A peptide capable of inhibiting bacterial biofilm / fibril formation

Abstract

The present invention provides a peptide capable of inhibiting and disintegrating bacterial biofilm / fibril formation. Particularly, the present invention provides a retro inverso peptide which can inhibit the biofilm formation as well as disintegrates the pre-formed biofilm of Staphylococcus aureus.

Description

[0001] A PEPTIDE CAPABLE OF INHIBITING BACTERIAL BIOFILM / FIBRIL FORMATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of peptide chemistry. Particularly, the present invention relates to peptide and its potential of inhibiting and disintegrating bacterial biofilm and fibril formation. More particularly, the present invention relates to retro-inverso peptides and their inhibitory and disintegrating effect on bacterial biofilm and fibrils.

[0004] BACKGROUND OF THE INVENTION

[0005] Antimicrobial resistance (AMR) is one of the top challenges being faced by the global health industry. Antimicrobial Resistance occurs when a pathogen such as a bacteria, virus or fungi develops drug resistance and no longer respond to antimicrobial medicines. As a result of the drug resistance, antimicrobial medicines such as antibiotics become ineffective and infections become difficult or impossible to treat.

[0006] One of the reasons for developing drug resistance in bacteria is formation of biofilms or fibrils. The self-aggregation of proteins and peptides forms amyloids into highly ordered fibrillar structures in bacteria. Amyloid aggregates are key components of biofilms of many bacterial species. These biofilms acts as protection layer for bacterial cells and minimizes impact of external agents such as antibiotics, chemotherapy, and disinfectants.

[0007] The amyloid form of proteins and peptides are also responsible for many human degenerative pathologies like Alzheimer’s, Parkinson’s, and Huntington's diseases. The high thermodynamic stability of such protein and peptide aggregates resists disruption of amyloids by proteases and detergents.

[0008] Biofilm formation is exhibited by both the free-floating microorganisms as well as microorganisms which grow on surface such as on medical devices. Medical device-associated biofilms also pose a serious threat to the life and health of patients. Microorganisms can adhere to almost all medical devices. Formation of biofilms around these microorganisms develops resistance to antimicrobial agents thereby leading to medical device-associated biofilm infections in patients.Among the various microorganisms exhibiting drug resistance, Staphylococcus aureus is posing a serious challenge. Staphylococcus aureus (S. aureus), a gram-positive bacterium, is frequently found in the nose, respiratory tract, and on the skin, and causes infections such as endocarditis, necrotizing pneumonia, and septic shock. S. aureus is also one of the most common microorganisms for medical device-associated infections. S. aureus triggered infections posing challenge in treatment options are primarily due to the formation of biofilm around the bacterial surface. Biofilms provide a protective safeguard shielding bacterial cells from host immune responses, antibiotics and disinfectants.

[0009] In Staphylococcus aureus biofilms, the amyloid fibrils are formed primarily by phenol-soluble modulins (PSMs), small amphipathic peptides that are classified into a-type (20-25 amino acids) and 0-type (44 amino acids). PSMs contribute to biofilm formation and exhibit cytolytic activity, with PSMa3, a 22-residue a-type peptide, exhibiting the highest cytotoxicity.

[0010] Disrupting biofilm integrity by targeting amyloid fibrils involves several strategies. Small molecule inhibitors, such as Congo Red, curcumin, and EGCG, can destabilize amyloid fibrils and reduce biofilm stability. Peptide-based approaches, including anti-amyloid or fibril-binding peptides, can block or destabilize amyloid fibrils, preventing biofilm formation. Enzymatic degradation using amyloid-degrading enzymes like proteinase K can also cleave fibrils and disrupt the biofilm matrix. Nanomaterials such as silver nanoparticles and carbon nanotubes interact with amyloid fibrils, destabilizing the biofilm structure. Physical methods like ultrasound (sonication) and electroporation can mechanically disrupt biofilms, including their amyloid components. Despite their potential, challenges such as ensuring specificity (to target bacterial amyloids without affecting human proteins), the high resistance of the biofilm matrix to antimicrobials and immune responses, and the need for effective in vivo translation remain significant obstacles for these strategies. Also, none of the existing methods have been investigated to disrupt the structural integrity of biofilms by targeting the unique cross-a amyloid fibrils within the biofilm matrix. The present invention addresses these shortcomings of the prior art and provides a new peptide which facilitates S. aureus biofilm dissolution by preventing both cross-a fibril formation and disassembling preformed fibrils from PSM-a3 peptide.

[0011] OBJECTIVE OF THE INVENTIONAn important objective of the present invention is to provide a retro inverse phenol soluble modulins alpha-3 (RI-PSMa3) peptide.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention provides a peptide of SEQ ID No. 1 or an analogue thereof, said peptide is capable of inhibiting and disintegrating bacterial biofilm / fibril formation, wherein the peptide is a retro-inverso peptide. Particularly, the present invention relates to a retro inverse phenol soluble modulins alpha-3 (RI-PSMa3) peptide and its effect on the cross-a amyloid assembly of native PSMa3, facilitating Staphylococcus aureus biofilm dissolution. More specifically, RI-PSMa3 peptide acts on the biofilm of S. aureus and stops or disintegrate the biofilm formation.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate exemplary embodiments and together with the description, serve to explain the disclosed invention.

[0016] Figure 1: (A) represents the topological relationship shown for a model peptide and its fully retro-inverso analogue. (B) Represent the sequence of PSMa3 and RI-PSMa3 peptides. (C) Shows the aggregation kinetics of L-PSMa3 and RI-PSMa3 were monitored by ThT fluorescence assay. The concentration of each peptide was 25 pM. (D) Shows the aggregation kinetics at an increased concentration (25 pM, 50 pM, 100 pM and 200 pM) of the RI-PSMa3 peptide as monitored by the same ThT fluorescence assay. (E) Shows the representative SEM images of the a) L-PSMa3, and b) RI- PSMa3 after 14 hours of incubation at 25 pM concentration, c) The same was taken for the RI-PSMa3 peptide at a concentration of 200 pM.

[0017] Figure 2: Shows analytical RP-HPLC profile (X = 214 nm) together with ESI-MS data (inset) of the crude and purified peptides. A linear gradient of 15%-75% buffer B in buffer A (buffer A = 0.1 % TFA in water; buffer B = 0.08% TFA in acetonitrile) at 40 °C over 10 minutes including 4 min equilibration time using Agilent Zorbax SB-C3, 5 pm, 4.6 x 150 mm, LC column with 0.9 mL / min flow rate was used for the chromatographic separation.Figure 3: (A) shows the kinetics of PSMa3 amyloid formation in the presence of the RI-PSMa3, monitored by ThT fluorescence. Keeping the proportion of the L-PSMa3 and RI-PSMa3 peptides same, the concentration of each peptide varied from 12.5pM to 50pM. (B) Shows the representative SEM images of PSMa3 fibrils after 14 hours of incubation in the presence of an equimolecular amount of the RI-PSMa3 peptide. (C) Represent the dose-dependent Kinetics of PSMa3 amyloid formation in the presence of the RI-PSMa3, monitored by ThT fluorescence. The molar equivalent of the RI-PSMa3 peptide varied from 0.05 to 1.00 with respect to the L-PSMa3 peptide. (D) Shows the zoomed-in version of Figure C visualizes the complete suppression of the L-PSMa3 ThT intensity when the equivalent amount of the RI-PSMa3 peptide was 0.25 and above. (E) Shows the representative SEM images of PSMa3 fibrils after 24 hours of incubation in the absence or presence of the different equivalent amounts of RI-PSMa3 peptide Figure 4: (A) illustrates Dose-dependent ThT kinetics for the disaggregation of preformed fibrils of PSMa3 in the presence of RI-PSMa3. The molar equivalent of the RI- PSMa3 peptide varied from 0.25 to 2.00 with respect to the L-PSMa3 peptide’s amount. (B) Shows Dose-dependent disaggregation of the preformed PSMa3 fibrils from end-point fluorescence data taken after 15 hours. (C) Shows the representative SEM images of pre-formed PSMa3 fibrils treated with different equivalents ofRI-PSMa3 peptides.

[0018] Figure 5: (A) Depicts the sequence of the chemically synthesized PSMa3 peptide and its analogues. (B) shows the schematic representation of two-component dynamic combinatorial disulphide formation of peptides. In the figure, the orange color bar is the A-type peptide and the blue color bar is the B-type peptide as shown in Table C. The disulphide-bonded complex can be detected after the guanidine-mediated denaturation and RP-HPLC ESI-MS analysis. (C) represent in the table, the A-type peptide represents the L-PSMa3 peptide having C-terminal cysteine residue while the B-type peptide represents any of the two other peptides having Cys-residue either at the N- terminus or the C-terminus (RI-PSMa3(Cys) and (Cys)RI-PSMa3). (D) Represent that an equal equivalent amount of peptide-A was mixed with peptide-B in the folding condition in the presence of redox reagent (20 mM phosphate, 1 mM GSH, 0.2 mM GSSG, pH 8.0). For experiments 1-3, the individual peptide was the only component whereas for experiments 4-5, the mixture of two peptides has been used. The column chart represents the quantity of individual dimer (percentage) obtained when the reaction reached equilibrium. The percentage of theproducts was quantified after the guanidine-mediated denaturation by the intensity of the peak (area under the curve) detected at 214 nm wavelength in analytical RP-HPLC. (E) Shows the far-UV CD spectrum (190nm-260nm) was obtained by measuring the ellipticity of the sample in the aqueous phosphate buffer (20 mM phosphate, pH 7.4). All the peptides showed characteristic alpha-helical signatures with L-peptides showing characteristic negative minima at 208 nm and 222 nm while the Rl-peptides showed positive maxima at the same two wavelengths. (F) Shows the DLS data of L-PSMa3 aggregating species were taken at different time points and represented as color traces. (G) Shows the DLS data of RI-PSMa3 peptide and buffer alone. Only the data point after 1 hour of the incubation of RI-PSMa3 is shown here. (H) Shows the DLS data of L-PSMa3 aggregating species taken after 1-hour incubation and the addition of the equivalent amount of the RI-PSMa3 peptide. The aggregating species at different time points are represented as color traces.

[0019] Figure 6: (A) Shows percentage MRSA biofilm inhibition by RI-PSMa3. Chloramphenicol was used as a positive control. All experiments were carried out in triplicate, and data represent the mean standard error (one-way ANOVA P < 0.0001). (B) Shows the micrographs of disrupted matured biofilms of the tested MRSA strain (ATCC 43300) formed on glass surfaces by the RI-PSMa3 at different concentrations by scanning electron microscopy (Carl Zeiss, Evo 18). (a, d) Growth control; (b, e) RI-PSMa3 (100 pg / ml); and (c, f) RI-PSMa3 (150 pg / ml).

[0020] Other embodiments, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description such changes and modifications are covered within the scope of the present invention.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. Particularly, the word “comprising” is intended to be used to cover within its ambit other possible ingredients or steps or features of an aspect or embodiment of the invention which are apparent to a skilled person after reading the present disclosure. The word “comprising” is intended to mean “including” but not necessarily “consisting of’ or “composed of.” It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

[0023] It is noted that the examples given in the description below are intended to clarify the invention and are not intended to limit the invention to those examples per se. Except in the operating and comparative examples, or where otherwise explicitly indicated, all numbers in this description and claims indicating amounts of material or conditions of reaction, physical properties of materials and / or use are to be understood as modified by the word “about”. Numerical ranges expressed in the format "from x to y" are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format "from x to y", it is understood that all ranges combining the different endpoints are also contemplated.

[0024] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology described herein are those well-known and commonly used in the art.

[0025] The present invention provides a peptide capable of dissolving and disintegrating the bacterial biofilm. The peptide of the invention has inhibitory potential on the amyloid formation of the native peptide, which is one of the components of the bacterial biofilm. Particularly, the present inventors identified peptide-based inhibitors which can impede the development of the extracellular polymeric substance (EPS) matrix and, more importantly, disassemble the fully formed EPS matrix within Staphylococcus aureus biofilms. These and other aspects, features and advantages of the invention will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims.The present invention provides a retro inverse peptide capable of inhibiting the formation of or disintegrating the formation of bacterial biofilm. Particularly, the invention provides a retro-inverso phenol soluble modulins alpha-3 (RI-PSMa3) peptide and explores the effect of the RI-PSMa3 on the cross-a amyloid assembly of PSMa3, facilitating Staphylococcus aureus biofilm dissolution.

[0026] The bacterial strain used for the present invention MRSA (Methicillin-resistant S. aureus ATCC 43300), was procured from the American Type Culture Collection (ATCC).

[0027] The team of inventors studied the property of the retro-inverso (RI) sequence of the cross-a amyloid-forming peptide PSMa3, which is among the most lytic members of the PSM family peptides of S. aureus. In retro-inverso peptide, the amino acid sequence is reversed (retro) and each amino acid is replaced withits enantiomer (inverse) (Figure 1A scheme). The inventors designed a peptide structure, which despite the reversed sequence and inversed chirality, can still interact with the same receptor as the parent peptide, as the side chain topology of the retro-inverso sequence is so designed that it mimics the original peptide's conformation. Additionally, retro-inverso peptide of the invention has theadvantage of increased resistance to proteolysis due to the presence of D-amino acids.

[0028] The sequence of the said retro-inverso peptide is represented by SEQ ID No. 1. Accordingly, in an embodiment, the present invention provides a retro inverse phenol soluble modulins alpha-3 (RI-PSMa3). The RI-PSMa3 peptide of the invention is capable of inhibiting or disintegrating the biofilm formation in bacteria. The invention thus provides a new therapeutic approach for biofilm-associated infections.

[0029] In a specific embodiment, the invention provides a peptide of SEQ ID No. 1 or an analogue thereof or a sequence >60% similar thereof, said peptide is capable of inhibiting and disintegrating bacterial biofilm / fibril formation, wherein the peptide is a retro-inverso peptide.

[0030] In a preferred embodiment, the present invention provides that the peptide is retro inverse phenol soluble modulins alpha-3 (RI-PSMa3).

[0031] In a preferred embodiment, the present invention provides that the sequence of the peptide consists of non-natural amino acids.In a preferred embodiment, the present invention provides that the peptide acts on the biofilm of Staphylococcus aureus and stops the biofilm formation and disintegrates pre-formed matured biofilms. In yet another preferred embodiment, the peptide disintegrates the biofilm by targeting the amyloid fibrils within the biofilm matrix.

[0032] In a preferred embodiment, the peptide selectively acts on the cross-a amyloid formation of PSMa3 peptide.

[0033] In yet another preferred embodiment, the peptide binds selectively to the native PSMa3 in an antiparallel fashion.

[0034] In a preferred embodiment, the present invention provides a composition comprising the said peptide along with pharmaceutically acceptable additives and carriers.

[0035] In yet another embodiment, the present invention provides a surface disinfectant comprising the said peptide along with pharmaceutically acceptable additives, carriers and / or other disinfectant agents.

[0036] Yet another embodiment of the invention provides that a method of synthesizing the said peptide comprising the step of:

[0037] i. coupling of amino acid Cysteine (0.25M) for 2-10 min at room temperature followed by 5-15 min at 45-65 °C on resin under an N2 atmosphere with vortex mixing using using coupling reagent and additives;

[0038] ii. coupling of all other amino-acids in the SEQ ID No 1 (0.25M) for 5-10 min at 60-70 °C under an N2 atmosphere with vortex mixing using coupling reagent and additives; iii. after every coupling cycle, carrying out Fmoc deprotection by 20% piperidine in DMF at 50 °C;

[0039] iv. after completion of the coupling of all residues, using a cocktail of TFA, phenol, water, DODT , and TIPS to cleave peptides from the resin;

[0040] v. evaporating TFA to reduce the solution volume;

[0041] vi. precipitating the cleaved synthesized peptide with diethyl ether, and lyophilizing. In another embodiment, the cocktail used for cleave peptides from the resin contains 85 % TFA, 5% phenol, 5%water, 2.5% DODT and 2.5% TIPS.In yet another embodiment the resin, coupling reagent and additives used namely are Rink amide aminomethyl, DIC and oxyma with DIEA respectively

[0042] The following examples are given by way of illustration and for better understanding of the present invention and should not be construed to limit the scope of present invention.

[0043] EXAMPLES:

[0044] Example 1: The method of synthesis of the RI-PSMa3 peptide

[0045] The present inventors prepared RI-PSMa3 peptides as well as the Cysteine-containing analogs using Fmoc chemistry solid phase peptide synthesis. The crude peptides were purified using reverse-phase HPLC (RP- HPLC). The purified peptide was then subjected to a pre-treatment protocol with a TFA-HFIP (1 : 1 jmixture. This pretreatment procedure was designed to effectively dissect all aggregates present in the sample of interest.

[0046] Chemicals used for the synthesis of the RI-PSM«3 peptide:

[0047] N,N-diisopropylethylamine (DIEA), Ethyl cyanohydroxyiminoacetate (Oxyma) and all the L-and D-amino acids for Fmoc-SPPS was obtained from Chem-Impex International, USA and Gyros Protein Technologies. The side-chain protecting groups used were, Glu(OtBu), Asp(OtBu), Asn(Trt), Lys(OtBu), Ser(tBu) and Cys(Trt). Dichloromethane (DCM), diethyl ether, N,N'-dimethylformamide (DMF), HPLC grade N,N' -diisopropylcarbodiimide (DIC), 1, 1,1, 3,3,3-Hexafluoroisopropanol (HFIP), 2,2,2 - Trifluoroethanol (TEE), phenol, 2,2 (Ethylenedioxy)diethanethiol (DODT), Triisopropylsilane (TIPS) and trifluoroacetic acid (TFA) were purchased from SRL chemicals India. The HPLC grade acetonitrile (CH3CN) for the peptide purification was purchased from Thermofisher Scientific, India. Piperidine was obtained from AVRA chemicals, India. Thioflavin T (stain for amyloid grade) was obtained from Sigma- Aldrich. The Rink amide aminomethyl resin (polystyrene resin with 1% cross-linked with divinylbenzene) was obtained from Supra Science Private Limited, India.

[0048] a) Peptide Synthesis:

[0049] Fmoc-SPPS was carried out using amino acids (AA) (0.25 M), DIC (0.25 M) as a coupling reagent, and oxyma (0.25 M) with DIEA (0.025 M) as additives. Peptides were synthesized on Rink amide aminomethyl resin with a loading capacity of 0.5 -0.6 mmol / g. Cysteine was coupled for 5 min atroom temperature followed by 10 min at 50 °C and all other amino-acid coupling was performed for 7 min at 65 °C under an N2 atmosphere with vortex mixing. Fmoc deprotection after every coupling cycle was carried out by 20% piperidine in DMF at 50 °C. After synthesis, a cocktail of TFA (85%), phenol (5%), water (5%), DODT (2.5%), and TIPS (2.5%) was used to cleave peptides from the resin. TFA was evaporated to reduce the solution volume, the cleaved peptide was precipitated with diethyl ether, and lyophilized. Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) was performed (Figure 2) to assess the purity of cleaved peptides on an Agilent HPLC instrument using an Agilent zorbax SB-C3 (5 pm), 4.6x150 mm reverse-phase silica column at a flow rate of 0.9 mL / min using a linear gradient of 15-75% solvent B (0.08% TFA in acetonitrile) in solvent A (0.1% TFA in H2O) in 10 minutes. The UV absorbance of the column eluent was monitored at 214 nm wavelength. Preparative (RP-HPLC) of crude peptides was performed on a Waters 1525 preparative HPLC system using Agilent ZORBAX-SB C3 (5 pm, 80 A, 9.4 x 250 mm) columns at 40 °C using a linear gradient of 25-55% solvent B (0.08% TFA in acetonitrile) in solvent A (0.1% TFA in H2O) in 60 minutes at 45 °C. Fractions containing the purified target peptide were identified by ESLMS (see Table 1). The deconvolution of the observed mass was carried out using Agilent MassHunter Qualitative Analysis software (version B.07.00), and the deconvoluted mass was reported with an uncertainty of ± 0.02 Da.

[0050] Table 1. Sequence of the wild type PSMa3 peptide and the synthesized analogs.

[0051]

[0052] **The capital letters denote L-amino acids and the small letters denote D-amino acids.

[0053] b) Peptide pre-treatment:

[0054] The lyophilized powder of the L-PSMa3 and RI-PSMa3 peptide analogs were reconstituted in a mixture of TFA-HFIP ( 1 : 1 ) to achieve a concentration of 2 mg / ml. Next, the solution was subj ected to sonication for a duration of 10 minutes at a temperature of 37°C. The solution was then allowed to evaporate in a chemical hood over a period of 2 days. To remove any remaining solvent, a highvacuum apparatus was used for solvent evaporation. In cases where immediate testing was not performed, the treated peptides were stored at a temperature of -80°C.

[0055] Example 2: Schematic representation of the structure of RI-PSMa3 peptide

[0056] A schematic exemplary structure providing the stereochemical information can be depicted as bellow:

[0057]

[0058] Example 3: Characterization of the structure of the prepared RI-PSMa3 peptide

[0059] The secondary structure of the RI-PSMa3 peptide in the buffer (20 mM phosphate, pH 7.4) was determined by recording far-UV circular dichroism (CD) spectra. The CD spectra revealed alphahelical nature of peptide, wherein the RI-PSMa3 peptide showing the reverse CD signature, as expected for a D-peptide (Figure 4E). To quantify the equilibrium secondary structure components in the buffer, they used a server-based algorithm BESTsel. The result indicated that RI-PSMa3 peptide displayed an equal proportion of alpha-helical content with the native one.

[0060] Example 4: Amyloid formation of the individual peptides

[0061] Thioflavin-T (ThT) aggregation kinetics assay was used for demonstrating the formation of amyloid aggregates under the experimental conditions. In the ThT fluorescence assay for the L-PSMa3 peptide at 25 pM concentration, a very short initial lag phase was observed, followed by a rapid growth phase, and further followed by the plateau phase, indicative of equilibrium between fibril formation and fibril dissociation or fragmentation (Figure 1C). However, at an equal concentration of RI-PSMa3, no change was observed in fluorescence signal in this assay, suggesting that, unlike L-PSMa3 peptide, RI-PSMa3 does not form cross-a type amyloid fibril (Figure 10.Moreover, the visualization of the samples using scanning electron microscopy (SEM) after 14 hours of incubation revealed the presence of the amyloid formation for the L-PSMa3 peptide (Figure lE-a), but not the RI-PSMa3 peptide (Figure lE-b). Importantly, it is observed that RIPS Ma3 did not exhibit any fibril formation even at a concentration as high as 200 pM (Figure ID & lE-c).

[0062] Example 5: Inhibition of L-PSMa3 amyloid formation byRI-PSMa3

[0063] The potential inhibitory effect of RI-PSMa3 on amyloid formation of the L-PSMa3 peptide, was assessed by conducting ThT assays in presence of RI-PSMa3.

[0064] Solutions containing L-PSMa3 were incubated with an equal proportion ofRI-PSMa3 in 10 mM sodium phosphate buffer containing 150 mM NaCl at physiological pH in the presence of 200 pM of ThT for 14hours. A complete suppression of the fluorescence signal was observed (Figure 3A).

[0065] Similar results were observed at varied peptide concentration used (12.5 pM, 25 pM, or 50pM) in the assay (Figure 3A). These ThT assay results were further substantiated by SEM analysis, which visually confirmed the inhibition of amyloid formation in the presence of RI-PSMa3 (Figure 3B).

[0066] Furthermore, the inventors conducted co-incubation experiments in which L-PSMa3 peptide was combined with increasing concentration (equivalent amounts: 0.05, 0.10, 0.15, 0.25, 0.37,0.50, 0.75, 1.00) of RI-PSMa3 peptide, while maintaining aconstant L-PSMa3 concentration of 25pM relative to the untreated L-PSMa3, considered as 100% aggregation. It was observed that the presence of RI-PSMa3 dramatically reduced the level of amyloid fibrils (Figure 3C). Even with a 0.25 molar equivalent of RI-PSMa3 peptide, no increase in the ThT fluorescence signal was observed during the 14-hour incubation period. The inhibition of L-PSMa3 fibril formation by RI-PSMa3 was found to be dose-dependent. The visualization of samples by SEM further underscores the dose-dependent reduction of amyloid formation in the presence of RI-PSMa3 (Figure 3E a-d).

[0067] These experiments comprehensively demonstrate the inhibitory efficacy of RI-PSMa3 on the cross-alpha amyloid formation of the L-PSMa3 peptide, highlighting the potential of RI- PSMa3 as a valuable peptidic agent for modulating amyloid formation by L-PSMa3 peptide.

[0068] Example 6: Disaggregation potential of RI-PSM«3 on pre-formed fibrilsTo assess the potential of the RI-PSMa3 peptide to disrupt pre-formed L-PSMa3 fibrils, the inventors conducted a series of in-vitro experiments to investigate its ability to disassemble these amyloid structures. At first, the L-PSMa3 peptide were allowed to form aggregates in 10 mM sodium phosphate buffer containing 150 mM NaCl at physiological pH. The progress of the fibril formation was monitored using the ThT assay. After 4.5 hours, when the ThT fluorescence intensity reached maximum (Figure 4A), varied concentrations of RI- PSMa3 peptide were introduced to the matured fibrils, followed by continuous monitoring of the ThT fluorescence intensity for the subsequent 11 hours.

[0069] The results revealed a striking difference inthe signal behaviour. In the absence of RI-PSMa3, the ThT signal remained at the plateau, indicating the stability of the pre-formed L-PSMa3 fibrils. However, upon immediate addition of RI-PSMa3, they observed a drastic reduction of the ThT signal, indicative of the disruption of the pre-formed fibrils and oligomers in a dose-dependent manner (Figure 4A). Notably, the most significant reduction of the ThT signal, approximately 80% disaggregation, was observed when a 1.5-fold excess of RI-PSMa3 was introduced (Figure 4B), which was further supported by the SEManalysis (Figure 4C). In untreated preformed L-PSMa3 assemblies, a fibrillar network characteristic of amyloid presence was evident (Figure 4C-a). However, in the presence of RI-PSMa3, the fibril density gradually diminished (Figure 4C b-d). The magnitude of this effect varied depending on the molar equivalent of the RI-PSMa3 peptide used in the study.

[0070] Interestingly, in the presence of one equivalent of RI-PSMa3 no trace of fibrils was detectable, suggesting an apparent transformation of the preformed fibrils into smaller soluble oligomers or monomers. These smaller species were not detectable by ThT assay, further emphasizing the disaggregation potential of RI-PSMa3.

[0071] Example 7: Study of dynamic combinatorial disulphide bond formation chemistry

[0072] To investigate the binding mode and affinity of the native L-PSMa3 and RI-PSMa3 peptides, the inventors used the dynamic combinatorial disulphide bond formation chemistry. Generally, the extent of disulphide bond formationbetween two peptides would reflect the extent of interaction between them, forming the thermodynamically stable homo- or hetero-dimer.

[0073] For this study, the inventors designed and chemically synthesized peptides with cysteine residueeither at the C-terminus or the N-terminus, enabling the use of dynamic disulfide bond formation chemistry with LC-MS as the read-out. Since all analogs share the same amino acid compositions, an additional (arbitrary) residue was introduced at either the N- or C-terminus of each peptide to distinguish them using mass- spectrometry (Figure 5A).

[0074] A schematic of the dynamic combinatorial disulphide bond formation chemistry has been illustrated in Figure 5B. The experiment relied on the disulphide bond formation between two peptides under GSH / GSSG mediated folding conditions (20mM phosphate, ImM GSH, 0.2mM GSSG, pH 8.0) followed by denaturation and reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with online electron spray mass spectrometry (ESI-MS) analysis.

[0075] The first three experiments in which individual peptides (Figure 5C, Expl-Exp3) were treated under redox folding conditions revealed a quantitative homo-dimer product, with traces of glutathione adduct formation (Figure 5D).

[0076] In the next two experiments (Figure 5C, Exp4-Exp5 the equimolecular amounts of both peptides was incubated under redox folding conditions. It was observed that when the C-terminal cysteine-containing peptide L-PSMa3 (Cys) and RI-PSMa3 (Cys) were incubated, the equilibrium product distribution followed mostly statistical pattern (Figure 5D,Expt. No. 4). However, when the C-terminal cysteine-containing L-PSMa3 (Cys) peptide was incubated with the N-terminal cysteine -containing (Cys) RI-PSMa3 peptide, the thermodynamic equilibrium distribution shifted nearly quantitatively towards the hetero-dimeric product (Figure 5D, Expt. No. 5).

[0077] With the help of the dynamic combinatorial disulphide bond formation chemistry described above, the inventors found that the RI-PSMa3 peptide preferentially binds with the L-PSMa3 peptide, indicating a strong interaction between the two peptides when properly oriented. The RI-PSMa3 peptide preferentially binds with the native PSMa3 peptide in an antiparallel fashion.

[0078] Example 8: Dynamic light scattering experiment for trapping of transient oligomeric species The inventors conducted Dynamic light scattering (DLS) experiments with L-PSMa3 and RI-PSMa3. In the first experiment, L-PSMa3 and RI-PSMa3 peptides were allowed to evolve independently to an equilibrium characterized by a maximally soluble higher oligomeric state. DLS data collection was done over the span of an hour, data being recorded at 10-minute intervals. Theinventors found that the population distribution of the L-PSMa3 peptide reached equilibrium, with significant enrichment of species exhibiting average hydrodynamic radii of 925.6 nm (Figure 4F). This equilibrium coexisted with minorspecies having hydrodynamic radii of 3.12 nm and 126.2 nm, indicating their participation in the complex peptide ensemble. In contrast, the population distribution of RI-PSMa3 remained skewed toward species with very low hydrodynamic radii, residing primarily at 2.0 nm. (Figure 4G)

[0079] In the subsequent experiment, the inventors observed a significant shift in the DLS profile when an equal proportion of RI-PSMa3 was added to the pre-equilibrated PSMa3 oligomeric species. The dynamics of the population distribution were monitored at four different time points within an hour (5 minutes, 15 minutes, 30 minutes, and 60 minutes). A remarkable finding was revealed during the experiment. The initially dominant population of species with hydrodynamic radii of 925.6nm underwent a profound shift, transitioning to significantly lower-order oligomers characterized by hydrodynamic radii of 342.3nm. At the same time, equilibrium was maintained with the remaining smaller species at 3.2nm, with both populations exhibiting nearly equal intensity (Figure 4H).

[0080] The dynamic light scattering (DLS) experiment of the peptide PSMa3 in the absence and presence of the RI-PSMa3 revealed that the RI- PSMa3 peptide possesses a remarkable ability to capture transient oligomeric species along the intricate cross-alpha amyloid formation pathway and drives the equilibrium towards specific lowly-populated oligomeric states, suggesting a dynamic and versatile role for RI-PSMa3 in modulating amyloid formation.

[0081] Example 9: In vitro experiment showing dose- dependent MRSA biofilm inhibition by RI-PSM«3 peptide

[0082] In vitro study was performed to show the inhibition of the formation of the extracellular polymeric substance (EPS) matrix and, dismantling the mature EPS matrix within S. aureus biofilms. A substantial reduction in the biofilm mass in the bacterial population (MRSA strain ATCC 43300) was observed in a crystal-violet staining assay when RI-PSMa3 peptide was applied to a matured biofilm- containing bacterial colony in a dose-dependent manner (Figure 6A). The RI-PSMa3 at 150pg / ml led to -50% inhibition in the biofilm biomass formation. This investigation serves asa compelling demonstration of the singular anti-biofilm potential exhibited by the RI-PSMa3 peptide.

[0083] Further, the SEM analysis of MRSA biofilm was conducted which revealed the inhibitory effect of RI-PSMa3 on >S’. aureus biofilm formation. Fewer bacterial cells and less extra-cellular polymeric substance (EPS) accumulation on glass coverslips(F7^«re 6B) were observed when treated with of RI- PSMa3. These results are in agreement with the results obtained from the crystal violet spectrophotometry assay.

[0084] Thus, the above in-vitro experiment established that the RI-PSMa3 peptide can disperse MRSA biofilm biomass.

[0085] While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the above teachings. Modification is possible. Accordingly, the spirit of the present invention should be understood in accordance with the claims set forth below, and all of its equivalents will fall within the scope of the present invention.

[0086] Advantages of the invention:

[0087] • The retro-inverso PSMa3 (RI-PSMa3) peptide, can be used as a therapeutic agent for treating biofilm-associated infections, particularly those caused by Staphylococcus aureus (e g., MRSA).

[0088] • RI-PSMa3 peptide, itself does not form fibrils even at higher concentrations. Therefore the peptide can be effectively used to inhibit the amyloid fibril formation of PSMa3 peptide uses higher doses.

[0089] • RI- PSMa3 can be used in combination with conventional antibiotics or disinfectants to increase their efficacy.

[0090] • The peptide could be applied as a coating to medical devices (e.g. catheters, implants, prostheses) to prevent biofilm formation and associated infections,

[0091] • RI- PSMa3, particularly useful in healthcare settings to reduce the incidence of devicerelated infections.• RI-PSMa3 could be incorporated into wound dressings, gels, or sprays to treat and prevent biofilm-related infections in wounds, particularly in burn victims, diabetics, or those with chronic wounds.

[0092] • The RI-PSMa3 peptide could be formulated into new types of disinfectants or surface disinfectants used in healthcare, food processing, and other industries where bacterial biofilms pose a significant risk.

[0093] • RI-PSMa3 peptide of the invention is capable of enhancing patient outcome, decrease in antimicrobial resistance, and boosting the efficacy of current antimicrobial medicines by inhibiting the production of amyloid fibrils and dissolving pre-existing biofilms.

[0094] • Since biofilms are known to shield bacteria from standard therapies, the RI-PSMa3 peptide of the invention is especially helpful in treating persistent and recurring infections.

[0095] Y1

Claims

Claims:

1. A peptide of SEQ ID No. 1 or an analogue thereof or a sequence >60% similar thereof, said pepide is capable of inhibiting and disintegrating bacterial biofilm / fibril formation, wherein the peptide is a retro-inverso peptide.

2. The peptide as claimed in claim 1, wherein the peptide is retro inverse phenol soluble modulins alpha-3 (RI-PSMa3).

3. The peptide as claimed in claim 1, wherein the sequence of the peptide consists of nonnatural amino acids.

4. The peptide as claimed in claim 1, wherein the secondary structure of the peptide is alphahelical.

5. The peptide as claimed in claim 1, wherein the peptide acts on the biofilm of Staphylococcus aureus and stops the biofilm formation and disintegrates pre-formed matured biofilms.

6. The peptide as claimed in claim 1, wherein the peptide disintegrates the biofilm by targeting the amyloid fibrils within the biofilm matrix.

7. The peptide as claimed in claim 1, wherein, the peptide selectively acts on the cross-a amyloid formation of PSMa3 peptide.

8. The peptide as claimed in claim 1, wherein the peptide binds selectively to the native PSMa3 in an antiparallel fashion.

9. A composition comprising the peptide as claimed in claim 1 along with pharmacetually acceptable additves and carriers.

10. A surface disinfectant comprising the peptide as claimed in claim 1 along with pharmacetually acceptable additves, carriers and / or other disinfectant agents.

11. A method of synthesizing the peptide as claimed in claim 1, wherein the method comprises the steps of:i. coupling of amino acid Cysteine (0.25M) for 2-10 min at room temperature followed by 5-15 min at 45-65 °C on resin under an N2 atmosphere with vortex mixing using using coupling reagent and additives;ii. coupling of all other amino-acids in the SEQ ID No 1 (0.25M) for 5-10 min at 60- 70 °C under an N2 atmosphere with vortex mixing using coupling reagent and additives;iii. after every coupling cycle, carrying out Fmoc deprotection by 20% piperidine in DMF at 50 °C;iv. after completion of the coupling of all residues, using a cocktail of TFA , phenol, water, DODT, and TIPS to cleave peptides from the resin;v. evaporating TFA to reduce the solution volume;vi. precipitating the cleaved synthesized peptide with diethyl ether, and lyophilizing.

12. The method as claimed in claim 11, wherein the cocktail used for cleave peptides from the resin contains 85 % TFA, 5% phenol, 5%water, 2.5% DODT and 2.5% TIPS.

13. The method as claimed in claim 11, wherein the resin, coupling reagent and additives used are Rink amide aminomethyl, DIC and oxyma with DIEA respectively.

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