WO2019211878A1 - Peptoid of formula i, pharmaceutical compositions and method for preparation thereof - Google Patents

Abstract

Alzheimer's disease (AD) is a complex multifactorial syndrome disease. In AD, two proteins play main role i,e, Aβ42 peptide and hyperphosphorylated microtubule associated protein (Tau) aggregation cause severe damages in both neuron cell membrane and key signal processing. There are other factors like loss of acetylcholine function, destabilization of microtubule also responsible for this disease. Therefore, the main challenge of the field is the multifactor property of this disease. Our main target is to develop a molecule which will target maximum factors of AD. Various attempts have been devoted towards this direction but clinical outcome is very less. Thus, the present invention provides a peptide of formula I, method of preparing the same and its pharmaceutical composition thereof.

Description

PEPTOID OF FORMULA I, PHARMACEUTICAL COMPOSITIONS AND

METHOD FOR PREPARATION THEREOF

FIELD OF THE INVENTION:

Alzheimer’s disease (AD) is one of the most neurodegenerative dementia around the globe nowadays (1. Bertram, L., and Tanzi, R. E. (2008) Thirty years of Alzheimer’s disease genetics: the implications of systematic meta-analyses. Nat. Rev. Neurosci. 9, 768-778).More than five million people in the world are suffering from AD. The present invention relates to a novel peptoid designed and synthesized to be used as a“multi-target-directed ligand” (MTDL) against this multifactorial complex disease. This peptoid targets most of the factors of AD like inhibition of Ab fibrillations, stabilization of microtubule, inhibition of AChE activity and also reduction of the intracellular reactive oxygen species (ROS). Moreover, peptoids are Oligomers of N-substituted glycine which overcomes the metabolic instability, so it has increased bioavailability.

BACKGROUND OF THE INVENTION:

Soto et al showed that LPFFD Peptide decreases neurotoxicity and fibrillation of A 42(Sigurdsson EM., Permanne B., Soto C., Wisniewski T., and FrangioneB., (2000) In Vivo Reversal of Amyloid-b Lesions in Rat Brain J. Neuropathol. Exp. Neurol.59, 11-17 and Soto C., Sigurdsson EM., Morelli L., R. Kumar A., Castano EM. and Frangione B. (1998) b-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: Implications for Alzheimer's therapy, Nat. Med. 4, 822 - 826.) The objective of adding lysine side chain at the C- terminal of the peptoid was to disrupt the salt bridge between Asp23 and Lys28 which is present in the amyloid fiber structure(Lu hrs* T., Ritter* C., Adrian† M., Riek-Loher* D., Bohrmann;!; B., belli H., Schubert* D. and Riek* R. (2005) 3D structure of Alzheimer’s amyloid-(l- 42) fibrils PNAS. 102, 17342-17347). But the common drawback of peptides composed of natural amino acids is metabolic instability(Prades R., Oller-Salvia B., Schwarzmaier SM., Selva J., Moros M., Balbi M., Grazffl V., Fuente J. M., Egea G., Plesnila N., Teixidy M. and Giralt E. (2015) Applying the Retro-Enantio Approach to obtain a Peptide Capable of Overcoming the Blood-Brain Barrier Angew. Chem. 54, 3967 -3972). Usually a serum protease digests peptide bonds within seconds or few minutes and thus they have very low half-life (i.e. bioavailability) in serum. Therefore peptides are considered as poor pharmacokinetics. Zuckermannei al discovered Oligomers of N-substituted glycine, or “peptoids” (Zuckermann R. N., Kerr J. M., Kent, S. B. H., and Moos, W. H. (1992) Efficient Method for the Preparation of peptoids [Oligo(N-substituted glycines)] by submonomer solid- phase synthesis. J. Am.Chem. Soc. 114, 10646-10647) which overcome the metabolic instability of peptides. So, their bioavailability increases and also the absence of hydrogen in amide groups in the peptoid’s backbone reduces the peptoid’s polarity and improves membrane permeability( Cho, S., Choi, J., Kim, A., Lee, Y., and Kwon, Y. U., (2010) Efficient solid- phase synthesis of a series of cyclic and linear peptoid dexamethasone conjugates for the cell permeability studies. J. Comb.Chem. 12, 32l-326).Other advantages using peptoids are the ingredients of peptoid synthesis are cheaper and also easy to synthesize. A various types of side chains can be inserted into the peptoid chain .So, diversity of peptoids sequence can be greater than peptides. Using of peptoids over peptides gives another advantages i.e, it reduces the cytotoxicity which may arise due to peptide breakdown. But due to similar type of side chain and spacing between sides chains of peptoids with peptides suggest peptoids may show similar kind of activity with better efficiency. The bioavailability of peptoids over peptides make them an important and promising class of therapeutic due to their proteolytic stability(Miller, S. M., Simon, R. J., Ng, S., Zuckermann, R. N., Kerr, J.M., and Moos, W.H.(l994) Proteolytic studies of homologous peptide and N-substituted glycine peptoid oligomers. Bioorg.Med.Chem. Lett. 4, 2657-2662).

Although there are lots of peptide based molecules like“NAPVSIPQ” (NQ) and“LPFFD” inhibit Ab42 fibrillations and also show potential in various in vivo studies. Recently,“Gly- His-Lys-Sr-Val-Sr-Phe-Sr”(Rajasekhar K., Madhu C., and Govindaraju T. (2016) Natural Tripeptide-Based Inhibitor of Multifaceted Amyloid b Toxicity. ACS Chem. Neurosci. 7, 1300-1310.) and“CPO Ab17-21 P”(Liu S., Park S., Allington G, Prellil F., Sun Y., Marta- Arizal M., Scholtzova H., Biswas G., Brown B., B.Verghese P., Mehta P.D., Kwon Y.U. and Wisniewski T. (2017) Targeting Apolipoprotein E / Amyloid b Binding by Peptoid CPO_ Ab 17-21 P Ameliorates Alzheimer’s Disease Related Pathology and Cognitive Decline. Sci. Rep. 7, 8009.) are also reported for reducing Ab toxicity. Despite of the enormous research efforts, the success rate for the discovery of new molecule for the treatment of AD is very unsatisfactory. This is probably due old classical drug discovery approach“one molecule, one target” or may be low bioavailability of peptides. OBJECTIVES OF THE INVENTION:

The main objective of the present invention is to provide anew class of compound“Peptoid” for treatment of Alzheimer’s disease.

Another objective of the present invention is to prepare the said Peptoid.

Yet anotherobjective of the invention is to target several factors of AD with a single molecule based on most popular strategy“one molecule, multiple targets”. Still another objective is to design a serum stable compound for the treatment of AD which can also cross the blood brain barrier.

Yet another and most important objective is, the compound should be economicaland easy to synthesize. The starting ingredients must be easily available. Moreover, the compound should be less cytotoxicto neuronal cells.

Still another objective of the present invention is to prepare pharmaceutical compositions comprising the Peptoid.

Yet another objective of the present invention is to use the Peptoid in treatment of Alzheimer Disease.

ABBREVIATIONS:

AD: Alzheimer Disease, Ab: Amyloid beta, PAS: Peripheral anionic site, AChE: Acetylcholinesterase, PC 12: Rat pheochromocytoma cells, NGF: Neuronal growth factor, MD: Molecular docking, RP-HPLC: Reverse phase high pressure liquid chromatography, Kb: Binding constant, DAPI: 4',6-diamidino-2-phenylindole, DMEM: Dulbecco’s Modified Eagle’ s Medium, DIC:A, A-DiisopropylcarbodiimideGTP:Guanosine 5'-Triphosphate,ThT : Thioflavin T, TEM: Transmission Electron Microscopy, FBS: Fetal Bovine Serum, DMSO: Dimethyl sulphoxide, MeOH:Methanol, MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide, ESI: Electrospray ionization, PDB:Protein Data Bank, IgG: Immunoglobulin G, DCFDA: 2’, 7’ -dichlorofluorescin diacetate, RPMLRoswell Park Memorial Institute, HFIP: l,l,l,3,3,3-Hexafluoro-2-propanol, ATC:acetylthiocholine, DTNB:5,5'-Dithiobis-(2-Nitrobenzoic Acid), UV-VIS:Eiltraviolet-visible. SUMMARY OF THE INVENTION

The present invention provides for a peptoid of formula I.

In an embodiment the peptoid have a molecular weight of 635.80.

In another embodiment the peptoid binds with microtubule lattice (K_b-6.576Xl0³ M ¹) in and stabilizes microtubule.

In yet another embodiment the peptoid inhibits amyloid fibril formation and oligomer formation.

In still another embodiment the peptoid reduces the activity of AChE, inhibits AChE induced amyloid fibril formation, reduces intra cellular ROS and shows neuroprotection against Ab induced toxicity.

In yet another embodiment the peptoid is able to cross Blood Brain Barrier (BBB) in mice model.

In still another embodiment, the present invention provides for a method of preparing the peptoid of formula I, comprising solid phase synthesis of peptoid using Rink Amide resin, purification of crude peptoid by RP-HPLC and characterization by ESI Mass Spectroscopy.

In yet another embodiment, the present invention provides for a pharmaceutical composition comprising the peptoid as along with pharmaceutically acceptable excipients.

In still another embodiment, the present invention provides for a method for treating Alzheimer’s disease by administering effective amount of peptoid as claimed in claim 1. In yet another embodiment the mode of administration generally of peptoids for treating Alzheimer’s disease is through intranasal and intraperitoneal route.

In still another embodiment the peptoid can be administrated with only water or saline solution.

BRIEF DESCRIPTION OF THE DRAWINGS:

Fig.l.describes(a) Structure of LPFFK peptoid and (b) Synthetic scheme for preparation of LPFFK peptoid in solid phase synthesis method.

Fig.2describes (a) Turbidity assay of LPFFK peptoid showing LPFFK helps to polymerise the tubulin (b) The rate of enhancement of DAPI fluorescence in presence of LPFFK peptoid is higher compare to control indicates that LPFFK promotes tubulin polymerization (c) Tryptophan fluorescence of tubulin was quenched upon gradual addition of LPFFK peptoid and graph shows the binding constant 6.576X10³. (d) MTT assay of the LPFFK peptoid showing non cytotoxic in nature.

Fig.3 describes theFT-IR spectra that indicates that LPFFK peptoid inhibits fibril formation of Ab42 (a) LPFFK peptoid at 0 day incubation (T)Ab42 Peptoid after 7 days incubation (c) LPFFK peptiod after 7 days Incubation (d) Ab peptide with LPFFK peptoid after 7 days incubation.

Fig.4 describes a) ThT assay reveals inhibition of Ab42 aggregation in presence of LPFFK peptoid. (b) Docking study shows that LPFFK peptoid binds with the most hydrophobic site of Ab42. TEM image reveals the inhibition of Ab42 aggregation by LPFFK peptoid (c) Ab42 peptide forms fiber after 7 days incubation at 37 °C. (d) Absence of fibril structure after co-incubation of Ab42 peptide and LPFFK peptoid at 37 °C for 7 days. Three independent experiments performed in triplicates to calculate ± SEM. The asterisk denotes statistically significant differences between indicated classes: *p<0.05.

Fig.5 describes (a) Bar diagram represents inhibition of AChE activity upon treatment with LPFFK peptoid. (b) ThT fluorescence assay for inhibition of AChE-induced Ab42 aggregation using (c) Lineweaver-Burk plots of AChE activity with different substrate concentrations in the presence of different concentrations of peptoid. (d) Docking image reveals that the peptoid binds with both PAS and CAS sites of AChE. Inhibition over-production of ROS in neuron cells (PC 12) by LPFFK peptoid. (e) MTT assay in the presence of FPFFK peptoid and H₂0₂ clearly reveals that with increasing peptoid concentrations cell viability increased (f) DCFDA assay performed in the presence of H₂0₂ shows that with increase in peptoid concentration, the production of H₂0₂ decreases indicating the antioxidant potential peptoid. Three independent experiments performed in triplicates to calculate ± SEM. The asterisk denotes statistically significant differences between indicated classes: *p<0.05 and **p<0.04.

Fig.6 describes microscopic images, which reveals that FPFFK peptoid stabilizes the intracellular microtubule networks (a) Differential Interference Contrast (DIC) (b),(c) and(d) Fluorescence image at 488 nm,405 nm and merged image of PC 12 cells in absence of FPFFK peptoid (control) (e) DIC (f) 488 nm (g) 405 nm and (h) merged image in presence of FPFFK peptoid of PC 12 cells.

Fig.7 describes microscopic images, which reveals that FPFFK peptoid helps to main the neuronal morphology of the Ab42 treated PC12 cells. Control cells (a) DIC (b) 488nm, (c) 405nm and (d) merged image. Ab42 treated PC 12 cells (e) DIC (f) 488nm, (g) 405nm and (h) merged image. Ab42 and FPFFK treated PC 12 cells (i) DIC (j) 488nm, (k) 405nm and (1) merged image.

Fig.8 describes LPFFK peptoid protects differentiated PC 12 cells against NGF deprivation. (a) DIC image of differentiated PC 12 cells (b) DIC image of PC 12 cells treated with Anti-NGF reveals cells are in spherical shape at NGF depriving condition (c) DIC image of PC 12 cells treated with Anti-NGF and LPFFK peptoid reveals healthy PC 12 cells indicating neuroprotective role of peptoid. (d) Neuron cell viability by MTT assay after anti-NGF treatment and anti-NGF in the presence of different concentrations of LPFFK peptoid. (e) Quantitative analysis of neurite out growth by LPFFK peptoid. (f) Bar diagram represents excellent serum stability of the peptoid in the presence of serum protease. Three independent experiments performed in triplicates to calculate ± SEM. The asterisk denotes statistically significant differences between indicated classes: *p<0.05 and

**p<0.04. Fig.9 describes microscopic images of MAP2 staining of the primary cortical neurons in the different channels (DIC, 56lnm, 405nm and merged)

Fig. 10 describes the HPLC chromatogram of LPFFK peptoid.

Fig.ll describes the Mass data of LPFFK shows 636 (635+H⁺) Da.

Fig .12describes the RP-HPLC chromatogram of crude solution of mice brain extract.

Fig.13 describes the mass spectrum of mice brain extract after purification by RP-HPLC.

Fig.14 describes the mass spectrum of control mice brain extract.

DETAILED DESCRIPTION OF THE INVENTION

Present invention provides a novel peptoid designed from the most hydrophobic core of Ab peptide which is mainly responsible for oligomerisationi.e toxicity in the AD brain. This peptoid(Fig. la) was synthesized through solid phase peptoid synthesis method (Fig. lb), followed by purification through RP-HPLC (Fig. 10) and characterization was done by ESI Mass spectroscopy (Fig. 11). The peptoid was used instead of peptide because of their metabolic stability and also the membrane permeability for peptoid is higher due to the lower polarity of peptoid as it does not contain hydrogens in amide groups. Another important advantage is that the peptoid is less cytotoxic but in case of peptide the toxicity may arise due to peptide breakdown.

Present invention also illustrates that this peptoid ‘LPFFK’ interacts with tubulin and microtubule in vitro study(Fig. 2a and 2b). Intrinsic tryptophan quenching experiment was performed to find the binding ability of the peptoid with tubulin (Fig. 2c) which showed very moderate binding energy (6.576X10³). Further, inventors also investigated whether the peptoid able to interact with intracellular tubulin/microtubule or not. For that purpose, first cell viability assay was performed. The results showed the peptoid was not cytotoxic (Fig. 2d).Then, the microtubule images (Fig. 6a-6d)show that microtubule network in PC 12 cells are healthier for LPFFK treated cells than the untreated cells (Fig. 6e-6h) (control cells). These results clearly indicate the peptoid binds and stabilizes with intracellular tubulin/microtubule. The ability of peptoid to inhibit Ab fibrillizations has been examined. Initially, FT-IR study was used to understand capability of the peptoid for the inhibition of Ab fibrillizations. For this experiment, Ab42 peptide was co-incubation with LPFFK for 7 days. FT-IR spectra revealed that Ab42 peptide(Fig. 3b) formed fibril structure (1734 cm^_1)but in presence of the peptoid, Ab42 peptide(Fig. 3d)failed to form the fibril structure (1655 cm ¹). FT-IR spectra also revealed that peptoid did not form any b-sheet structure at 0 day (Fig. 3a) and 7 days (Fig. 3c) incubation. Next, inventors interested to know the quantity of fibril inhibition by the peptoid. For that purpose, Thioflavin T(ThT) (Fig. 4a) was performed. The figure showed that at 15mM the fibril formation was decreased upto 55%.TEM image also revealed that after 7 days ΐh^I^ΐΐohAb peptide formed fibrils (Fig. 4c) but in similar condition in presence of peptoid Ab42 peptide did not form fibrils (Fig. 4d) .These data clearly indicate that LPFFK inhibits amyloid fibrillizations in vitro. Next, molecular docking was used to find how LPFFK interacts with Ab peptide. Docking study (Fig. 4b) shows that LPFFK interacts with the most hydrophobic ooΐoίAb 17-21 ) of Ab (PDB ID: HYT)(Crescenzi, O., Tomaselli, S., Guerrini, R., Salvadori, S., D'Ursi, A. M., Temussi, P. A. and Picone, D. (2002) Solution structure of the alzheimer amyloid b-peptide (1-42) in an apolar microenvironment. Eur. J. Biochem. 269, 5642-5648) strongly through both hydrophobic and H-bonding interaction. H-bonding interaction between NH₂ group of peptoid terminal amide group with -C=0 group of Glul l. Further, -C=0 group of peptoid backbone interacts with -NFb group of Glnl5 and -NFb group of Lysl6. It is well reported before that Ab fibrillation occurs through antiparallel interaction between one monomer with another monomer(Gordon, D. J., Sciarretta, K. L., and Meredith, S. C. (2001) Inhibition of beta- Amyloid (40) Fibrillogenesis and disassembly of beta-amyloid (40) fibrils by short beta-amyloid congeners containing N-methyl amino acids at alternate residues. Biochemistry 40, 8237-8245).These docking experiment shows that LPFFK peptoid binds to the hydrophobic region and inhibits fibril formation by blocking hydrophobic site of Ab.

Inventors checked the inhibitory effect of LPFFK peptoid on Acetylcholinesterese (EeAChE) which is the main reason for the loss of cognitive function in AD. Ellman protocol was used to check the inhibitory effect. At 5mM concentration, the peptoid was able to decrease 25% activity of AChE(Fig. 5a). It is known that any compound that binds with AChE can block the propagation of the Ab42 fibrilization as the PAS helps to aggregate Ab42ί.e increase the neurotoxicity in brain. Therefore, Inventors checked AChE induced Ab42 aggregation by thioflavin T method. The figures(Fig. 5b) showed that the fluorescence intensity of AChE induced Ab42 aggregation with peptoid decreases with increase time whereas fluorescence intensity for the control experiment increases. Two different concentrations of peptoid were used and there is clear evidence for dose dependent effect on inhibition of AChE induced Ab42 aggregation. Next the mechanism of inhibition was studied by lineweaverburk plot(Fig. 5c) which showed noncompetitive inhibition as the Vmax value changed but the Km value remained the same. Docking study also revealed that the peptoid bound with the CAS and PAS of AChE(Fig. 5d).

Various in vitro assays showed promising behavior towards multifactorial AD. Therefore, inventor checked the ability of LPFFK peptoid for targeting multifactorial factors in cellular level. Next, inventor assessed the antioxidant property of LPFFK peptoid. PC 12 cells were exposed to 200mM H₂0₂ with LPFFK peptoid after the incubation of cells with H₂0₂. H₂0₂ exposed Cells showed viability about 21 % with respect to control cells in absence of LPFFK peptoid in MTT reduction method. Three different concentrations of peptoid were used in this experiment and at 25 mM concentration the peptoid helped to protect the cells about three times higher than H₂0₂ treated cells(Fig. 5e). Next DCFDA assay(Fig. 5f) was performed with three different concentrations. With increase in peptoid concentration the production of H₂0₂ decreases which also supported the antioxidant property of peptoid. Ab42 peptide directly interacts with b-tubulin and promotes the apoptotic cell death (Saha A., Mohapatra S., Kurkute P., Jana B., Mondal P., Bhunia D., Ghosh S. and Ghosh S.* (2015) Interaction of Ab peptide with tubulin causes inhibition of tubulin polymerization and apoptotic death of cancer cells . Chem. Commun., 51, 2249-2252). Therefore inventor checked the ability of the peptoid towards the inhibition of apoptotic cell death caused by Ab42. Ab42 (2mih) was treated with PC 12 cells in presence of peptoid (20 mih). The figure(Fig. 7e-7h) showed that the only Ab42 treated cells become in spherical shape i.e. no differentiation was observed for these cells. But in presence of peptoid(Fig. 7i-7l), the differentiation of the cells were observed and cells were healthy as like the control cell(Fig. 7a-7d). Present invention also investigated whether LPFFK exhibited any neuroprotection against anti NGF toxicity. This neuroprotection study is performed in neuronal cell line PC 12. Previously, it was reported that withdrawal of NGF from differentiated PC 12 cells helped to produce of Ab peptide(Matrone, C, Di Luzio, A., Meli, G., D’Aguanno, S., Severini, C., Ciotti, M. T., Cattaneo, A., and Calissano, P. (2008) Activation of the amyloidogenic route by NGF deprivation induces apoptotic death in PC 12 cells. J. Alzheimer’s Dis. 13, 81-96) which is the main cause for toxicity or cell death in differentiated PC12 cells. This study has been widely used to study the mechanism of death of neurons in AD. Hence, this model was used to know the neuroprotective potential of LPFFK. At first PC 12 cells were differentiated in NGF containing medium for 5 days. Then the differentiated cells were treated for overnight with anti-NGF along with different doses of LPFFK. The results(Fig. 8d) suggested that the LPFFK was able to provide protection to the cells compared with the control cells. At 5mM concentration LPFFK helped to protect 94% cells with respect to control cells. Microscopic images(Fig. 8a-8c) also revealed that in presence of only anti- NGF PC 12 cells become spherical shape but in presence of LPFFK PC 12 cells maintain their normal morphology. Inventor examined the serum stability of peptoid in Horse Serum in 37 °C up to 24h. RP-HPLC was used to determine the amount of peptoid intact at different time intervals (0, 4, 12, and 24 h). The figure (Fig. 8f) showed that the peptoid was stable in serum protease. The lower susceptibility of the peptoid towards serum makes this peptoid a good candidate for using this peptoid as a drug. Finally, in-vivo mice model experiment with peptoid was performed to check its blood-brain barrier BBB crossing permeability. Intra-cardiac injections of peptoid (2.5 mM, 100 pL) was given into the healthy mice after 6h, mice were sacrificed to separate out brains .Then blood vessel and meninges were removed using stereo microscope. Brains were taken into a mortar and crushed througly in liquid nitrogen with pestle . then the crushed brain were mixed with 1 : 1 acetonitrile and water soluble part was taken after centrifugation and analyzed through ESI mass spectrocoscopy(Fig. 13) after purification through RP-HPLC(Fig. 12). The ESI mass spectrum is actually matches with the expected mass. For control experiment, in place of peptoid only PBS was injected into the mice. The mass spectrum(Fig. 14) of the control experiment showed no mass around the molecular weight of the peptoid. This result indicates that LPFFK able to cross the BBB.

Inventors also checked the toxicity of LPFFK peptoid in primary cortical neurons. For that purpose primary cortical neurons have been cultured following optimized protocol(Adak A, Das G, Barman S, Mohapatra S, Bhunia D, Ghosh S, Biodegradable Neuro-Compatible Peptide Hydrogel Promotes Neurite Outgrowth, Shows Significant Neuroprotection, and Delivers Anti- Alzheimer Drug (2017) ACS ApplMater Interfaces. 9, 5067- 5076). Anti-MAP2 antibody was used as a marker for staining the dendrites of neurons of the primary cortical neurons. LPFFK was treated in cultured primary cortical neurons. Microscopic images(Fig. 9e-9f) revealed that there were no significant morphological change with the control image (Fig. 9a- 9d) which confirmed that LPFFK did not have toxic effect on primary neurons. It is within the confines of the present invention that the formulations of the peptoid of formula I may be further associated with a pharmaceutically-acceptable carrier, thereby comprising a pharmaceutical composition. The pharmaceutically-acceptable carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. The formulations of the present invention may be prepared by methods well known in the pharmaceutical art.

Examples:

Example 1: Synthesis of Peptoid:

Solid phase peptoid synthesis method was used to prepare LPFFK peptoid using Rink Amideresin. Crude peptoid was purified by RP-HPLC and characterized by ESI Mass Spectroscopy.

Peptoid was synthesized by solid phase synthesis method. Rink amide resin was used as solid support. Bromoacetic acid was used as a building block or acylating agent. Various amines of the corresponding amino acids were used. DIC was used as a coupling reagent. TFA (95%) in water was used as a cleavage cocktail solution. The purity of the peptoid was checked by RP- HPLC showing 100% purity. The mass of the peptoid was checked by ESI-mass, which matched with the expected mass with H⁺. This purified peptoid was used for the various bio physical study and cell based studies without further modification. The mode of administration was intra-peritoneal to check the blood brain barrier. The cytotoxicity of the peptoid in the PC 12 cells upto 100mM has been checked and found non cytotoxic.

Example 2: Tubulin Polymerization Assay/Tubulin turbidity assay:

Tubulin turbidity assay was performed using 4mM GTP, 20mM tubulin, 10% dimethyl sulfoxide(DMSO was used to initiate the polymerization) in Brinkley Reassembly Buffer 80 (BRB 80). Mixing of the above components with peptoid were done in ice. The turbidity of the solution was measured by measuring absorbance at 350 nm for 40 min in the UV-Vis Spectrophotometer (G6860A Cary 60 UV-Vis Spectrophotometer, Agilent Technologies) at 37°C. For control experiment the same procedure was performed in the absence of LPFFK peptoid. Example 3: Microtubule assembly assay:

A solution containing 10 mM DAPI, 100 mM tubulin, 10 mM GTP in BRB80 with peptoid buffer was prepared. The solution was excited at 355 nm wavelength at 37 °C and the emission spectra of the solution was recorded from 400 nm to 600 nm wavelength for 60 min in five minute time interval in Quanta Master Spectrofluorometer (QM-40), which is equipped with Peltier for controlling the temperature during experiment. Then the data was calculated in origin Pro 8.5 software.

Example 4: Determination of binding affinity of LPFFK Peptoid with the tubulin by fluorescence intensity quenching study of intrinsic Tryptophan residue of tubulin: The intensity of intrinsic Tryptophan fluorescence of tubulin was measured in presence of different concentration of LPFFK. Then the binding constant was calculated using a modified Stern- Volmer equation. The fluorescence emission spectra was measured from 310 to 450 nm and excitation of the sample was done at 295 nm at room temperature in Quanta Master Spectrofluorometer (QM-40) equipped with Peltier for controlling the temperature.

Example 5: Ellman’s assay was performed in the following procedure: Five different concentrations of the LPFFK peptoid were used to know the inhibitory effect. At first enzyme was incubated with different concentration of peptoid for 1 hour at 37°C. Then 0.01 M DTNB and 0.075 M ATC solution were mixed with enzyme and exactly after 1.5 minute of mixing absorption was measured at 4l2nm. For the reference value, LPFFK peptoid was replaced by phosphate buffer. For determining the non-enzymetic reaction the enzyme solution was replaced by phosphate buffer.

Example 6: Kinetics Study: For the kinetics measurement, different substrate concentrations were used. We also used five different concentration of LPFFK peptoid. For this experiment, the above procedure was followed. Origin Pro 8.5 software was used to draw the Lineweaver- Burk plot.

Example 7: AChE Induced b-Amyloid Aggregation Testing: The stock solution of thioflavin T and AChE were prepared using phosphate buffer to get the final concentration at 15 mM and 2.5 unit/mL. Ab42 was suspended in HFIP and the solvent was removed under steam of nitrogen. Then NH₄OH solution was added and a fixed amount of phosphate buffer was used to make the concentration 25mM of Ab42. AChE(40 pL, final concentration lU/mL) was mixed with LPFFK peptoid(5 pF), Ab42 (5 pF final concentration 25 mM) and phosphate buffer. The resulting solution was incubated at room temperature for upto 24 h. Thioflavin T solution was added to the mixed solution and fluorescence was measured using 435nm as an excitation wavelength and 485nm as an emission wavelength.

Example 8: Fourier Transform Infrared Spectroscopy:

Ab42 Peptide and Ab42 Peptide with peptoid solution were prepared in a similar manner as described above. From the stock, an aliquot of 20 pF was taken out and lyophilization was done after mixing with KBr. Then pellet was prepared. The spectrums of samples were recorded after the required time interval (i.e immediately after sample preparation or after 7 days).

Example 9: Thioflavin T Fluorescence Assay:

Stock solution of Thioflavin T (ThT) of concentration 50 mM in PBS was prepared and stored in 4 °C freeze with dark cover to prevent degradation from light. To prepare Ab42 peptide stock solution, Ab42 peptide was dissolved in PBS to get 50 pMconcentration. Ab42 peptide (25 mM) with five different peptoid concentrations were prepared. Then these solutions were incubated at 37 °C on a water bath. To perform the fluorescence study, 40 pF of sample solution was taken out from the stock solution and was mixed with 200 pF of thioflavin T solution (50 mM) and final volume was made up to 400 pF with PBS.

Example 10: Transmission electron microscopy (TEM):

A solution of 10 mM of Ab42 and another solution of 10 mM of Ab42 with 20 mM peptoid were incubated at 37 °C for 7 days. A 10 pF aliquot of the incubated solutions were placed on a 300 mesh copper grid. The solution was kept for 1 min then the excess solution was removed and staining was done with 2% uranyl acetate in water. Then the grid was washed with milliQ water. Images were taken using a TECNAI G2 SPIRIT BIOTWIN CZECH REPUBFIC 120 kV electron microscope operating at 120 kV.

Example 11: PC12 Cells Culture:

Rat adrenal phenochromocytoma line (PC 12 cells) was a generous gift from Dr. SuvendraNath Bhattacharyya (Principal Scientist, Molecular & Human Genetics Division, and CSIR-IICB Kolkata). RPMI medium was used to grow the cells containing 10% horse serum (HI-MEDIA) and 5% fetal bovine serum (GIBCO) under 5% C0₂ at 37-°C atmosphere temperature. The PC12 cells were then differentiated into neurons using 1% Horse Serum and 100 ng/ml NGF. All further experiments have been performed using the differentiated neurons.

Example 12: Cell Viability Assay (Cytotoxicity Assay):

PC 12 cells were treated with different concentrations of peptoid in a 96 well plate except the control cells. After 24 h of treatment medium was removed from the plate and 50 pL of MTT (3-(4, 5)-dimethyl-thiahiazol-2-y 1-3, 5-di-phenytetrazolium bromide) was added and incubated further for 4h. Then MeOH: DMSO (1: 1) was added to dissolve formazan and absorbance was measured in multimode micro plate reader at 570nm.

Example 13: Cytotoxicity Assay with H2O2:

PC 12 cells were seeded in 96 well plate one day before the treatment. Cells were incubated with different concentration of peptoid. After 6h of peptoid pre-treatment, 200mM H₂0₂ was added and incubated for another 24h. Then cells were washed and cell viability was measured using MTT assay. Results were expressed as percent viability = [(A570 (treated cells)- background)/ (A570 (untreated cells)-background)] x 100.

Example 14: Cytotoxicity Assay with H₂0₂by DCFDA:

A fluorometric assay was done based on intracellular oxidation of (2, 7-H₂DCFDA) to measure the ROS level. PC 12 cells were grown on black 96 well plate for 24 h prior to treatment. PC 12 cells which have been preincubated with different concentrations of peptoid for 6h were further exposed to 200 mM H₂0₂ another 24h. After that, 50 pFof 100 mM 2, 7-H₂DCFDA was added and incubated for 30 min. Then DCF fluorescence was measured in microplate spectrofluorometer with excitation at 485nm and emission at 535nm.

Example 15: NGF Study:

Rat pheochromocytoma cells (PC 12) cells were cultured in RPMI medium with 10% horse serum (HS) and 5% fetal bovine serum. These cells were incubated with NGF (100 ng/mF) containing 1% horse serum medium for 5 days for neuronal differentiation before the treatment. Then cells were treated with anti-NGF (2 pg/mF) for another 20 h along with different concentrations of peptoid. Then MTT assay was done to check cell viability and the images of cells were observed under microscope.

Example 16: Cell shrinkage study in presence of Ab42 by microscope:

PC 12 cells were grown on confocal dishes and differentiated into neurons for another 5 days. The differentiated neurons were pre-treated with various concentrations of peptoid and further treated with 10 mM Ab42 for 48 h in serum free RPMI. The cells were then fixed with 4% formaldehyde and permeabilized using 0.2% Triton-X. The fixed cells were then incubated overnight at 4 °C with primary antibody (alpha-Tubulin, Novus Biologicals) in 1:300 concentration and was then incubated at 37 °C with mouse IgGfluor(Millipore) for 2 h. The cells were washed with PBS and incubated at 37°C for lh with 1% Hoechst 33258(calbiochem). The cells were again washed with PBS and fluorescence microscopy was performed.

Example 17: Microtubule stability:

PC 12 cells were grown on confocal dishes and differentiated into neurons for another 5 days. The differentiated neurons were treated with various concentrations of peptoid for 24h. The cells were then fixed with 4% formaldehyde and permeabilized using 0.2% Triton-X. The fixed cells were then incubated overnight at 4 °C with primary antibody (alpha-Tubulin, Novus Biologicals) in 1:300 concentration and was then incubated at 37 °C with mouse IgG fluor(Millipore) for 2 h.The cells were washed with PBS and incubated at 37°C for lh with 1% Hoechst 33258(Calbiochem). The cells were again washed with PBS and fluorescence microscopy was performed.

Example 18: Effect of LPFFK peptiod on primary cortical neuron culture: Primary cortical from the neocortex of El 8 day embryonic rat brains were cultured following the previously described method(Beaudoin M J G., Lee S.H., Singh D., Yuan Y., Ng Y.,Reichardt F L. and Arikkath J. (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat. Protoc. 7, 1741-1754). Briefly, the brains from E18 embryosof Sprague Dawley Ratswere isolated. Brain cortices were then dissected, digested and filtered. The cells were then suspended in MEM medium that contains 10% horse serum and glucose (0.6% wt/vol). The suspended cells (3-5 x l0⁵/mL) were taken and cultured on coverslips coated with poly-D-lysine at 37 °C with 5% C0₂ environment. The culture medium was changed toneurobasal media supplemented with 2% B27, 1% Pen/Strep and 0.5 mMGlutaMAX after 4 h of incubation. Once the neurons have attached and neuronal morphology appeared, the cells were treated with 10 mM of peptiod to observe their effect on cellular morphology.

Example 19: Blood Brain Barrier crossing experiment with peptoid(Alonso E., Vieira AC., Rodriguez L, Alvarino R., Gegunde S., Fuwa H., Suga Y., Sasaki M., Alfonso A., Cifuentes JM, Botana LM.(20l7) Tetracyclic Truncated Analogue of the Marine Toxin Gambierol Modifies NMDA, Tau, and Amyloid b Expression in Mice Brains: Implications in AD Pathology acschemneuro. , 8(6), 1358-1367)

Healthy C57BL/6J female mice were used for the study and divided in two groups (3mice/group). Then, inter-cardiac injections were performed with 100 pL of sample (concentration of 10 mg/Kg body weight of mice). For control experiment, injections were performed with 100 pL of water. After 6h, mice were sacrificed to separate out brains and using stereo microscope blood vessel, meninges were removed. Then, the brains were crushed using mortar pestle in liquid nitrogen and dissolved in l: l(water: Acetonitrile). After centrifugation, soluble part was taken for RP-HPLC and ESI mass spectrum.

Example 20: Primary cortical neurons staining with MAP2:

After treatment of LPFFK peptoid with primary cortical neurons, cells were fixed with 4 % formaldehyde and permeabilized using 0.3 % Triton-X. Then, the fixed cells were treated with primary Mouse anti-MAP2 and incubated for overnight at 2-8 °C .Cells were washed with DPBS, then incubated with secondary antibody Alexa Fluor 594 and then was further stained using Hoechst 33258. The cell morphology was then observed under confocal microscope having a 60X objective (Olympus).

Example 21: Docking:

To find the binding mode of LPFFK peptoid with AChE docking study was performed. For docking study inventors took crystal structure of AChE (PDB ID: 2CKM). The PDB file was downloaded from RCSB protein data bank. All the water molecules and non-protein atoms were removed from protein for docking study. The docking study was carried out in Autodock- Vina (software version 1.1.2). Autodock-Vina (software version 1.1.2) was used to know the binding mode of LPFFK with

Amyloid beta protein (PDB ID: 1IYT). Blind docking was performed for this case. The Protein-

Peptoid interaction was presented in 2D interaction plot using the help of Ligplot.

Example 22: Data Analysis:

Microscopic images were analysed using Image J software.

ADVANTAGES:

The main advantages are:

1. A novel peptoid targeting multifactorial functions against Alzheimer’s disease

2. Peptoid based therapy advantageous over peptide as peptoid has higher bioavailability than peptide.

3. Peptoids are stable in human serum protease.

4. Due to their serum stability and lower molecular weight, peptoids may cross the Blood Brain Barrier (BBB).

5. Peptoids are easy to synthesize and the starting materials for their synthesis are very economical.

6. The structural diversity of the peptoid is greater than the peptide.

7. This single peptoid targets the multifactor of the Alzheimer’s disease like stabilization of microtubule, inhibits amyloid fibril formation, inhibits AChE acitivity and AChE induced amyloid fibril inhibition , reduces Intracellular ROS and shows significant neuro -protection against Ab induced toxicity.

8. Therefore, this peptoid can be treated as a potential drug candidate for multifactorial Alzheimer’s disease.

Claims

CLAIMS:

1. A peptoid of formula I.

Form mumla I

2. The peptoid as claimed in claim 1, having a molecular weight of 635.80.

3. The peptoid as claimed in claim 1, wherein the peptoid binds with microtubule lattice (K_b-6.576Xl0³M^_1)and stabilizes microtubule.

4. The peptoid as claimed in claim 1 , wherein the peptoid inhibits amyloid fibril formation and oligomer formation.

5. The peptoid as claimed in claim 1, wherein the peptoid reduces the activity of AChE, inhibits AChE induced amyloid fibril formation and reduces intra cellular ROS.

6. The peptoid as claimed in claim 1, wherein the peptoid shows neuroprotection against Ab induced toxicity.

7. The peptoid as claimed in claim 1, wherein the peptoid is able to cross Blood Brain Barrier (BBB) in mice model.

8. The method of preparing the peptoid as claimed in claim 1, comprising solid phase synthesis of peptoid using Rink Amide resin, purification of crude peptoid by RP-HPLC and characterizationby ESI Mass Spectroscopy.

9. The pharmaceutical composition comprising the peptoid as claimed in claim 1 along with pharmaceutically acceptable excipients.

10. The method of treating Alzheimer’s disease by administering effective amount of peptoid as claimed in claim 1.