally substituted C6-C10 aryl, ether, C2-C10 alkenyl, C6-C10 heteroaryl, C3- C10 cycloalkyl, C3-C10 heterocycloalkyl, Ci-Ce alkoxy, optionally substituted amino, and/or Ci-Ce alkyl amino; and
R3 is selected from the group comprising
Figure imgf000061_0001
2. The inhibitor of acyl protein thioesterase (APT) of formula (II) according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein the ring A is a thiophene ring represented by formula:
Figure imgf000061_0002
3. The inhibitor of acyl protein thioesterase (APT) of formula (II) according to any one of claims 1 -2, or a pharmaceutically acceptable salt thereof, wherein each R1 , R2, R4, R5 is hydrogen.
4. The inhibitor of APT of formula (II) according to any one of claims 1 -3, selected from the group comprising
Figure imgf000062_0001
any pharmaceutically acceptable salt thereof.
10
5. An inhibitor of APT of formula (II) according to any one of claims 1 -4, or a pharmaceutically acceptable salt thereof, for use as a medicament.
6. An inhibitor of APT of formula (II) according to any one of claims 1 -4, or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
7. An inhibitor of APT of formula
Figure imgf000063_0001
or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of a viral and/or a bacterial infection.
8. The inhibitor of APT for use according to any one of claims 6-7, wherein said viral infection is caused by a virus selected from the group comprising Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Togaviridae, Rhabdoviridae, Filoviridae, Pneumoviridae, Retroviridae, Herpesviridae, Flaviviridae, Hepeviridae, and/or Bunyaviridae.
9. The inhibitor of APT for use according to claim 8, wherein said Coronaviridae comprises SARS-CoV-2, SARS-CoV-1 , MHV, TGEV, SADS, MERS-CoV, HCoV-HKU1 , HCoV-229E, HCoV-NL63 and/or HCoV-OC43.
10. The inhibitor of APT for use according to any one of claims 6-7, wherein said bacterial infection is caused by bacterial strains selected from the group comprising Bacillaceae, Vibrionaceae, Pectobacteriaceae Yersiniaceae, Staphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae, Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales and/or Pasteurellaceae.
11 . The inhibitor of APT for use according to any one of claims 6-10, wherein said viral infection is selected from the group comprising COVID, SARS, flu, rheumatic diseases, encephalitis, haemorrhagic fevers, acquired immunodeficiency syndrome (AIDS), sarcoma and/or leukemia; and/or wherein said bacterial infection is selected from the group comprising Typhoid fever, Salmonellosis, bacteria Gastroenteritis intestinal infection, bacteremia, Shigellosis, Bacteria pneumonia, endophthalmitis, bacteremia, septicemia, sepsis, endocarditis, salpingitis, skin infections, and/or meningitis.
12. A pharmaceutical composition comprising at least one inhibitor of APT according to any one of claims 1 -4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
13. The pharmaceutical composition according to claim 12, further comprising one or more additional therapeutic agents selected from the group comprising antibacterial agents and/or antiviral agents.
14. The pharmaceutical composition according to any one of claims 12-13, wherein said pharmaceutical composition is administered by oral, inhalation, nebulization, intranasal, intrapulmonary, intradermal and/or intramuscular route of administration.
15. A kit comprising an inhibitor of APT according to any one of claims 1 -4, and information for use thereof.
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