CN106749558B - Broad-spectrum HIV inhibiting lipopeptides, derivatives thereof, pharmaceutical compositions thereof and uses thereof - Google Patents

Abstract

The invention discloses a lipopeptide for inhibiting HIV in a broad spectrum, a derivative thereof, a pharmaceutical composition thereof and application thereof. The lipopeptide is a) or b) as follows: a) the lipopeptide comprises a polypeptide with antiviral activity, a connecting arm connected with the carboxyl terminal of the polypeptide, an amino acid X residue connected with the connecting arm and a lipophilic compound connected with the amino acid X residue; b) the lipopeptides include polypeptides having antiviral activity, a lipophilic compound attached to the carboxy terminus of the polypeptide; the virus is any one of the following v1-v 7: v1, HIV-1, HIV-2 and SIV; v2, HIV-1 and HIV-2; v3, HIV-1 and SIV; v4, HIV-2 and SIV; v5, HIV-1; v6, HIV-2; v7, SIV; the amino acid X is K or C or S or T or Y. The present invention may be used for the treatment and/or prevention of HIV infection.

Description

Broad-spectrum HIV inhibiting lipopeptides, derivatives thereof, pharmaceutical compositions thereof and uses thereof

Technical Field

The invention relates to lipopeptide for inhibiting HIV in a broad spectrum manner in the field of biomedicine, a derivative thereof, a pharmaceutical composition thereof and application thereof.

Background

Human Immunodeficiency Virus (HIV) causing Acquired Immune Deficiency Syndrome (AIDS) is classified into two types, type 1 and type 2. At present, the number of HIV infection is about 3600 ten thousand worldwide, HIV-1 is the main pathogen, and about 100-. However, HIV-2 has gradually spread to regions such as Europe, Asia, and North America, posing a serious threat to human health [1 ]. The vaccine is the best means for preventing AIDS, but the effective AIDS vaccine has great breakthrough in short term. Therefore, the development of drugs for blocking different replication stages of viruses is a key strategy for preventing and controlling AIDS at present. Because the prior AIDS treatment drugs for clinical application are designed according to HIV-1, the activity of a plurality of drugs to HIV-2 is limited, and the drugs cannot be used for clinical treatment of HIV-2 infection.

HIV entry into a target cell is mediated by envelope glycoprotein (Env) on its surface [2] which is composed of surface subunit gp120 and transmembrane subunit gp41 linked by a non-covalent bond and is in a trimeric structure in its natural state. gp120 has a major function of binding to cell receptor CD4 and co-receptors (such as chemokine receptor CCR5 or CXCR4, etc.), and gp41 mainly mediates membrane fusion of virus and cell. studies have found that gp41 the extracellular domain comprises several important functional regions including hydrophobic Fusion Peptide (FP), N-terminal helix repeat (NHR), C-terminal helix repeat (CHR). As early in 1997, by resolving the crystal structure of a polypeptide complex derived from NHR and CHR, the core structure of gp41 is found to be a six-stranded helix bundle (6-HB) in which the N-helix composed of three NHRs interacts with amino acid residues at a and d positions to form a centrally located helix through interaction with each other, amino acid residues at e and g positions expose to the six helix bundles (6-HB), and the N-helix bundle is found to form a central helix bundle, which is exposed, and when the three-helix-binding to the target protein is found to the cell is stabilized by interaction with the three-loop motif of the three-helix motif of the protein binding to the protein of the protein involved in the protein binding to the protein of the cell receptor CD 355 and the protein, which is found to form a structural domain of a three-protein, which is found to form a structural domain of a structural motif of a loop binding to form a loop binding to a loop after HIV binding to a loop binding to a structural motif which is found to a structural motif which is known to a structural motif which is found to.

The polypeptides derived from gp41NHR and CHR have significant anti-HIV activity, mainly by competitively blocking the formation of 6-HB of the virus itself by binding to the corresponding NHR or CHR [7,8 ]. The U.S. FDA approved AIDS therapeutic drug T-20(Enfuvirtide, Fuzeon) was obtained in 2003 as a 36 amino acid residue polypeptide derived from CHR. T-20 is the first and only viral membrane fusion inhibitor currently approved for clinical therapy, but it induces resistance easily and has poor activity against HIV-2, greatly limiting its widespread use [9 ]. The polypeptide T1249 with the length of 39 amino acids is a second generation product of T20, has higher capability of inhibiting HIV-1 and HIV-2, but stops clinical trials due to the problems of dosage form and cost. Therefore, much research has been devoted to the development of a new generation of HIV membrane fusion inhibiting polypeptides. Since T-20 does not contain the essential NHR pocket binding sequence (PBD), another CHR polypeptide, C34, is widely used as a template for the design of novel inhibitors [7 ]. However, most of the newly designed polypeptides have a long amino acid sequence as T-20 and T1249, e.g., T2635 has 38 amino acids and SC35EK has 35 amino acids. The Cifuwei peptide (SFT) and the Abofovir peptide (ABT) in the Chinese clinical test have 36 and 34 amino acids respectively. Some polypeptides designed using HIV-2 and/or SIV sequences are also equally long, e.g., P3 and C34EHO each contain 34 amino acids [10 ]. Meanwhile, one of the obvious defects of the polypeptide drugs is the short biological half-life, such as the half-life of T-20 is only 3.8 hours, so that the treatment of patients needs to adopt high-dose frequent injection (90 mg/time and 2 times/day) clinically. In recent years, Chong H and the like focus on the structure, function and inhibitor research of HIV fusion protein gp41, and a new concept of designing a targeted NHR hydrophobic pocket short polypeptide HIV membrane fusion inhibitor based on an M-T hook structure and a salt bridge helical structure is proposed [11,12 ]. Recently, polypeptide 2P23 designed by introducing HIV-2/SIV sequence such as Xiong S has only 23 amino acids, and has high antiviral activity, broad spectrum, and strong inhibitory activity against HIV-1, HIV-2 and Simian Immunodeficiency Virus (SIV) [13 ]. Recent studies have also shown that lipid chemical modification of polypeptides, so-called "lipopeptides", can not only improve the antiviral targeting and activity of polypeptides, but also significantly improve the stability and biological half-life of polypeptides, such as cholesterol-modified C34(C34-Chol) [14] and palmitic acid (C16) -modified HP23 short peptide (LP-11) [15 ]. These research advances have all laid a solid theoretical foundation and technological route for designing new HIV membrane fusion inhibitors.

Reference documents:

1.de Silva TI,Cotten M,Rowland-Jones SL.HIV-2:the forgotten AIDSvirus.Trends Microbiol 2008,16:588-595.

2.Colman PM,Lawrence MC.The structural biology of type I viralmembrane fusion.Nat Rev Mol Cell Biol 2003,4:309-319.

3.Tan K,Liu J,Wang J,Shen S,Lu M.Atomic structure of a thermostablesubdomain of HIV-1gp41.Proc Natl Acad Sci U S A 1997,94:12303-12308.

4.Chan DC,Fass D,Berger JM,Kim PS.Core structure of gp41from the HIVenvelope glycoprotein.Cell 1997,89:263-273.

5.Chan DC,Kim PS.HIV entry and its inhibition.Cell 1998,93:681-684.

6.Chan DC,Chutkowski CT,Kim PS.Evidence that a prominent cavity inthe coiled coil of HIV type 1gp41is an attractive drug target.Proc Natl AcadSci U S A1998,95:15613-15617.

7.He Y.Synthesized peptide inhibitors of HIV-1gp41-dependent membranefusion.Curr Pharm Des 2013,19:1800-1809.

8.Eckert DM,Kim PS.Mechanisms of viral membrane fusion and itsinhibition.Annu Rev Biochem 2001,70:777-810.

9.Xu L,Pozniak A,Wildfire A,Stanfield-Oakley SA,Mosier SM,RatcliffeD,et al.Emergence and evolution of enfuvirtide resistance following long-termtherapy involves heptad repeat 2mutations within gp41.Antimicrob AgentsChemother2005,49:1113-1119.

10.Borrego P,Calado R,Marcelino JM,Pereira P,Quintas A,Barroso H,Taveira N.An ancestral HIV-2/simian immunodeficiency virus peptide withpotent HIV-1and HIV-2fusion inhibitor activity.AIDS 2013,27:1081-1090.

11.Chong H,Qiu Z,Su Y,Yang L,He Y.Design of a highly potent HIV-1fusion inhibitor targeting the gp41pocket.AIDS 2015,29:13-21.

12.Chong H,Yao X,Qiu Z,Sun J,Zhang M,Waltersperger S,et al.Short-peptide fusion inhibitors with high potency against wild-type andenfuvirtide-resistant HIV-1.FASEB J 2013,27:1203-1213.

13.Xiong S,Borrego P,Ding X,Zhu Y,Martins A,Chong H,Taveira N,He Y.Ahelical short-peptide fusion inhibitor with highly potent activity againstHIV-1,HIV-2and simian immunodeficiency virus.J Virol.2016，Dec 16；91(1).pii：e01839-16PMID：27795437.

14.Ingallinella P,Bianchi E,Ladwa NA,Wang YJ,Hrin R,Veneziano M,etal.Addition of a cholesterol group to an HIV-1peptide fusion inhibitordramatically increases its antiviral potency.Proc Natl Acad Sci U S A 2009,106:5801-5806.

15.Chong H,Wu X,Su Y,He Y.Development of potent and long-acting HIV-1fusion inhibitors.AIDS 2016,30(8):1187-1196.

disclosure of Invention

The technical problem to be solved by the invention is how to improve the antiviral activity of the polypeptide with HIV-1 and/or HIV-2 and/or SIV inhibiting activity on HIV-1 and/or HIV-2 and/or SIV, and/or prolong the duration of the antiviral activity.

In order to solve the above technical problems, the present invention provides lipopeptides having anti-HIV-1 and/or HIV-2 and/or SIV activity.

The lipopeptide (compound) with anti-HIV-1 and/or HIV-2 and/or SIV activity provided by the invention is a) or b) shown in the specification:

a) the lipopeptide comprises a polypeptide with antiviral activity, a connecting arm connected with the carboxyl terminal of the polypeptide, an amino acid X residue connected with the connecting arm and a lipophilic compound connected with the amino acid X residue; the amino acid X is K, C, S, T or Y;

b) the lipopeptides include polypeptides having antiviral activity, a lipophilic compound attached to the carboxy terminus of the polypeptide;

in the a) or b), the virus is any one of the following v1-v 7:

v1, HIV-1, HIV-2 and SIV;

v2, HIV-1 and HIV-2;

v3, HIV-1 and SIV;

v4, HIV-2 and SIV;

v5、HIV-1；

v6、HIV-2；

v7、SIV。

in the lipopeptides having anti-HIV-1 and/or HIV-2 and/or SIV activity as described above, the antiviral activity may also be referred to as viral inhibitory activity, in particular, inhibition of viral cell fusion and/or inhibition of viral entry into cells and/or inhibition of viral replication.

In the above lipopeptides having HIV-1 and/or HIV-2 and/or SIV inhibiting activity, said linker arm in a) or b) may be a flexible linker arm, such as Fmoc-NH-PEGn-CH₂CH₂COOH, n is 2, 3,4, 6, 8, 10, 11 or 12, said linking arms can also be replaced by other flexible linking arms known in the art; and/or the presence of a gas in the gas,

the lipophilic compound may be a fatty acid (fatty acid) or cholesterol (cholestrol) containing 8 to 20 carbon atoms, dihydrosphingosine (dihydrosphingosine); the lipophilic compound may be vitamin e (tocopherol) and the like.

In the above lipopeptides having HIV-1 and/or HIV-2 and/or SIV inhibitory activity, said fatty acid having from 8 to 20 carbon atoms may be palmitic acid (also known as palmitic acid) (C16) or stearic acid (C18).

In the lipopeptide with HIV-1 and/or HIV-2 and/or SIV inhibiting activity, all amino acids in the polypeptide with HIV-1 and/or HIV-2 and/or SIV inhibiting activity can be L-type amino acids, and one or more of the amino acids can also be replaced by D-type amino acids, artificially modified amino acids, naturally existing rare amino acids and the like, so as to improve the bioavailability, stability and/or antiviral activity of the polypeptide. Wherein the D-form amino acid means an amino acid corresponding to the L-form amino acid constituting the protein; the artificially modified amino acid refers to common L-type amino acid which is modified by methylation, phosphorylation and the like and forms protein; the rare amino acids existing in nature include unusual amino acids constituting proteins and amino acids not constituting proteins, such as 5-hydroxylysine, methylhistidine, gamma-aminobutyric acid, homoserine and the like.

In the lipopeptide with HIV-1 and/or HIV-2 and/or SIV inhibiting activity, the polypeptide in a) or b) is any one of P1-P20:

p1 and a polypeptide shown in a sequence 1 in a sequence table; i.e., the polypeptides represented by amino acid residues 1-23 of LP-19, LP-20, LP-21, LP-22, and LP-23 of FIG. 1;

p2, a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 1 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p3 and a polypeptide shown in a sequence 2 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-31 of LP-31 in FIG. 1;

p4, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 2 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p5 and a polypeptide shown in a sequence 3 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-31 of LP-32 in FIG. 1;

p6, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 3 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p7 and a polypeptide shown in a sequence 4 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-34 of LP-29 in FIG. 1;

p8, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 4 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p9 and a polypeptide shown in a sequence 5 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-25 of LP-25 in FIG. 1;

p10, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 5 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p11 and a polypeptide shown in a sequence 6 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-25 of LP-24 in FIG. 1;

p12, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 6 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p13 and a polypeptide shown in a sequence 7 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-31 of LP-28 in FIG. 1;

p14, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 7 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p15 and a polypeptide shown in a sequence 8 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-30 of LP-30 in FIG. 1;

p16, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 8 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p17 and a polypeptide shown in a sequence 9 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-27 of LP-26 in FIG. 1;

p18, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 9 in the sequence table, wherein the derivative polypeptide has the antiviral activity;

p19 and a polypeptide shown as a sequence 10 in a sequence table; i.e., the polypeptide represented by amino acid residues 1-28 of LP-27 of FIG. 1;

p20, and a derivative polypeptide obtained by adding or substituting more than 1 amino acid residue at any site of the polypeptide shown in the sequence 10 in the sequence table, wherein the derivative polypeptide has the antiviral activity.

In the above lipopeptide having activity of inhibiting HIV-1 and/or HIV-2 and/or SIV, the derivative polypeptide consists of 20 to 34 amino acid residues.

In the above lipopeptide having activity of inhibiting HIV-1 and/or HIV-2 and/or SIV, in a) or b), in order to improve stability, the lipopeptide further comprises an amino-terminal protecting group and/or a carboxy-terminal protecting group, wherein the amino-terminal protecting group is linked to the amino terminus of the polypeptide and the carboxy-terminal protecting group is linked to the carboxy terminus of the polypeptide.

In the above lipopeptides having HIV-1 and/or HIV-2 and/or SIV inhibiting activity, the lipopeptide is af) or bf):

af) consisting of the polypeptide with antiviral activity, a linker arm connected to the carboxyl terminus of the polypeptide, an amino acid X residue connected to the linker arm, and a lipophilic compound and a protecting group connected to the amino acid X residue; the amino acid X is K, C, S, T or Y; the protecting group is an amino-terminal protecting group and/or a carboxy-terminal protecting group, the amino-terminal protecting group is attached to the amino terminus of the lipopeptide, and the carboxy-terminal protecting group is attached to the carboxy terminus of the lipopeptide;

bf) consisting of said polypeptide having antiviral activity, a lipophilic compound attached to the carboxy terminus of said polypeptide and a protecting group; the protecting group is an amino-terminal protecting group attached to the amino terminus of the lipopeptide and/or a carboxy-terminal protecting group attached to the carboxy terminus of the lipopeptide.

The amino terminal protecting group can be any one of acetyl, amino, maleoyl, succinyl, tert-butyloxycarbonyl or benzyloxy or other hydrophobic groups or macromolecular carrier groups; the carboxyl terminal protecting group may be any of amino, carboxyl, amido or tert-butyloxycarbonyl or other hydrophobic or macromolecular carrier groups.

In the above lipopeptide having activity of inhibiting HIV-1 and/or HIV-2 and/or SIV, the linker arm is Fmoc-NH-PEG8-CH₂CH₂COOH。

Fmoc-NH-PEG8-CH₂CH₂The English name of COOH is 1- (9H-fluoro-9-yl) ester or 5,8,11,14,17,20,23,26-Octaoxa-2-azanonacosanedioic acid, C₃₄H₄₉NO₁₂). Fmoc-NH-PEG8-CH during chemical synthesis of specific lipopeptides₂CH₂COOH can be formed by two Fmoc-NH-PEG4-CH₂CH₂COOH (British name is Fmoc-15-amino-4,7,10, 13-tetraoxapentadecanoic acid) in series.

The lipopeptide having HIV-1 and/or HIV-2 and/or SIV inhibitory activity may specifically be any one of the 14 lipopeptides LP-19 to LP-32 in FIG. 1.

The polypeptide of any one of the above P1-P20, its pharmaceutically acceptable salt, or its derivative also belong to the protection scope of the present invention.

The derivative of the polypeptide can be at least one of the following 1) to 5):

1) the amino terminal of the polypeptide is connected with an amino terminal protecting group and/or the carboxyl terminal of the polypeptide is connected with a connector obtained by a carboxyl terminal protecting group;

2) the carboxyl terminal of the polypeptide is connected with an oligopeptide or a lipophilic compound to obtain a connector;

3) the amino terminal of the polypeptide is connected with a linker obtained by oligopeptide or lipophilic compound;

4) the amino terminal and the carboxyl terminal of the polypeptide are connected with oligopeptide or lipophilic compound to obtain a connector;

5) the polypeptide is a modifier obtained by modifying protein, polyethylene glycol and maleimide.

Multimers of PM1 or PM2 are also within the scope of the invention:

PM1, multimers formed by the lipopeptides, pharmaceutically acceptable salts thereof, or derivatives thereof;

PM2, multimers formed by the polypeptide, pharmaceutically acceptable salts thereof, or derivatives thereof.

The following compositions are also within the scope of the present invention: a composition comprising C1) and C2): C1) is C11), C12) or/and C13); said C11) is said lipopeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof; said C12) is said polypeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof; said C13) is said multimer;

C2) a pharmaceutically acceptable carrier or adjuvant;

the composition has at least one of the following functions F1) -F5):

F1) resisting viruses;

F2) treating and/or preventing and/or adjunctively treating diseases caused by viral infection (such as AIDS);

F3) inhibiting cell fusion of the virus;

F4) inhibiting the entry of a virus into a cell;

F5) inhibiting viral replication;

F1) -F5), the virus is any one of the following v1-v 7:

v1, HIV-1, HIV-2 and SIV;

v2, HIV-1 and HIV-2;

v3, HIV-1 and SIV;

v4, HIV-2 and SIV;

v5、HIV-1；

v6、HIV-2；

v7、SIV。

the application of the above C11), C12), C13) or/and C14) in preparing at least one product of E1) to E5) also belongs to the protection scope of the invention:

said C14) is said composition;

e1) is an antiviral product, such as a drug or vaccine;

e2) is a product for treating and/or preventing and/or assisting in treating diseases (such as AIDS) caused by virus infection, such as drugs or vaccines;

e3) is a product that inhibits cell fusion of viruses, such as drugs or vaccines;

e4) is a product that inhibits the entry of a virus into a cell, such as a drug or vaccine;

e5) is a product that inhibits viral replication, such as a drug or vaccine;

the E1) -E5), the virus is any one of the following v1-v 7:

v1, HIV-1, HIV-2 and SIV;

v2, HIV-1 and HIV-2;

v3, HIV-1 and SIV;

v4, HIV-2 and SIV;

v5、HIV-1；

v6、HIV-2；

v7、SIV。

in order to solve the above technical problems, the present invention provides a pharmaceutical compound.

The medicinal compound provided by the invention is the C11), the C12) or the C13).

The medicinal compound has at least one function of F1) -F5):

F1) resisting viruses;

F2) treating and/or preventing and/or adjunctively treating diseases caused by viral infection;

F3) inhibiting cell fusion of the virus;

F4) inhibiting the entry of a virus into a cell;

F5) inhibiting viral replication;

F1) -F5), the virus is any one of the following v1-v 7:

v1, HIV-1, HIV-2 and SIV;

v2, HIV-1 and HIV-2;

v3, HIV-1 and SIV;

v4, HIV-2 and SIV;

v5、HIV-1；

v6、HIV-2；

v7、SIV。

the following methods for treating and/or preventing virus-infected animals are also within the scope of the present invention:

a method of treating or/and preventing viral infection of an animal comprising administering said C11), said C12), said C13), or/and C14) to a recipient animal to inhibit viral infection of the animal;

said C14) is said composition;

the virus is any one of the following v1-v 7:

v1, HIV-1, HIV-2 and SIV;

v2, HIV-1 and HIV-2;

v3, HIV-1 and SIV;

v4, HIV-2 and SIV;

v5、HIV-1；

v6、HIV-2；

v7、SIV。

the lipopeptide or polypeptide, the derivative thereof or the pharmaceutically acceptable salt thereof, the polymer, the composition or the pharmaceutically acceptable compound provided by the invention can be used for treating and/or preventing HIV (HIV-1 and/or HIV-2) and/or SIV infection. In practice, the lipopeptide or polypeptide of the present invention, its derivative, or a pharmaceutically acceptable salt thereof, the multimer, the composition or the pharmaceutical compound can be administered as a medicament directly to a patient, or can be administered to a patient after mixing with a suitable carrier or excipient for the purpose of treating and/or preventing HIV infection. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections and the like. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired. The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; for luminal administration, such as rectally and vaginally; administration to the respiratory tract, e.g., nasally; administration to the mucosa. The above route of administration is preferably by injection.

The lipopeptide or polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, the multimer, the composition or the pharmaceutical compound thereof can be administered in an amount depending on many factors, such as the nature and severity of the disease to be prevented or treated, sex, age, body weight and individual response of the patient or animal, the specific active ingredient used, administration route and administration frequency, and the like. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g. two, three or four dosage forms.

The lipopeptide or polypeptide, the derivative thereof or the pharmaceutically acceptable salt thereof, the polymer, the composition or the pharmaceutically acceptable compound can be directly and independently used for treating and preventing HIV infected people, and can also be used together with one or more anti-HIV medicines so as to achieve the aim of improving the overall treatment effect. Such anti-HIV agents include, but are not limited to, reverse transcriptase inhibitors, protease inhibitors, invasion inhibitors, integration inhibitors, maturation inhibitors, and the like. The reverse transcriptase inhibitor can be one or more of AZT, 3TC, ddI, d4T, ddT, TDF, Abacavir, Nevirapine, Efavirenz, Delavirdine and the like; the protease inhibitor can be one or more of Saquinavir mesylate, Idinavir, Ritonavir, Amprenavir, Kaletra and Nelfinavir mesylate; the invasion inhibitor can be one or more of Maraviroc, TAK-779, T-20, T2635, Cifuwei peptide, Ebosvir peptide, VIRIP (VIR-576), etc.; the integration inhibitor can be one or more of Raltegravir, Dolutegravir, Elvitegravi and the like.

For any particular patient, the specific therapeutically effective dose level will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration and rate of excretion of the particular active ingredient employed; the duration of treatment; drugs used in combination or concomitantly with the specific active ingredient employed; and similar factors known in the medical arts. For example, it is common in the art to start doses of the active ingredient at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved. In general, the lipopeptides or polypeptides, derivatives thereof, or pharmaceutically acceptable salts thereof, the multimers, the compositions or the pharmaceutical compounds of the present invention can be administered to a mammal, particularly a human, at a dose of between 0.001-1000 mg/kg body weight/day, such as between 0.01-100 mg/kg body weight/day, and such as between 0.1-10 mg/kg body weight/day.

The lipopeptide LP-19 of the invention has the advantages of broad spectrum, strong effect and long effect:

1. LP-19 has stronger broad-spectrum anti-HIV-1, HIV-2 and SIV activities, and especially shows strong advantageous effects on HIV-2 and SIV, LP-19 has strong inhibition effect on HIV-1, HIV-2 and SIV mediated cell membrane fusion, which is obviously higher than three control polypeptides T-20, 2P23 and LP-11 (figure 3); LP-19 had a potent inhibitory effect on both HIV-1 and SIV-mediated cell invasion, significantly higher than the three control polypeptides T-20, 2P23 and LP-11 (FIG. 4). The inhibitory activity of LP-19 on infectious HIV-1 and HIV-2 was significantly higher than that of the three control polypeptides T-20, 2P23 and LP-11 (FIG. 5).

2. The inhibitory activity of LP-19 against various subtypes of HIV-1 virus was also significantly higher than that of the three control polypeptides T-20, 2P23 and LP-11 (FIG. 6).

3. The activity of LP-19 for inhibiting the T-20 drug-resistant strain is 10613.53 times, 6.18 times and 2.18 times higher than that of T-20, 2P23 and LP-11 respectively; the activity of LP-19 in inhibiting 2P23 drug-resistant strain was 173.63 times, 45.09 times and 6.83 times higher than that of T-20, 2P23 and LP-11, respectively (FIG. 7).

4. LP-19 shows significant potent and long-lasting activity in monkeys, with a subcutaneous peak inhibition value of over 100 times that of T-20 and a venous peak inhibition value of over 2000 times that of T-20. LP-19 inhibited peak serum maximal dilutions 66-fold and 40-fold, respectively, even at 60 and 72 hours of subcutaneous injection, similar to the performance of T-20 at 2 and 4 hours (FIG. 8).

The derivatives LP-20 to LP-32 of LP-19 have a broad spectrum of activity, being effective against both HIV-1 and HIV-2 and SIV (FIG. 1).

The lipopeptide, the derivative thereof, or the pharmaceutically acceptable salt thereof, the polymer, the composition or the pharmaceutical compound provided by the invention can be used for treating and/or preventing HIV (HIV-1 and/or HIV-2) and/or SIV infection. In practice, the lipopeptides, derivatives thereof, or pharmaceutically acceptable salts thereof, the multimers, the compositions or the pharmaceutical compounds of the present invention may be administered as a medicament directly to a patient, or mixed with a suitable carrier or excipient and administered to a patient for the purpose of treating and/or treating an HIV infection.

Drawings

FIG. 1 shows the structure of lipopeptides having HIV-1 and/or HIV-2 and/or SIV inhibitory activity and their antiviral activity.

FIG. 2 shows the structure and function of HIV fusion protein gp41 and its polypeptide membrane fusion inhibitor. Wherein FP refers to gp41 fusion peptide; NHR refers to the N-terminal repeat; CHR refers to the C-terminal repeat; 6-HB refers to the hexamer helix. Amino acids in the polypeptide sequence, marked in bold, are residues that form M-T hooks, and the underlined portion is the pocket binding region (PBD). In FIG. 2, all of the polypeptides or lipopeptides are protected at their amino terminus by an acetyl group and at their carboxyl terminus by an amino group.

FIG. 3 is a graph showing the inhibition of HIV-1, HIV-2 and SIV mediated cell membrane fusion by LP-19 and control polypeptides.

FIG. 4 is a graph showing the inhibition of HIV-1 and SIV pseudovirus cell entry by LP-19 and control polypeptides.

FIG. 5 is a graph showing the inhibitory effect of LP-19 and control polypeptides on HIV-1 and HIV-2 infection.

FIG. 6 shows the inhibition of various subtypes of HIV-1 by LP-19 and control polypeptides.

FIG. 7 shows the inhibition of 2P 23-resistant strain by LP-19 and control polypeptide T-20.

FIG. 8 shows the antiviral activity of the sera of cynomolgus monkeys injected with LP-19 and control polypeptides. In the figure, M248, M249, M250, M252, M253 and M254 are monkey numbers.

FIG. 9 is a circular dichroism analysis of 2P23 and LP-19 self-helix structures and their interaction with NHR. In the figure, NA indicates that the Tm value (nottappable) cannot be determined accurately because the helical structure is not sufficiently dissociated in the measurement temperature range.

The abbreviations for the amino acids in the present invention have the meaning well known in the art, E is glutamic acid, M is methionine, T is threonine, W is tryptophan, K is lysine, V is valine, L is leucine, I is isoleucine, C is cysteine, Q is glutamine, N is asparagine, Y is tyrosine, S is serine, T is threonine, a is alanine; in FIGS. 1-9, Ac is acetyl, PEG8 is Fmoc-NH-PEG8-CH₂CH₂COOH, C16 palmitic acid, C18 stearic acid, NH₂Is amino, Chol is bileSterol, Dih is sphinganine and Toc is vitamin E. In FIGS. 3-5, polypeptide concentration refers to the concentration of lipopeptide or polypeptide.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Of the lipopeptides having HIV-1 and/or HIV-2 and/or SIV inhibitory activity of the present invention, the most preferred embodiments are those of af) and bf) as defined above. Wherein the polypeptide with HIV-1 and/or HIV-2 and/or SIV activity is the polypeptide shown in the sequence 1 in the sequence table, namely the polypeptide shown in the 1 st-23 rd amino acid residues of LP-19, LP-20, LP-21, LP-22 and LP-23 in figure 1, namely 2P23), P3 (the polypeptide shown in the sequence 2 in the sequence table, namely the polypeptide shown in the 1 st-31 th amino acid residues of LP-31 in figure 1), P5 (the polypeptide shown in the sequence 3 in the sequence table, namely the polypeptide shown in the 1 st-31 th amino acid residues of LP-32 in figure 1), P7 (the polypeptide shown in the sequence 4 in the sequence table, namely the polypeptide shown in the 1 st-34 th amino acid residues of LP-29 in figure 1), P9 (the polypeptide shown in the sequence 5 in the sequence table, i.e., the polypeptide represented by the amino acid residues at positions 1 to 25 of LP-25 in FIG. 1), P11 (the polypeptide represented by sequence No. 6 in the sequence Listing, i.e., the polypeptide represented by the amino acid residues at positions 1 to 25 of LP-24 in FIG. 1), P13 (the polypeptide represented by sequence No. 7 in the sequence Listing, i.e., the polypeptide represented by the amino acid residues at positions 1 to 31 of LP-28 in FIG. 1), P15 (the polypeptide represented by sequence No. 8 in the sequence Listing, i.e., the polypeptide represented by the amino acid residues at positions 1 to 30 of LP-30 in FIG. 1), P17 (the polypeptide represented by sequence No. 9 in the sequence Listing, i.e., the polypeptide represented by the amino acid residues at positions 1 to 27 of LP-26 in FIG. 1), and P19 (the polypeptide represented by sequence No. 10 in the sequence No. 1 to 28 of LP-27 in FIG. 1 in. The lipophilic compound is palmitic acid (also known as palmitic acid) (C16), stearic acid (C18), cholesterol (cholestrol), dihydrosphingosine (dihydrosphingosine) or a vitaminElement e (tocopherol). The connecting arm is Fmoc-NH-PEG8-CH₂CH₂COOH. The protecting groups are amino terminal protecting groups and carboxyl terminal protecting groups. The amino terminal protecting group is acetyl, and the carboxyl terminal protecting group is amino. These lipopeptides having HIV-1 and/or HIV-2 and/or SIV inhibitory activity are designated by the names LP-19 to LP-32, respectively, and their structures are shown in FIGS. 1 and 2, have broad spectrum activity (effective against both HIV-1 and HIV-2 and SIV) and are long-acting.

The lipopeptide with the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV has stronger binding capacity to NHR target sequences from HIV-1, HIV-2 and SIV, has extremely strong inhibition effect on viruses, and shows obvious long-acting activity in non-human primates (monkeys).

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. All of the polypeptides in the examples below have amino acids in the L-form.

Example 1 design of lipopeptides

This example designs lipopeptides having HIV-1 and/or HIV-2 and/or SIV inhibitory activity, designated by the names LP-19, LP-20, LP-21, LP-22, LP-23, LP-24, LP-25, LP-26, LP-27, LP-28, LP-29, LP-30, LP-31 and LP-32, respectively, and their structures are shown in FIG. 1.

Example 2 preparation of Polypeptides and lipopeptides

1. Preparation of polypeptides

The amino terminal of the polypeptides T-20 and 2P23 is connected with acetyl as amino terminal protecting group, the carboxyl terminal is connected with amino as carboxyl terminal protecting group, and the structures of the polypeptides are shown in figure 2. These polypeptides were synthesized manually from the carboxy-terminal to the amino-terminal using standard solid-phase polypeptide synthesis (Fmoc/tBu strategy). All polypeptide sequences are as normal as for polypeptide synthesisThe C-terminus is amidated and the N-terminus is acetylated. Amino protecting group Fmoc is removed by using amino acid protected by Fmoc and Rink resin (with a substitution constant of 0.44mmol/g) as a solid phase carrier and using DMF solution of 25% (volume percentage content) piperidine, wherein the removal steps are carried out twice each time, and the time duration is respectively 8min and 10 min. The condensation method adopted in the peptide-joining reaction is a DIPC/HOBt method and a PyBOP method, 3 times of equivalent of amino acid and activating reagent are adopted, the reaction time is 1 hour, and the reaction process is monitored by ninhydrin qualitative color development (Kaiser method). If the condensation reaction of a certain amino acid is not complete, the reaction time is prolonged properly or the condensation is repeated once until the desired target peptide segment is obtained. Cleavage reagents (trifluoroacetic acid: 1, 2-ethanedithiol: thioanisole: phenol: H)₂O: triisopropylsilane-68.5: 10:10:5:3.5:1, v/v) the target polypeptide was cleaved from the resin and the side chain protecting groups were removed (cleavage at 30 ℃ for 4 hours). Filtering, adding the filtrate into cold anhydrous diethyl ether to precipitate polypeptide, and centrifuging. Washing with diethyl ether for several times, and drying to obtain crude polypeptide product.

2. Preparation of lipopeptides

LP-19, LP-20, LP-21, LP-22, LP-23, LP-24, LP-25, LP-26, LP-27, LP-28, LP-29, LP-30, LP-31, and LP-32, each of which has the structure shown in FIG. 1, are prepared according to the following procedure.

Modification of the polypeptide with palmitic acid (LP-19, LP-26 to LP-30), stearic acid (LP-20, LP-24, LP-25, LP-31, LP-32), dihydro (neuro) sphingosine (LP-22) or vitamin E (LP-23) is accomplished by amidation of the side chain amino group of Lys at the C-terminus of the polypeptide (ref. 15: Chong H, Wu X, Su Y, He Y. development of and cloning HIV-1fusion inhibitors AIDS 2016,30(8):1187-1196 in the background). The modification of the polypeptide by cholesterol (LP-21) is via a highly chemoselective thioether-forming reaction between the sulfhydryl group of the C-terminal Cys side chain of the polypeptide and cholesteryl bromoacetate onto the polypeptide chain (see background references 14: Ingallinella P, Bianchi E, Ladwa NA, Wang YJ, Hrin R, Venezianano M, et al.Addition of a cholesterol group to an HIV-1peptide fusion inhibitor and catalysis of recombinant microorganisms involved in the construction of polypeptide molecules and protein molecules. Proc Natl Acad Sci U S A2009,106: 5801. fig.5 shows that the modification of the polypeptide by cholesterol is carried out by a thiosulfide-forming reaction.

The synthesis of lipopeptides is illustrated below by way of example LP-19:

chemical reagents used, such as Rink Amide MBHA resin, Fmoc-PEG4-OH (Fmoc-15-amino-4,7,10, 13-tetraoxanonetacanoic acid), various Fmoc amino acids, palmitoyl chloride (palmityl chloride), N '-Diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), trifluoroacetic acid (TFA), Ethanedithiol (EDT), ninhydrin, piperidine (PIPE), phenol, N' -Dimethylformamide (DMF), chromatographically pure acetonitrile, and the like, were purchased from major chemical suppliers and were not further purified prior to use.

The synthesis was performed by standard manual solid phase Fmoc method, starting with Rink Amide MBHA resin (substitution constant 0.34mmol/g) from the C-terminus to the N-terminus. The Fmoc protecting group on Rink resin was removed with 25% piperidine/DMF (vol/vol) and then the first C-terminal amino acid residue was introduced by grafting with 2-fold equivalent of Fmoc-Lys (Dde) -OH/HOBt/DIC to the resin. Then, the N-terminal Fmoc protecting group was removed again with 25% piperidine/DMF (volume ratio) to make the N-terminal free amino group. This was repeated to connect two PEG4 and each amino acid residue in sequence.

The raw materials and the amounts used correspond to Fmoc-PEG4-OH (1.5eq), Fmoc-PEG4-OH (1.5eq), Fmoc-Lys (Boc) -OH (3eq), Fmoc-Leu-OH (3eq), Fmoc-Leu-OH (3eq), Fmoc-Glu (OtBu) -OH (3eq), Fmoc-Ile-OH (3eq), Fmoc-Lys (Boc) (3eq), Fmoc-Lys (3eq), Fmoc-Glu (OtBu) -OH (3eq), Fmoc-Leu-OH (3eq), Fmoc-Glu (OtBu) (3eq), Fmoc-Glu-OH (3eq), Fmoc-Val-OH (3eq), Fmoc-Lys (Boc) -OH (3eq), Fmoc-Glu (OtBu) -OH (3eq), Fmoc-Trp (Boc) -OH (3eq), Fmoc-Glu (OtBu) -OH (3eq), Fmoc-Trp (Boc) -OH (3eq), Fmoc-Thr (tBu) -OH (3eq), Fmoc-Met-OH (3eq), and Fmoc-Glu (OtBu) -OH (3 eq). Finally, N-terminal acetylation capping (3 times equivalent Ac) is carried out₂O, 6 times equivalent diisopropylethylamine), completing the synthesis of the backbone. Wherein the reaction time of each step is as follows: deprotection for 8min, twice; the grafting time for the conventional amino acid was 60 minutes and for the PEG4 was 180 minutes.

The resin was treated with 2% hydrazine hydrate/DMF solution (vol/vol) to remove the protecting group of the side chain of the C-terminal Lys, and then mixed with 3-fold equivalent of palmitoyl chloride and 6-fold equivalent of diisopropylethylamine and subjected to amidation reaction (60 min) with the side chain amino group of the C-terminal Lys, thereby achieving palmitoylation modification on the C-terminal Lys residue.

After each step of reaction, the resin is washed by DMF more than six times, the reaction process is monitored by ninhydrin qualitative color development (Kaiser method), and if the condensation reaction of a certain amino acid is incomplete, the condensation is repeated once until the required target peptide segment is obtained.

Cleavage and side chain protection removal: after the synthesis of the lipopeptides was complete, the resin was dried in vacuo. The cleavage reagent (trifluoroacetic acid: 1, 2-ethanedithiol: thioanisole: phenol: H) was added to the dried resin₂O: triisopropylsilane ═ 68.5:10:10:5:3.5:1, v/v), cleavage conditions at 30 ℃ for 3 hours cleaved the target lipopeptide from the resin and the side chain protecting groups removed. Filtering, adding the filtrate into cold anhydrous diethyl ether to precipitate lipopeptide, and centrifuging. Washing with ether for several times, and drying to obtain lipopeptide crude product.

3. Purification of polypeptides and lipopeptides

The purification and characterization of polypeptide and lipopeptide are carried out by reversed-phase high performance liquid chromatograph, the column material of the chromatographic column is reversed-phase C18 silica gel with 10 micron particle size, the pore diameter is 100 angstroms, and the size of the chromatographic column is 50 × 250 mm.

The chromatographic operation conditions are as follows: linear gradient elution, eluent composed of mobile phase A and mobile phase B. The mobile phase A is an aqueous solution of trifluoroacetic acid and acetonitrile, the concentration of the trifluoroacetic acid is 0.05 percent by volume, and the concentration of the acetonitrile is 2 percent by volume. Mobile phase B was 90% (volume percent) acetonitrile in water. The linear gradient elution was from 20% B to 40% B for 20 min at a flow rate of 25 ml per minute and a UV detection wavelength of 220 nm. The solvent was lyophilized to obtain a fluffy lipopeptide pure product, the chemical structure of which was characterized by MALDI-TOF mass spectrometry, and the purity of which was given by analytical high performance liquid chromatography (flow rate: 1 ml/min). Wherein, the model of the analytical high performance liquid chromatograph: shimadzu CBM-10A VP PULS, the type of the column used was Agela 4.6X 250mm C18. The chromatographic operation conditions are as follows: linear gradient elution, eluent composed of mobile phase A and mobile phase B. The mobile phase A is an aqueous solution of trifluoroacetic acid and acetonitrile, the concentration of the trifluoroacetic acid is 0.05 percent by volume, and the concentration of the acetonitrile is 2 percent by volume. The mobile phase B is an aqueous solution of trifluoroacetic acid and acetonitrile, the volume percentage concentration of the trifluoroacetic acid is 0.05 percent, and the volume percentage concentration of the acetonitrile is 90 percent. The linear gradient eluted from 25% B to 45% B over 20 min. The obtained polypeptide and lipopeptide pure products are characterized by analytical reversed phase high performance liquid chromatography, which shows that the purity of LP-19, LP-20, LP-21, LP-22, LP-23, LP-24, LP-25, LP-26, LP-27, LP-28, LP-29, LP-30, LP-31LP-32, T-20 and 2P23 is more than 95%.

Example 3 detection of broad-spectrum antiviral Activity of lipopeptides

3.1 Experimental materials and methods

3.1.1 inhibition of HIV-and SIV-mediated cell fusion by lipopeptides

Materials and methods for HIV-1, HIV-2 and SIV-mediated inhibition of cell fusion experiments are described in the literature published by Xiong S et al (reference 13 in the background). Among them, HEK293T cells (abbreviated as 239T cells) were purchased from American Type Culture Collection (ATCC); u87CD4+ CXCR4+ cells were provided by NIH AIDS reagent and reference (catalog No. 4036); plasmid pROD of ROD molecular clone of HIV-2 strain was given by professor Nuno Taveira, university of California; expression of SIV Strain SIV_pbjAnd SIV₂₃₉Plasmids for the envelope proteins (pSIVpbj-Env and pSIV239, respectively) were offered by professor Xun Qing, university of Redding; fluorescent reporter system plasmid DSP_1-7And DSP_8-11The plasmid for expressing the envelope protein of HIV-1 strain NL4-3 is a recombinant expression plasmid obtained by inserting the envelope protein (ENV) encoding gene of HIV-1 strain NL4-3 into a vector pcDNA3.1(-) and is prepared by adding 239T cells (effector cells) into a 96-well cell culture plate (1.5 × 10)⁴One/well), U87CD4+ CXCR4+ (target cells) were added to 24-well cell culture plates (8 × 10)⁴One/hole), both at 37 ℃ and 5% CO, respectively₂Cultured under the conditions ofAnd (4) at night. The next day, the plasmid expressing the envelope protein (Env) of the strain and DSP_1-7The plasmid was prepared as follows 1: 1 mixing and transfecting 239T cells, and mixing the DSP_8-11The plasmid transfected U87CD4+ CXCR4+ cells. 5% CO at 37 deg.C₂After culturing for 48 hours under the conditions of (1), the DSP is added_8-11Plasmid transfected U87CD4+ CXCR4+ cells were resuspended in 300 microliters of prewarmed medium and 0.05 microliters of Enduren viable cell substrate (Promega) was added. The test substance T-20, 2P23, LP-11, or LP-19 was dissolved in dimethyl sulfoxide (DMSO) and diluted with a cell culture solution, and then added to effector cell wells of a 96-well plate at a 3-fold dilution, and then 75. mu.l of target cells were transferred to the effector cells. After gentle centrifugation to bring the effector and target cells into sufficient contact, the cells were incubated at 37 ℃ for 1 hour, and luciferase activity (relative fluorescence units, RLU) was measured. The inhibition rate of each concentration sample is calculated, and half effective inhibition dose (IC) is calculated by utilizing GraphPad Prism Software 2.01 Software₅₀Value).

3.1.2 inhibition of HIV-1 and SIV pseudoviruses

The inhibition of the polypeptide on the entry of the virus into target cells was evaluated by using HIV recombinant pseudovirus system, and the method was described in reference to the literature published by XiongS et al [ background reference 13]The plasmids expressing HIV-1 or SIV envelope proteins were as described in 3.1.1 above. The basic steps comprise (1) pseudovirus preparation: will express HIV-1NL4-3, SIV_pbjOr SIV₂₃₉The plasmid of strain envelope protein and HIV skeleton plasmid pSG3 △ ENV (expressing all proteins except ENV in HIV genome, provided by NIH AIDS reagent and reference item in USA, catalog number is 11051), transfect 293T cell according to the mass ratio of 1:2, and set a control only transfect pSG3 △ ENV with the same quantity at 37 ℃ and 5% CO₂After 6 hours of incubation in the cell incubator, the medium was changed and incubation was continued for 48 hours to allow secretion of pseudovirus into the supernatant. The supernatant in the cell culture flask or cell culture plate was aspirated as much as possible by a pipette, filtered through a 0.45 μm filter or centrifuged at 1000g for 10 minutes to collect the supernatant, and Fetal Bovine Serum (FBS) was added thereto to give a final concentration of 20%, and transferred to a polypropylene tube to be stored at-80 ℃ for later use or directly subjected to virus titration. (2) Titration of HIV pseudovirus: virus was plated in 96-well plates5-fold dilution, setting 4 multiple wells with 8 gradients, final volume of 100 microliter, trypsinizing and counting TZM-bl cells, diluting the cells to 1 × 10 with DMEM complete medium⁵Per ml, 100. mu.l cells (containing 15. mu.g/ml DEAE-dextran) were added to each well at 37 ℃ in 5% CO₂Culturing for 48 hours, then taking out a 96-well plate from a cell culture box, sucking supernatant from a sample loading hole, adding 30 microliters of cell lysate, placing for 10 minutes, adding 100 microliters of luciferase detection reagent, sucking 100 microliters of liquid from each hole by using a pipette, adding the liquid into a corresponding 96-well white plate, reading a luminescence value by using a microplate luminometer, calculating the virus titer by using a Reed-Muench method, (3) detecting the antiviral activity, namely diluting and paving the to-be-detected object (T-20, 2P23, LP-11 or LP-19 (dissolved by DMSO and diluted by cell culture solution)) into the 96-well plate according to a multiple ratio (3 times), wherein the final volume is 50 microliters, and the to-be-detected object is replaced by 50 microliters of DMEM culture medium as a negative control, adding 100 microliters of 1 × 10 concentration⁵Each ml of TZM-bl target cells (containing 15. mu.g/ml DEAE-dextran) was added to 50. mu.l of the HIV pseudovirus obtained above (equivalent to 100TCID per well)₅₀) 5% CO at 37 deg.C₂After 48 hours of incubation under conditions, relative fluorescence units (RLU) per well were determined using luciferase assay reagent (Promega). Calculation of% inhibition and IC₅₀The value is obtained.

3.1.3 inhibition of infectious HIV-1(NL4-3) and HIV-2(ROD) strains

For inhibition of replication of HIV-1(NL4-3) and HIV-2(ROD) strains, reference is made to the publication by Xiong S et al [ background Art reference 13]. Molecular cloning plasmid pNL4-3 encoding HIV-1 strain NL4-3 was provided by the NIH AIDS reagent and reference project (catalog No. 114); molecular cloning of HIV-2 strain ROD plasmid pROD was as described in 3.1.1 above. The plasmid was prepared using a plasmid extraction kit from QIAGEN, and the plasmid was transfected into 293T cells using Lipofectamine (TM) 2000 transfection reagent from Invitrogen at 37 ℃ and 5% CO₂After 6 hours of incubation in the cell incubator, the medium was changed and the incubation was continued for 48 hours. Gently collecting supernatant from cell culture flask or cell culture plate with pipette, filtering with 0.45 micrometer filter to obtain supernatant, adding 20% FBS, and packaging in polypropylene tubeAnd storing at-80 ℃ for later use or directly carrying out virus titration by the method similar to the HIV pseudovirus. To determine antiviral activity, the test substance (T-20, 2P23, LP-11 or LP-19 (dissolved in DMSO and diluted in cell culture)) was diluted in duplicate (3-fold) into 96-well plates in a final volume of 50 microliters, with 50 microliters of DMEM medium replacing the test substance as a negative control. 100 microliters of TZM-bl cells (10) were added⁵Cells/ml, containing 15. mu.g/ml DEAE-dextran), 50. mu.l of the virus obtained, corresponding to 100TCID per well, are added₅₀. After 48 hours of incubation, relative fluorescence units (RLU) per well were determined using luciferase assay reagent (Promega). Calculation of% inhibition and IC₅₀The value is obtained.

3.2 Experimental results and analysis

3.2.1 inhibition of HIV-1, HIV-2 and SIV mediated cell fusion by LP-19

First, the inhibitory activity of LP-19 against various virus-mediated cell fusions was analyzed, and T-20, 2P23, and LP-11 were used as controls. The results are shown in FIG. 3, and LP-19 has a potent inhibitory effect on HIV-1, HIV-2 and SIV mediated cell membrane fusion, which is significantly higher than the three control polypeptides. Inhibition of HIV-1 Strain NL4-3 mediated cell fusion by LP-19 IC₅₀0.14nM and inhibition IC of T-20, 2P23 and LP-11₅₀Values were 7.17nM, 0.28nM and 0.78nM, respectively; inhibition IC of ROD-mediated cell fusion of LP-19 to HIV-2 strain₅₀2.27nM and inhibition IC of T-20, 2P23 and LP-11₅₀Values were 569.8nM, 12.25nM and 20.96nM, respectively; LP-19 pairs SIV_pbjInhibition of cell fusion mediated IC₅₀0.67nM and inhibition IC of T-20, 2P23 and LP-11₅₀Values were 5.44nM, 1.91nM and 4.18nM, respectively; LP-19 pairs SIV₂₉₃Inhibition of cell fusion mediated IC₅₀2.28nM and inhibition IC of T-20, 2P23 and LP-11₅₀Values were 219nM, 2.64nM and 11.91nM, respectively.

3.2.2 inhibition of HIV-1 and SIV pseudoviruses by LP-19

The inhibitory effect of LP-19 and control polypeptides on pseudovirus-mediated cell invasion was further evaluated, and the results are shown in FIG. 4. T-20, 2P23, LP-11 and LP-19 on HIV-1NInhibition of L4-3 mediated cell invasion IC₅₀78.78nM, 0.78nM, 0.21nM and 0.12nM, respectively, for SIV_pbjInhibition of cell invasion mediated IC₅₀246.41nM, 10.56nM, 17.38nM and 0.48nM, respectively, for SIV₂₃₉Inhibition of cell invasion mediated IC₅₀402.77nM, 3.67nM, 2.9nM and 0.58nM, respectively. LP-19 has a potent inhibitory effect on both HIV-1 and SIV-mediated cell invasion, significantly higher than the three control polypeptides T-20, 2P23 and LP-11.

3.2.3 inhibition of HIV-1 and HIV-2 infection by LP-19

Meanwhile, inhibitory activity against infectious HIV-1 and HIV-2 was examined, and the results are shown in FIG. 5. Inhibition IC of T-20, 2P23, LP-11 and LP-19 against HIV-1 strain NL4-3₅₀Inhibition IC of ROD of HIV-2 strain at 112.75nM, 0.62nM, 0.22nM and 0.16nM, respectively₅₀353.68nM, 16.21nM, 8.09nM and 1.66nM, respectively. The inhibitory activity of LP-19 on infectious HIV-1 and HIV-2 was significantly higher than that of the three control polypeptides T-20, 2P23 and LP-11. The combination of the experimental results of 3.2.1, 3.2.2 and 3.2.3 shows that LP-19 has stronger broad-spectrum anti-HIV-1, anti-HIV-2 and anti-SIV activities, and particularly shows strong advantageous effects on HIV-2 and anti-SIV.

3.2.4 inhibition of various subtypes of HIV-1 by LP-19

AIDS is mainly caused by HIV-1 worldwide, and various subtypes are generated due to virus variation, including A-D, F-H, J, K subtype and the like. Among them, subtypes A, B and C are the major viruses responsible for the world's HIV epidemic. In China, however, the prevalence of B/C and A/E recombinant viruses dominates. To further evaluate the activity against LP-19, a set of 29 HIV-1 pseudoviruses was prepared, including the international representative strains and the HIV strains currently circulating in China, among which there were subtype A3 strain, subtype B6 strain, subtype B' 3 strain, subtype C6 strain, subtype G1 strain, recombinant A/C1 strain, recombinant A/E4 strain, and recombinant B/C5 strain. These pseudovirus strains were maintained in the laboratory of professor hominis, institute of pathogenic biology, academy of Chinese medical sciences, see background art reference 13. The antiviral activity of T-20, 2P23, LP-11 and LP-19 was determined by reference to the method in 3.1.2 above (inhibition of HIV-1 and SIV pseudoviruses). Results of antiviral experimentsIt was shown that T-20, 2P23, LP-11 and LP-19 inhibit the mean IC of various subtype HIV-1 strains₅₀34.01nM, 5.22nM, 1.29nM and 0.47nM, respectively. It can be seen that the inhibitory activity of LP-19 against various subtypes of HIV-1 virus was also significantly higher than that of the three control polypeptides T-20, 2P23 and LP-11 (FIG. 6).

3.2.5 inhibition of LP-19 against T-20 and 2P23 resistant strains of virus

T-20 is the only HIV membrane fusion inhibitor approved for clinical treatment at present, however, the activity of the inhibitor is obviously lower than that of the polypeptide of the new generation, and the inhibitor is easy to induce drug resistance mutation, so that the clinical antiviral treatment fails. 2P23 is a helical short peptide which is newly designed by the inventor and only has 23 amino acids and contains an M-T hook structure, mainly targets an NHR hydrophobic pocket structure of a fusion protein gp41, has good inhibitory activity on HIV-1, HIV-2 and SIV, but also presents certain cross resistance on drug-resistant mutation sites induced by some short peptides. To more fully evaluate the antiviral broad spectrum and superiority of LP-19, we generated pseudoviruses carrying NHR mutations, containing drug resistance sites corresponding to T-20 and 2P23, respectively (FIG. 7). These plasmids for pseudovirus production were obtained by the inventors by site-directed mutagenesis based on the plasmid pNL4-3-Env expressing the envelope protein of HIV-1 strain NL4-3, and were stored and used routinely in the laboratory of professor Hoyu, institute of pathogenic biology, academy of Chinese medicine. The T-20 resistant strains in FIG. 7 are HIV-1 4-3 mutant strains in Table 3 of (Chong H, Yao X, Zhang C, Cai L, Cui S, Wang Y, He Y. Biophysic Property and Anti-HIV Activity of Albrevilide, a 3-Maleimidopapironic Acid-modified peptide Fusion inhibitor PLoS one.2012,7(3): e 32599), respectively, and the subscript of the strain name in FIG. 7 is the strain name in Table 3 in this document. 2P23 resistant strains are HIV-1NL4-3 mutant strains in Table 1 of (SuY, Chong H, Xiong S, Qiao Y, Qiu Z, He Y. genetic pathway of HIV-1resistance level fusion inhibition of the Gp41pocket. J Virol.2015, 89(24): 12467) 12479) respectively, and the subscript of the strain name in FIG. 7 of the present invention is the strain name in Table 1 of the document. The antiviral activity of T-20, 2P23, LP-11 and LP-19 was determined by reference to the method described in 3.1.2 above. The results of antiviral experiments show that T-20, 2P23, LP-11 and LP-19 inhibit T-20 drug resistanceAverage IC of strains₅₀1804.3nM, 1.05nM, 0.37nM and 0.17nM, respectively (FIG. 7). As can be seen, the activity of LP-19 for inhibiting the T-20 drug-resistant strain is 10613.53 times, 6.18 times and 2.18 times higher than that of T-20, 2P23 and LP-11 respectively; average IC of LP-19 for inhibiting 2P23 drug-resistant strain₅₀134.4nM, 36.52nM, 5.53nM and 0.81nM, respectively. It can be seen that the activity of LP-19 for inhibiting the 2P23 drug-resistant strain is 165.93 times, 45.09 times and 6.83 times higher than that of T-20, 2P23 and LP-11 respectively.

Example 4 in vivo antiviral Activity assay of LP-19

The results of the above-described multiple in vitro cell-based antiviral experiments indicate that LP-19 is a broad-spectrum, highly active antiviral lipopeptide. To determine whether LP-19 also has potent antiviral activity in vivo, LP-19, as well as control polypeptides T-20 and 2P23, were injected subcutaneously or intravenously into monkeys, and serum antiviral activity was measured in vitro using blood samples taken at various time points. The method can not only know the in vivo antiviral activity of the object to be detected, but also is helpful for analyzing the stability and half-life period of the object to be detected in vivo. The specific method comprises the following steps: 6 experimental macaques (rhesus monkeys) are selected, half male and half female, the age is 3-4 years, and the weight is 3.4-4.7 kg. The test substances T-20, 2P23 or LP-19 (all dissolved in sterile distilled water) are injected subcutaneously at the rate of 3mg (3mg/Kg) per kilogram of body weight, 0.4ml of venous blood specimen is extracted before (0 hour) injection and 1,2, 4, 6, 8, 12, 18, 24, 36, 48, 60 and 72 hours after injection respectively, and the serum is separated according to the conventional method. LP-19 was injected by intravenous route (dissolved in sterile distilled water) in addition to subcutaneous route at a dose of 3mg/Kg body weight. Each experiment was separated by more than 2 weeks to ensure that there was no residue from the last injection of test substance. Serum was tested for the activity of inhibiting HIV-1 pseudovirus NL4-3 mutant D36G (see Table 2 of reference 11 in the background art) using an antiviral assay according to the method in example 3. Serum was diluted 3-fold. The results of the experiment are shown in FIG. 8, T-20 injected subcutaneously showed peak inhibition at 2 and 4 hours, with the maximal dilution of serum inhibiting infectivity of 50% NL4-3 being 45-fold and 46-fold, respectively; subcutaneous injection of short peptide 2P23 showed peak inhibition at 1 and 2 hours, with maximal serum dilution of 60 and 68 fold, respectively; the subcutaneous injection of the short peptide LP-19 shows inhibition peak values at 6 and 8 hours, and the maximum dilution times of serum are 5396 times and 4720 times respectively; intravenous LP-19 showed peak inhibition at 1 and 2 hours with 99107-fold and 76346-fold maximal serum dilutions, respectively. It can be seen that LP-19 exhibits significant potent and long-lasting activity in monkeys, with a subcutaneous peak inhibition value of over 100 times that of T-20, and a venous peak inhibition value of over 2000 times that of T-20. LP-19 inhibited peak serum maximal dilutions 66-fold and 40-fold, respectively, even at 60 and 72 hours of subcutaneous injection, similar to the performance of T-20 at 2 and 4 hours.

Example 5 analysis of the Structure of LP-19 and its interaction with NHR target sequences

5.1 Experimental materials and methods

Secondary structure and helix stability (Tm value) of polypeptides were determined using Circular Dichroism (CD), experimental method reference [14 ]. The circular dichroism instrument is Jasco-815 produced by Nissan products. The self-helix content and Tm of LP-19 and its template 2P23 were first determined at different concentrations (20, 40, 80, 160, and 320. mu.M, respectively). Then, the helix content and Tm value of 6-HB formed by LP-19 or 2P23 and NHR polypeptide N36(Ac-SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH2) were determined. 2P23, LP-19 and N36 were each dissolved in phosphate buffered saline (PBS, pH 7.2) and the concentration was determined by UV absorption at 280 nm. And judging the interaction condition and the helix content among the polypeptides according to the CD signals. The stability of the hexahelix structure formed by the polypeptide inhibitor with N36 was determined by CD temperature scanning.

5.2 Experimental results and analysis

The CD test results are shown in FIG. 9. first, unmodified polypeptide 2P23 as a control contains a typical α -helix structure (A in FIG. 9) with helix contents of 76.4%, 81.4%, 83.8%, 84% and 79.7% at 20 μ M, 40 μ M, 80 μ M, 160 μ M and 320 μ M concentrations, respectively, and Tm values of 48.1 deg.C, 54.7 deg.C, 59.9 deg.C and 67.8 deg.C (B in FIG. 9) at 20 μ M, 40 μ M, 80 μ M and 160 μ M concentrations, respectively, when the concentration reaches 320 μ M, its helix is not sufficiently helicated at the measured maximum temperature (98 deg.C) to result in an inaccurate determination of Tm, suggesting a high thermal stability of its helix.A lipopeptide LP-19 at 20 μ M, 40 μ M, 80 μ M, 160 μ M and 320 μ M concentrations, respectively, is 70%, 71.1%, 71.9%, 76.2% and 320 μ M concentrations, respectively, and a further showing a significant increase in the stability of the polypeptide forming a multimer-helix complex (P) as a multimer-multimer structure forming a further increase in the typical helix-helix binding of the polypeptide, which is shown in FIGS. 2P-9, 3. A-9, 2P-9, 3,2, and 3-9. the stability of the highest temperature of the complexes is not significantly increased by comparison of the highest complexes (98. A-9, 3. A-9, 3. A, 3. the exemplary phosphomer.

Example 6 antiviral Activity of LP-19 derived lipopeptides

As can be seen from the results of the above examples, LP-19 lipopeptides have the advantages of broad spectrum, potent and long-lasting activity. Further replacement of palmitic acid in LP-19 with other lipophilic compounds synthesized a new set of lipopeptides (FIG. 1). Wherein LP-20, LP-24 and LP-25 are modified with stearic acid (C18), LP-21 is modified with cholesterol (Chol), LP-22 is modified with dihydro (neuro) sphingosine (Dih), and LP-23 is modified with vitamin E (Toc). At the same time, lipopeptides (LP-26 to LP-32) were synthesized that did not contain PEG8 but instead corresponded to amino acid sequences of different lengths. Their inhibitory activities against HIV-1NL4-3 strain pseudovirus, infectious HIV-2ROD strain and SIV239 strain pseudovirus were examined with reference to antiviral experiments of 3.1.2 and 3.1.3 in example 3. The plasmid for virus production was as described above. The results are shown in FIG. 1, where these lipopeptides all have a very potent antiviral activity. They are effective against both HIV-1 and HIV-2 and SIV.

<110> institute of pathogenic biology of Chinese academy of medical sciences

<120> broad-spectrum HIV inhibiting lipopeptides, derivatives thereof, pharmaceutical compositions thereof and uses thereof

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<170>PatentIn version 3.5

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Claims (4)

1. A lipopeptide, or a pharmaceutically acceptable salt thereof, characterized by: the lipopeptide is formed by sequentially connecting polypeptide with antiviral activity, a connecting arm connected with the carboxyl terminal of the polypeptide, an amino acid X residue connected with the connecting arm and a lipophilic compound connected with the amino acid X residue; the amino acid X is K;

the connecting arm is Fmoc-NH-PEGn-CH₂CH₂COOH, n is 8; and

the lipophilic compound is palmitic acid; the polypeptide is shown as a sequence 1 in a sequence table.

2. A multimer formed from the lipopeptide or pharmaceutically acceptable salt thereof of claim 1.

3. A composition comprising C1) and C2): C1) is C11) or/and C13); c11) is the lipopeptide of claim 1 or a pharmaceutically acceptable salt thereof; the C13) is the multimer of claim 2;

C2) a pharmaceutically acceptable carrier or adjuvant;

the composition has at least one of the following functions F1) -F5):

F1) resisting viruses;

F2) treating and/or preventing and/or adjunctively treating diseases caused by viral infection;

F3) inhibiting cell fusion of the virus;

F4) inhibiting the entry of a virus into a cell;

F5) inhibiting viral replication;

F1) -F5), the virus is any one of the following v1-v 7:

v1, HIV-1, HIV-2 and SIV;

v2, HIV-1 and HIV-2;

v3, HIV-1 and SIV;

v4, HIV-2 and SIV;

v5、HIV-1；

v6、HIV-2；

v7、SIV。

4, C11), C13) or/and C14) for the preparation of at least one of the products E1) -E5):

c11) is the lipopeptide of claim 1 or a pharmaceutically acceptable salt thereof; the C13) is the multimer of claim 2; said C14) is the composition of claim 3;

the E1) is an antiviral product;

e2) is a product for treating and/or preventing and/or assisting in treating diseases caused by virus infection;

e3) is a product for inhibiting cell fusion of viruses;

e4) is a product for inhibiting the invasion of cells by viruses;

e5) is a product that inhibits viral replication;

the E1) -E5), the virus is any one of the following v1-v 7:

v1, HIV-1, HIV-2 and SIV;

v2, HIV-1 and HIV-2;

v3, HIV-1 and SIV;

v4, HIV-2 and SIV;

v5、HIV-1；

v6、HIV-2；

v7、SIV。

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