POTENT PEPTIDE INHIBITORS OF PROTEIN AGGREGATION

Abstract

The present invention provides a novel peptide as potent inhibitor of protein aggregation. A peptide for an inhibition of protein aggregation having the general Formula 1, [X.sub.iSACX.sub.1].sub.mHHHH[X.sub.2X.sub.3CGG].sub.m is provided. The m is 0 or 1; Xi is an acetyl group; X.sub.1 is a hydrophilic polar uncharged amide group containing amino acid; X.sub.2 is His or Leu; and X.sub.3 is His or Ser. The present invention specifically relates to peptide-based inhibitors useful in treatment of amyloid related disorders.

Claims

1-8. (canceled)

9. A peptide useful in inhibition of protein aggregation having formula: [X.sub.iSACX.sub.1].sub.mHHHH[X.sub.2X.sub.3CGG].sub.m, wherein m is 1; Xi is an acetyl group; X.sub.1 is a hydrophilic polar uncharged amide group containing amino acid; X.sub.2 is His or Leu; X.sub.3 is His or Ser; and C at 3.sup.rd position and 11.sup.th position is selected from the group consisting of cysteine and cystine.

10. The peptide as claimed in claim 9, wherein the C-terminus amino acid is amidated.

11. A method for preparing the peptide of claim 1, comprising: a. providing His.sub.6-tagged α-Syn bound on Co.sup.++-NTA resin at a range of 4-7 .Math.g; b. providing a phage library encoding from about 2x10.sup.11 to about 7×10.sup.11 pfu displaying cyclic peptide; c. incubating about 10.sup.11 titre of said phage library obtained in step (b) with the His.sub.6-tagged α-synbound on Co.sup.++-NTA resin obtained at step (a) for up to 1 hour; d. eluting any bound phages obtained step (c) with a buffer at a pH of from 1 to 3; e. neutralizing the pH of the product of step (d) immediately after elution; f. amplifying any phages obtained at step (d); g. selecting the strong interacting peptides of α-syn; h. performing repeated washing with the strong interacting peptides obtained at step (f) with Tween 20 and NaCl; i. eluting the phages bound to α-syn obtained at step (h); and j. isolating said peptide.

12. The peptide as claimed in claim 9, wherein said peptide inhibits aggregation amyloidogenic protein of α-synuclein.

13. The peptide as claimed in claim 9, wherein said peptide inhibits of α-sheet polymerisation of amyloidogenic proteins.

14. The pharmaceutical composition comprising the peptide of claim 9 and a pharmaceutically acceptable excipient thereof.

15. A method for treating amyloid associated disorders comprising administering a therapeutically effective amount of the peptide of claim 9 to a subject with an amyloid associated disorder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts model describing screening of potential peptides against α-syn fibrillation using phage display library. Phage display library encoding C-7 peptides were passed through His.sub.6-tagged α-syn bound Co.sup.++-NTA resin. After 3 rounds of biopanning, individual interacting phages in the form of plaques were amplified and screened for inhibiting α-syn fibrillation. Potent inhibitors were sequenced and further validated by in vitro fibrillation.

[0014] Table 1 shows phage titre- 5 .Math.g of His.sub.6-tagged α-syn was bound on Co.sup.++-NTA resin and mentioned titre of phage library was bound to resin for 1 hour and eluted according to manufacturer’s protocol. After 3 rounds of biopanning, eluted library was loaded without amplification. Individual plaques were screened from 5.sup.th and 6.sup.th eluent.

[0015] FIG. 2 depicts phage encoding Pep.3 inhibits in vitro α-syn fibrillation. Phages showing repeated binding to α-syn were further amplified according to manufacturer’s protocol (NEB #E8110S). The α-syn fibrillation was carried out in the presence and absence of various screened phages. Phages inhibited α-syn fibrillation with varying efficiency. The phage encoding Pep.3 nearly completely inhibited α-syn fibrillation.

[0016] FIG. 3 shows purified Pep.3 inhibits α-syn fibrillation. (A) In vitro α-syn fibrillation assay in the presence and absence of equimolar concentrations of linear and cyclized (cycPep.3) forms of peptide Pep.3. The assay was carried out as described in Example 4. (B) TEM images from samples collected after 3 h of α-syn fibrillation in the presence and absence of Pep.3 or cycPep.3.

[0017] FIG. 4 depicts Pep.2d shows inhibition of α-syn fibrillation among different truncations, α-syn (400 .Math.M) was incubated as mentioned above with and without Pep.2 and its derivatives and ThT fluorescence was measured after regular interval of time. As seen by lower fluorescence intensity, Pep.2d inhibits α-syn fibrillation.

[0018] FIG. 5 depicts MTT assay with PC12 cells shows reduction in α-syn associated toxicity in presence of Pep.2 and CycPep.2. 10 .Math.M of α-syn, with and without indicated peptides in equimolar ratio, and peptides alone were incubated with PC12 cells in 96 well plate for 24 hours. The peptides alone were taken as control to examine the toxicity of peptides to PC12 cells.

[0019] MTT assay was carried out in triplicate as described in Example 6 and absorbance was measured at 570 nm to check viability of cells. Increase in absorbance in presence of Pep.2 and CycPep.2 indicated reduced α-syn associated toxicity in PC12 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Many of the amyloid based disorders are due to higher tendency of associated intrinsically disordered protein. One of the primary challenges in the design of therapeutics against amyloid diseases has been to develop inhibitor that could prevent the formation of amyloid fibrils. Thus, the main objective of present study is the identification of small biomolecules such as peptides that could inhibit the fibrillation of intrinsically disordered proteins.

[0021] Commercially available phage display library from New England Biolabs (NEB) encoding cyclic 7 peptides with phage titre 2×10.sup.11-7×10.sup.11 plaque forming units (pfu), calculated as per manufacturer’s protocol, was used to screen novel interacting sequences with purified α-syn as target. α-syn was quantitated using BCA protein estimation kit as per manufacturer’s protocol (#23227 Thermo Scientific). Here 4-7 .Math.g His.sub.6-tagged α-syn was immobilized over cobalt metal affinity resin and phage library was incubated with it. FIG. 1 show the protocol used in brief. Interacting peptides were selected after 5-6 rounds of biopanning, and further screened for inhibition of fibrillation assay.

[0022] Using ThT assay, we show that α-syn, upon incubation at 37° C. under continuous shaking speed of 900 rpm forms fibrils. ThT is a fluorescent dye which is known to bind specifically to β-sheet rich fibrils, and such binding further increases its fluorescence intensity. The fibrillation assay was carried out with α-syn in the presence and absence of above identified interacting phages. As shown in FIG. 2, different phages inhibited α-syn fibrillation to different extent. Phage encoding peptide Pep.3 was found to be most potent in inhibiting α-syn fibrillation.

[0023] The genomic DNA was isolated from potential phages, and gene encoding corresponding displayed peptides were identified using DNA sequencing. The identified sequence was used to synthesize corresponding peptide. Both linear and cyclized version of peptides were synthesized. In vitro fibrillation assay was setup with linear as well as cyclic peptide. Cysteine refers to linear peptide and cystine refers to cyclic form of peptide. As evident from FIG. 3A, peptides at equimolar concentration as of α-syn were able to inhibit α-syn fibrillation. In order to further confirm that the decrease in ThioflavinT fluorescence in the presence of peptides was due to the ability of peptides to inhibit α-synfibrillation, samples were imaged using TEM. The samples containing α-syn incubated at 37° C. in the presence and absence of equimolar concentration of peptides was collected after 5 h of fibrillation reaction, and mounted on Carbon-coated 200 mesh grids followed by visualization through Transmission Electron Microscope using phosphotungstate staining. FIG. 3B shows that the co-incubation of the peptides inhibits formation of α-syn fibrils. In the peptide, the “C” at 3.sup.rd position and 11.sup.th position is selected from the group consisting of cysteine and cystine respectively.

[0024] Several truncated derivatives of Pep.2 were constructed by deleting residues from N-terminal as well as C-terminal of the peptide. 7 variants of Pep.2 were commercially synthesised (GL Biochem, China) and were examined for their anti-fibrillation activity by ThT assay. Peptide variants were incubated with α-syn in equimolar concentration along with ThT. As seen in FIG. 4, the designed variants show range of inhibition activities (TABLE A). Pep.2d was able to reduce α-syn fibrillation comparable with Pep.2. Pep.2g showed approximately 80% inhibition of fibrillation.

TABLE-US-00001 showing the designed variants of the peptide SEQ ID NOs Internal Designation SEQ ID NO 1 Pep.2d SEQ ID NO 2 Pep.2 SEQ ID NO 3 Pep.2g SEQ ID NO 4 Pep.2f SEQ ID NO 5 Pep.2e SEQ ID NO 6 Pep.2c SEQ ID NO 7 Pep.2b SEQ ID NO 8 Pep.2a

[0025] MTT ((4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) cell toxicity experiments were performed using Rat phaeochromocytoma (PC12) neuronal-like cells to assess toxicity of α-syn and the protective effects of the Pep.2 and CycPep.2 generated in this study. MTT assay (FIG. 5) was performed with PC12 cells incubated with α-syn fibrillation end products in presence and absence of peptides. As seen, the presence of α-syn leads to loss of cellular viability by about 50%. However, with similar concentration of α-syn, the presence of Pep.2 and CycPep.2 improved cellular viability by 30% and >99% respectively. The peptides alone were also examined for their toxicity at even relatively higher concentration (20 mM) and as seen in the FIG. 5 peptides are not toxic to PC12 cells.

[0026] Based on the sequences obtained we undertook in-vitro fibrillation screen and the peptide having Formula no.1: [X.sub.iSACX.sub.1].sub.mHHHH[X.sub.2X.sub.3CGG].sub.m was designed in accordance with the potent inhibitors. The above peptides can be used in pharmaceutical compositions such as lipidic formulations like liposomes, solid lipid nanoparticles with peptide stability and penetration enhancers.

EXAMPLES

[0027] The following examples are given by way of illustration therefore should not be construed to limit the scope of the Invention.

Example 1

Expression and Purification of His.SUB.6.-Tagged α-Syn

[0028] The N-terminal His.sub.6-tagged human wild-type α-syn was expressed in E. coli Rossetta (DE3) (#70954 Novagen) using pET29a based expression vector. After IPTG induction bacterial cell pellets were harvested by centrifugation and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM Pierce protease inhibitor cocktail. Protein purification was performed with minor changes in protocol as mentioned in van Raaij et al., 2006 (van Raaij, M.E., Segers-Nolten, I.M.J., and Subramaniam, V. (2006). Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synmutants. Biophysical journal 91, L96-98). Briefly, cell lysis was carried out by sonication, lysate was boiled at 95° C. for 30 minutes, followed by centrifugation at 10,000 × g for 30 minutes at 4° C. Streptomycin sulfate and Glacial acetic acid precipitated DNA was removed by centrifugation at 13,500 × g for 30 minutes at 4° C. The supernatant was incubated with 50% ammonium sulfate on ice to selectively precipitate α-synuclein. The pellet was separated by centrifugation at 13,500 × g for 30 min. at 4° C., and further washed with equal volume of 100 mM ammonium acetate followed by equal volume of ethanol. Pellet was dissolved in 10 mM HEPES, 50 mM NaCl, pH 7.4 and dialysed extensively to remove ammonium sulphate. Protein purity was confirmed on 15% SDS-PAGE. Protein was quantitated using BCA protein estimation kit as per manufacturer’s protocol (#23227 Thermo Scientific).

Example 2

Phage Display Screening

[0029] 10.sup.11 titre of Phage library (#E8120S NEB) each encoding 7 residue cyclic peptide was incubated with 5 .Math.g of His.sub.6-tagged α-syn bound on Co.sup.++-NTA resin for 1 hour. Unbound phages were removed with wash buffer (150 mM NaCl, 0.02% Tween20). The bound phages were eluted using buffer of pH 2.2, and the pH was neutralized immediately after elution. Eluted library was amplified using E. coli ER2738 strain (#E8110S NEB). The above process was further repeated three times with eluted phages to select only strong interactors of α-syn. In 4.sup.th round, washing was done with 0.05% Tween20 and eluted phages were directly bound to α-syn without amplification in order to increase specificity. The process was repeated twice with eluted phages.

Example 3

Peptide Synthesis

[0030] Peptides were synthesized at peptide synthesizing facility at Institute of Microbial Technology, Chandigarh, India. Peptides were synthesized by solid phase peptide synthesis strategy using Fmoc (N-(9-fluronyl)-methoxycarbonyl) chemistry in 0.01 mmole scale on a Protein Technologies Inc, USA, PS-3 peptide synthesizer as described elsewhere (Gautam, A., Sharma, M., Vir, P., Chaudhary, K., Kapoor, P., Kumar, R., Nath, S.K., and Raghava, G.P.S. (2015). Identification and characterization of novel protein-derived arginine-rich cell-penetrating peptides, European Journal of Pharmaceutics and Biopharmaceutics 89, 93-106).

Example 4

In Vitro Fibrillation Assay

[0031] Thioflavin T (ThT) (4 mM) was added to 400 .Math.M of purified α-syn with and without phages (10.sup.11) or purified peptides at equimolar concentration in a 96 microwell plate. The plate was incubated at 37° C. with a shaking speed of 900 rpm in linear mode in a multimode plate reader (TECAN infinite M200 PRO). Fluorescence kinetics was measured after every 15 minutes with emission wavelength of 482 nm upon excitation at 442 nm. Each experiment was repeated at least three times.

Example 5

TEM Imaging

[0032] The morphology of the oligomeric species was assessed using a JEM-2100 transmission electron microscope (TEM) (Jeol, Ltd.). For TEM experiments, samples were adsorbed onto carbon-coated grids and negatively stained with 1% Phosphotungstate for 30 seconds.

Example 6

MTT Assay

[0033] MTT experiments were performed using Rat phaeochromocytoma (PC12) cells to assess cytotoxic effect of α- syn. Inhibition of PC12 cell redox activity is specific and early indicator of cell death. In MTT assay, water soluble MTT dye (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (#M2003 Sigma-Aldrich) is converted to formazan, and the conversion is monitored via absorbance measurement at 570 nm. The conversion of the MTT dye to formazan is dependent of redox status of the cell and thus the change in absorbance can be used as an indicator of the cellular viability in the assay.

[0034] PC12 cells were maintained in growth media RPMI 1640 supplemented with 10% horse serum, 5% fetal bovine serum, and antibiotics penicillin and streptomycin. The overnight grown cells (50,000 cells/well) were used for MTT assay in 96 well plate. The cells were incubated with reaction mixture (1.2 .Math.l) comprised of α-syn (400 .Math.M) fibrils formed in the presence and absence of peptides (400 .Math.M). After incubation of 24 h at 37° C. in 5% CO.sub.2 atmosphere, 10 .Math.l of MTT (5 mg/ml in PBS) was added and incubated further for 4 h. For cell lysis, and solubilization of formazan crystal, 100 .Math.L of the solubilization buffer (20% SDS in 50% DMF) was added to each well and incubated for 90 minutes under mild shaking conditions. The absorbance was measured at 570 nm using a multimode plate reader (TECAN infinite M200 PRO). Each experiment was performed in triplicate.

TABLE-US-00002 Rounds Input Phage Titre Wash Buffer Eluted Phage Titre Amplified Phage Titre 1 10.sup.11 150 mM Nacl 0.02% Tween20 1.1×10.sup.8 2×10.sup.12 2 2×10.sup.11 150 mM NaCl 0.02% Tween20 5.5×10.sup.8 1.2×10.sup.13 3 1.2×10.sup.12 150 mM NaCl 0.02% Tween20 2.5×10.sup.7 3.1×10.sup.12 4 3.1×10.sup.11 150 mM NaCl 0.05% Tween20 8.6×10.sup.7 N.A 5 8.6×10.sup.7 150 mM NaCl 0.05% Tween20 1.3×10.sup.5 N.A 6 1.3×10.sup.5 250 mM NaCl 0.05% Tween20 9×10.sup.3 N.A

ADVANTAGES OF THE INVENTION

[0035] 1. The identified peptide inhibits α-syn fibrillation and thus acts as potent therapeutics against number of neurodegenerative diseases collectively known as α-synucleinopathies.

[0036] 2. As amyloid formed of diverse sequences share similar structure, identified peptides has the potential to inhibit amyloid fibril formation from other pathogenic intrinsically disordered proteins, and thus could act as potential therapeutics against various diseases such as Parkinson’s Disease (PD), Alzheimer’s Disease (AD), Huntington’s Disease (HD), diabetes and α-synucleinopathies.