DESC:FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. TITLE: CONFORMATIONALLY RESTRICTED CATIONIC PEPTIDES AS
ANTIMICROBIAL AGENTS AGAINST MULTIDRUG RESISTANT BACTERIA
2. APPLICANT DETAILS:
(a) NAME: Biotide Solutions LLP
(b) NATIONALITY: Indian
(c) ADDRESS: Biotide Solutions LLP, B-23, Geetanjali Enclave, Opposite Aurobindo
college, New Delhi- 110017.
PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the nature of this invention and the manner in
which it is to be performed.
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CONFORMATIONALLY RESTRICTED CATIONIC PEPTIDES AS ANTIMICROBIAL
AGENTS AGAINST MULTIDRUG RESISTANT BACTERIA
FIELD OF THE INVENTION
The present invention relates to novel antimicrobial peptides (AMPs) used for treating multi-drug
resistant bacterial strains. Particularly, the present invention relates to conformationally restricted
cationic peptides exhibiting high activity against both gram-positive and gram-negative bacteria
with acceptable hemolytic activity and a longer half-life. More particularly, the present invention
discloses a method of synthesizing cationic helical antimicrobial peptides by introducing a, ß
dehydrophenylalanine, and positively charged residues in various key positions. Further, the
present invention also discloses a pharmaceutical composition used for treating multi-drugresistant
bacterial strains.
BACKGROUND AND PRIOR ART OF THE INVENTION
Antibiotic resistance has become a great concern in terms of public health, and the delayed
development of new antibiotics to hinder the growth of antibiotic resistance makes the problem
more serious. Therefore, new antimicrobial strategies or alternative drugs are required to revive
the potency of traditional antibiotics and to guard human health.
Antimicrobial peptides (AMPs) represent such a new class of antibiotics. Many researchers have
contributed to the understanding of the structure-activity relationship of AMPs. AMPs are highly
variable, short peptides with 12–100 amino acids. AMPs are positively charged, with a net charge
of +2 – +9 due to the presence of basic amino acids (Lys, Arg). AMPs possess approximately 50%
hydrophobic residues, which favours an amphipathic conformation upon interaction with
membranes.
Nowadays, more than 1000 AMPs have been identified with antimicrobial activity. The general
features of AMPs include: (1) Diversity; AMPs have been discovered in most species, from
bacteria to mammals, in addition to designed, modified, and synthesized AMPs, (2) Uniqueness;
each AMP differs from others by unique size and sequence, (3) Secondary structure; AMPs are
either present or fold into a category of secondary structures, such as a-helix or ß-sheet, (4)
Physiological significance; AMPs not only exhibit the ability to kill or inhibit pathogenic
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microorganisms but also play an important role in modulating the immune system in-vivo, (5)
Wide spectrum of killing activity; AMPs have been reported to kill or inhibit a variety of organisms
or cells, including Gram-negative and Gram-positive bacteria, viruses, protozoa, parasites, fungi,
and even cancer cells.
Traditional antibiotics generally target a particular physiological process of bacteria, such as cell
wall synthesis, DNA replication, etc.; however, due to mutations and adaptation in bacteria, the
traditional antibiotics eventually become inefficient. In contrast, most AMPs target the bacterial
cell membrane without specific receptors and thus, become an ideal approach to overcome the
resistance resulting from bacterial mutations.
There are certain concerns of AMPs in clinical applications including toxicity, immunogenicity,
drug resistance, hemolytic activity, and other side effects. These peptides can exert enormous toxic
side effects on mammalian cells in long-term use. Certain AMPs have also been reported for their
hemolytic activity. For instance, Indolicidin, a 13-residue short cationic peptide-rich with
tryptophan (Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH), exhibits a broad
spectrum of antimicrobial activity (gram-positive and gram-negative bacteria, fungi, viruses), but
exhibit a hemolytic activity that limits its clinical application.
Even though these peptides are small and don't possess any immunogenicity, but are still more or
less toxic to human cells. These peptides are also having a very low propensity of developing
resistance and have a shorter half-life. These characteristics make this kind of peptide rarely used
in drug development.
Indian Patent Application No. 1327/DEL/2010 discloses amphipathic antibacterial peptides, which
are of cationic peptides and comprise dehydrophenylalanine and tryptophan residues.
Puniti Mathur et al., in a research study published in Biopolymers. 2004 titled “Peptide design
using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins” disclosed the
incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded
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conformations: beta-turns in short peptides and 3(10)- helices in larger ones. The basic principle
for designing peptides containing a,ß-dehydrophenylalanine amino acids. However, the article
does not disclose the new conformationally restricted cationic peptides as designed & developed
by the applicant.
Indian Patent Application No. 201611008172 discloses the spontaneous self-assembly of a
dipeptide NH2-Leu- ?Phe-COOH containing a, ß-dehydrophenylalanine into a mechanically
strong and proteolytically stable hydrogel. However, the patent application does not disclose the
new conformationally restricted cationic peptides as designed & developed by the applicant.
Sarika Pathak et al., in a research study published in American Society for Microbiology, 2011
titled “Rationale-Based, De Novo Design of Dehydrophenylalanine-Containing Antibiotic
Peptides and Systematic Modification in Sequence for Enhanced Potency” disclosed an approach
to design short, nonhemolytic, potent, and broad-spectrum antibiotic peptides with increased
serum stability. These peptides were designed to attain an amphipathic structure in helical
conformations. However, the article does not disclose the new conformationally restricted cationic
peptides as designed & developed by the applicant.
US Patent No. 7563764 discloses novel antimicrobial peptides that comprise hydrophobic and
cationic residues based on monomeric tri-peptide units. These peptides exhibit high antibacterial
activity and lower hemolytic activity, with a shorter half-life. However, the patent does not disclose
the new conformationally restricted cationic peptides as designed & developed by the applicant.
However, the patent does not disclose the new conformationally restricted cationic peptides as
designed & developed by the applicant.
Indian Patent No. 270452 discloses conformationally restricting a,ß-dehydrophenylalanine residue
and its use in the design, of peptide nanotube. In particular, it’s a highly ordered and directional
tubular structure formed by the self-assembly of a conformationally constrained dipeptide, -Lphenylalanine-
a,ß-dehydrophenylalanine. However, the patent does not disclose the new
conformationally restricted cationic peptides as designed & developed by the applicant.
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Indian Patent No. 363582 discloses an amphipathic dipeptide comprising a, ß-
dehydrophenylalanine (APhe), an unnatural amino acid at the C-terminus end and a charged amino
acid (Arg- APhe (RAF, SEQ ID 1), Glu- APhe (EAF, SEQ ID 2), Lys- APhe (KAF, SEQ ID 3),
Asp- APhe (DAF, SEQ ID 4)) at the N-terminal end wherein said dipeptide forms nanoparticles.
However, the patent application does not disclose the new conformationally restricted cationic
peptides as designed & developed by the applicant.
Indian Patent Application No 1735/DEL/2008 discloses novel antimicrobial peptides that
comprise hydrophobic and cationic residues, based on monomeric tri-peptide units. The peptides
of the present invention exhibit high antibacterial activity and low hemolytic activity. However,
the patent application does not disclose the new conformationally restricted cationic peptides as
designed & developed by the applicant.
Pooja C Dewan et al., in a research study published in Biochemistry. 2009 titled “Antimicrobial
Action of Prototypic Amphipathic Cationic Decapeptides and Their Branched Dimers” disclosed
the incorporation of a,ß dehydrophenylalanine (?Phe) residue in peptides. However, the article
does not disclose the new conformationally restricted cationic peptides as designed & developed
by the applicant.
Rudresh Acharya et al., in a research study published in BMC Structural Biology, 2007 titled
“Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the
crystal structure of a chiral undecapeptide” disclosed the de novo design of peptides, substitution
with ß-dehydrophenylalanine (?Phe). The basic principle for designing peptides containing a,ß-
dehydrophenylalanine amino acids. However, the article does not disclose the new
conformationally restricted cationic peptides as designed & developed by the applicant.
Thota CK, et al., in a research study published in Nature, 2016 titled “A novel highly stable and
injectable hydrogel based on a conformationally restricted ultrashort peptide” disclosed an
ultrashort peptide containing a, ß-dehydrophenylalanine, Leu?Phe, spontaneously forms strong
and stable hydrogel under physiological conditions. The gel efficiently entrapped a number of
hydrophobic and hydrophilic drug molecules and released them in a controlled manner. The gel
entrapped and released mitoxantrone and significantly controlled tumor growth in an in-vivo
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mouse model. However, the article does not disclose the new conformationally restricted cationic
peptides as designed & developed by the applicant.
On analysing the literature pertaining to cationic peptides, there appears need in the art to provide
cationic peptides with acceptable hemolytic activity and a longer half-life.
OBJECT OF INVENTION
Accordingly, the main objective of the present invention is to provide conformationally restricted
cationic peptides used for treating multi-drug resistant bacterial strains with an acceptable
hemolytic activity and a longer half-life.
Another objective of the present invention is to provide a method of synthesizing cationic helical
cationic peptides by introducing a, ß dehydrophenylalanine in key positions, positively charged
residues to provide overall cationic nature and other amino acids having high propensity of
occurrence in natural and synthetic antimicrobial peptides to acquire helical secondary structure
and high broad spectrum antimicrobial activity with acceptable toxicity.
Further, another object of the present invention is to provide a pharmaceutical composition used
for treating multi-drug resistant bacterial strains.
SUMMARY OF THE INVENTION
Accordingly, the present invention discloses cationic helical antimicrobial peptides (cAMPs) with
a longer half-life. In other words, the present invention has designed, synthesized, and
characterized a series of conformationally restricted cationic peptides containing a, ß-
dehydrophenylalanine amino acids, and positively charged residues in key positions with a
propensity to form helical structures.
Accordingly, the present invention provides conformationally restricted cationic peptides for
treating multi-drug resistant bacterial strains with an acceptable hemolytic activity and a longer
half-life.
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In an embodiment of the present invention, the conformationally restricted cationic peptides
exhibit strong enzymatic stability towards proteases.
In another embodiment of the present invention, the conformationally restricted cationic peptides
exhibit a helical configuration.
In still another embodiment of the present invention, the conformationally restricted cationic
peptides comprising a, ß dehydrophenylalanine, and positively charged residues in various key
positions.
In an embodiment of the present invention, the conformationally restricted cationic peptides are
designed based on a known lead peptide template VS2.
In another embodiment of the present invention, the lead peptide template VS2 is:-
Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2 (Seq ID No: 1).
In still another embodiment of the present invention, the conformationally restricted cationic
peptides are designed by replacing some key positioned amino acid residues of lead peptide
template VS2 (Seq ID No: 1) with another amino acid residue having a high propensity of
occurrence in natural and synthetic antimicrobial peptides and containing the net charge and the
conformation constraining element (?Phe) as constant.
In yet another embodiment of the present invention, the conformationally restricted cationic
peptides are represented as VS2-1- VS2-32, and include
VS2-1 Ac – K – W – ?F – W – K – ?F – G – K – ?F – G – K – NH2 (Seq ID No: 3)
VS2-2 Ac – K – W – ?F – W – K – ?F – L – K – ?F – L – K – NH2 (Seq ID No: 4)
VS2-3 Ac – K – W – ?F – W – K – ?F – I – K – ?F – I – K – NH2 (Seq ID No: 5)
VS2-4 Ac – K – V – ?F – W – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 6)
VS2-5 Ac – K – W – ?F – V – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 7)
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VS2-6 Ac – K – L – ?F – L – K – ?F – V – K – ?F – V – K – NH2 (Seq ID No: 8)
VS2-8 Ac – K – L – ?F – W – K – ?F – V – K – ?F – V – K – NH2 (Seq ID No: 9)
VS2-12 Ac – K – W – ?F – K – I – ?F – K – I – ?F – I – K – NH2 (Seq ID No: 10)
VS2-11 Ac – K – W – ?F – K – W – ?F – K – L – ?F – L – K – NH2 (Seq ID No: 11)
VS2-13 Ac – K – W – ?F – K – L – ?F – K – L – ?F – L – K – NH2 (Seq ID No: 12)
VS2-14 Ac – K – V – ?F – L – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 13)
VS2-15 Ac – K – V – ?F – L – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 14)
VS2-16 Ac – R – V – ?F – W – R – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 15)
VS2-31 Ac – R – P – ?F – G – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 16)
VS2-32 Ac – R – G – ?F – P – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 17)
In an embodiment of the present invention, the conformationally restricted cationic peptides VS2-
VS5 show activity against both gram-negative and gram-positive bacterial strains with a growth
inhibition percentage of up to 100%.
In another embodiment of the present invention, the conformationally restricted cationic peptides
along with one or more pharmaceutically acceptable excipients can be used for preparing a
pharmaceutical composition in a solid, liquid dosage forms using conventional methods.
In yet another embodiment of the present invention, the pharmaceutical composition can be used
for treating bacteria including Acinetobacter baumannii, Escherichia Coli, Staphylococcus aureus,
Streptococcus pneumonia, and Bacillus cereus.
DETAILED DESCRIPTION OF THE DRAWINGS
The invention has other advantages and features which will be more readily apparent from the
following detailed description of the invention and the appended claims, when taken in conjunction
with the accompanying drawings, in which:
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Fig 1[A-G]: depict the antimicrobial activity of the new analogues in various concentrations
ranging from 5µM-150µM against bacteria Escherichia coli (E. coli).
Fig 2[A-H]: depict the antimicrobial activity of the new analogues in various concentrations
ranging from 5µM-150µM against bacteria Staphylococcus aureus (S. aureus).
Fig 3: depict the antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3, VS-
4, VS2-5 and VS2-13) in various concentrations ranging from 5µM-150µM against bacteria
Acinetobacter baumanii (A.baumanii).
Fig 4: depict the antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3, VS-
4, VS2-5 and VS2-13) in various concentrations ranging from 5µM-150µM against bacteria
Bacillus cereus (B.cereus).
Fig 5: depict the antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3, VS-
4, VS2-5 and VS2-13) in various concentrations ranging from 5µM-150µM against bacteria
Klebsiella Pneumoniae (K.pneumoniae).
Fig 6: depict the antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3, VS-
4, VS2-5 and VS2-13) in various concentrations ranging from 5µM-150µM against bacteria
Proteus vulgaris (P.vulgaris).
Fig 7: depict theantimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3, VS-
4, VS2-5 and VS2-13) in various concentrations ranging from 5µM-150µM against bacteria
Pseudomonas aeruginosa (P.aeruginosa).
Fig 8: depict the antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3, VS-
4, VS2-5 and VS2-13) in various concentrations ranging from 5µM-150µM against bacteria
Staphylococcus epidermis (S.epidermis).
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Fig 9: depict the antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3, VS-
4, VS2-5, and VS2-13) in various concentrations ranging from 5µM-150µM against bacteria
Staphylococcus haemolyticus (S.haemolyticus).
Fig 10: depict the haemolysis activity screening of VS2 and the shortlisted analogues in various
concentrations ranging from 5µM-150µM.
Fig 11: depict the RP-HPLC spectra for [A] VS2-S after 0hr of treatment with serum, [B] VS2-S
after 24hr of treatment with serum, [C] VS2-2 after 0hr of treatment with serum, [D] VS2-2 after
24hr of treatment with serum, [E] VS2-3 after 0hr of treatment with serum, [F] VS2-3 after 24hr
of treatment with serum.
Fig 1a: depict RP-HPLC profile of VS2.
Fig 2a: depict RP-HPLC profile of VS2-S.
Fig 3a: depict RP-HPLC profile of VS2-1.
Fig 4a: depict RP-HPLC profile of VS2-2.
Fig 5a: depict RP-HPLC profile of VS2-3.
Fig 6a: depict RP-HPLC profile of VS2-4.
Fig 7a: depict RP-HPLC profile of VS2-5
Fig 8a: depict RP-HPLC profile of VS2-6.
Fig 9a: depict RP-HPLC profile of VS2-8.
Fig 10a: depict RP-HPLC profile of VS2-11.
Fig 11a: depict RP-HPLC profile of VS2-11.
Figure-12 : depict RP-HPLC profile of VS2-13.
Figure-13: depict RP-HPLC profile of VS2-14.
Figure-14 : depict RP-HPLC profile of VS2-15.
Figure-15: depict RP-HPLC profile of VS2-16.
Figure-16 : depict RP-HPLC profile of VS2-31.
Figure-17 : depict RP-HPLC profile of VS2-32.
Figure-18: : depict Antimicrobial activity of the new analogues against Escherichia coli (E.
coli).
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Figure-19 : : depict Antimicrobial activity of the new analogues against Escherichia coli (E.
coli).
Figure-20: depict Antimicrobial activity of the new analogues against Escherichia coli (E. coli).
Figure-21: depict Antimicrobial activity of the new analogues against Escherichia coli (E. coli).
Figure-22: depict Antimicrobial activity of the new analogues against Escherichia coli (E. coli).
Figure-23: depict Antimicrobial activity of the new analogues against Escherichia coli (E. coli).
Figure-24 : depict Antimicrobial activity of the new analogues against Escherichia coli (E. coli).
Figure-25 : depict Antimicrobial activity of the new analogues against Escherichia coli (E. coli).
Figure-26: depict Antimicrobial activity of the new analogues against Staphylococcus Aureus (S.
Aureus).
Figure-27 : depict Antimicrobial activity of the new analogues against Staphylococcus Aureus (S.
Aureus).
Figure- 28 : depict Antimicrobial activity of the new analogues against Staphylococcus Aureus
(S.
Aureus).
Figure-29 : depict Antimicrobial activity of the new analogues against Staphylococcus Aureus (S.
Aureus).
Figure-30: depict Antimicrobial activity of the new analogues against Staphylococcus Aureus (S.
Aureus).
Figure-31: depict Antimicrobial activity of the analogues against Staphylococcus Aureus (S.
Aureus).
Figure-32: depict Antimicrobial activity of the new analogues against Staphylococcus Aureus (S.
Aureus).
Figure-33 : depict Antimicrobial activity of the new analogues against Staphylococcus Aureus (S.
Aureus).
Figure-34 : depict Haemolysis results for the shortlisted peptide analogues.
Figure-35 : depict Cellular toxicity of VS2 and the shortlisted analogues in HEK293T cells.
Figure-36 : depict Antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3,
VS-4, VS2-5 and VS2-13) against Acinetobacter Baumanii.
Figure-37: depict Antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3,
VS-4, VS2-5 and VS2-13) against Bacillus Cereus.
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Figure-38: depict Antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3,
VS-4,
VS2-5 and VS2-13) against Klebsiella Pneumoniae.
Figure-39: depict Antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3,
VS-4, VS2-5 and VS2-13) against Proteus Vulgaris.
Figure-40 : depict Antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3,
VS-4, VS2-5 and VS2-13) against Pseudomonas aeruginosa.
Figure-41 : depict Antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3,
VS-4, VS2-5 and VS2-13) against Staphylococcus epidermis.
Figure-42 : depict Antimicrobial activity of VS2 and the shortlisted analogues (VS2-1, VS2-3,
VS-4, VS2-5 and VS2-13) against Staphylococcus Haemolyticus.
Figure-43 : depict Proteolytic stability of peptide analogues in the presence of 10% serum
containing a cocktail of proteases (A) VS2-S at 0hr (B) VS2-S at 24hr of treatment with 10%
serum (C) VS2-2 at 0hr (D) VS2-2 at 24hr of treatment with 10% serum (E) VS2-3 at 0hr and (F)
VS2-3 at 24hr of treatment with 10% serum.
Figure-44: depict TEM image of gram-positive bacteria i.e., S. aureus (a) untreated (b) treated
with VS2-5 showing disruption of cell membrane after treatment with antimicrobial peptide VS2-
5.
Figure-45 : depict TEM image of gram-positive bacteria i.e. E. coli (a) untreated (b) treated with
VS2-5 showing disruption of cell membrane after treatment with antimicrobial peptide VS2-5.
Figure-46: depict Membrane permeabilization activity of cationic antimicrobial peptide VS2-5 in
gram-positive bacteria S. aureus (a) untreated cells (b) Cells treated with FITC-VS2-5 and PI
in red channel showing permeability of cell impermeable dye (PI) into the cells (c) Cells treated
with FITC-VS2-5 and PI in FITC channel showing entry of FITC-VS2-5 into the cells.
Figure-47: depict Membrane permeabilization activity of cationic antimicrobial peptide VS2-5 in
gram-negative bacteria E. coli (a) untreated cells (b) Cells treated with FITC-VS2-5 and PI in
red channel showing permeability of cell impermeable dye (PI) into the cells (c) Cells treated
with FITC-VS2-5 and PI in FITC channel showing entry of FITC-VS2-5 into the cells.
Figure-48 : depict Confocal images showing interaction of VS2-5 peptide with bacterial DNA
resulting in DNA fragmentation of gram-positive cells S. aureus (a) Control non-treated cells
showing intact DNA stained with DAPI in blue channel (b) Cells treated with FITC-VS2
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showing fragmentation of DNA stained with DAPI in blue channel (c) Cells treated with FITCVS2
showing internalisation of VS2-5 peptide inside the cells in green channel.
Figure-49 : depict Confocal images showing interaction of VS2-5 peptide with bacterial DNA
resulting in DNA fragmentation of gram-negative cells E. coli (a) Control non-treated cells
showing intact DNA stained with DAPI in blue channel (b) Cells treated with FITC-VS2
showing fragmentation of DNA stained with DAPI in blue channel (c) Cells treated with FITCVS2
showing internalisation of VS2-5 peptide inside the cells in green channel.
Figure-50 : depict Flowchart sowing the dosing of animals for in-vivo acute toxicity study.
Figure-51 : depict Bar graph representing the fold change of the proinflammatory cytokines
assessed through q-PCR for SA (Staphylococcus Aureus) (A, B and C) or PA (D, E and F) infected
cells and VS2-2 treated cells. Cells (1st bar in all graphs) and Cells with peptide (2nd bar in all
graphs) were treated as control. It is observed that the cytokine levels have reduced in the peptide
treated cells (4th bar in all graphs) when compared to the infected cells (3rd bar in all graphs).
Figure-52 Images captured through slit-lamp shows the significant reduction in the opacity in
the VS2-2 treated eye when compared to infected eye indicating the effectiveness of the
peptide.
Figure-53 Clinical scores of (Purple) infected eye and (Green) VS2-2 treated eye. Data is
presented as mean ± SEM (n=3).
Figure-54 Colony Forming Units of infected and peptide treated mice eyeballs. Data shown
represents mean ± SEM (n=3). VS2-2 treated eyes post 6-hour infection has no viable bacteria
while the eyes to which peptide was injected post 24-hour infection had drastic reduction in
bacterial load.
Figure-55 Hematoxylin and Eosin (H&E) stained eyeball sections of mice eyeballs of (A, D)
Control, (B, E) Staphylococcus aureus and Pseudomonas aeruginosa infected respectively, (C,
F) infected eye with VS2-2 treated 6-hour post infection.
Figure-56 Myeloperoxidase (MPO) stained eyeball sections of mice eyeballs of (A, D)
Control, (B, E) Staphylococcus aureus and Pseudomonas aeruginosa infected respectively, (C,
F) infected eye treated with VS2-2 6-hour post infection.
Figure-57 Glial Fibrillary Acidic Protein (GFAP) stained eyeball sections of mice eyeballs of
(A, D) Control, (B, E) Staphylococcus aureus and Pseudomonas aeruginosa infected
respectively, (C, F) infected eye with VS2-2 treated 6-hour post infection.
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DETAILED DESCRIPTION OF THE INVENTION:
While the invention has been disclosed with reference to certain embodiments, it will be
understood by those skilled in the art that various changes may be made and equivalents may be
substituted without departing from the scope of the invention. In addition, many modifications
may be made to adapt to a particular situation or material to the teachings of the invention without
departing from its scope.
Throughout the specification and claims, the following terms take the meanings explicitly
associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the"
include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings,
like numbers indicate like parts throughout the views. Additionally, a reference to the singular
includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.
The tables, figures and protocols have been represented where appropriate by conventional
representations in the drawings, showing only those specific details that are pertinent to
understanding the embodiments of the present invention so as not to obscure the disclosure with
details that will be readily apparent to those of ordinary skill in the art having benefit of the
description herein.
As used herein, the terms “conformationally restricted cationic peptides”, when used in the context
of the present invention, refer to the peptides that are rigid and do not rotate and hence maintains
the configurations.
Accordingly, to accomplish the objectives of the present invention, the inventors propose
antimicrobial peptides used for treating multi-drug-resistant bacterial strains. Accordingly, the
present invention provides conformationally restricted cationic peptides exhibiting strong
antibacterial activities and a longer half-life.
In an embodiment of the present invention, the conformationally restricted cationic peptides
contain several non-protein amino acids, further enhancing their ability to acquire helical
conformation.
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In another embodiment of the present invention, the conformationally restricted cationic peptides
comprising a, ß dehydrophenylalanine, and positively charged residues in various key positions.
In still another embodiment of the present invention, the a, ß dehydrophenylalanine not only
induces conformational constraints (preferring helical structures) in the peptide backbone but also
renders enzymatic stability towards proteases, resulting in their longer half-life compared to their
naturally occurring, saturated counterparts.
In yet another embodiment of the present invention, the conformationally restricted cationic
peptides exhibit strong enzymatic stability towards proteases.
In an embodiment of the present invention, the conformationally restricted cationic peptides are
designed based on a known lead peptide template VS2.
In another embodiment of the present invention, the lead peptide template VS2 is:-
Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2 (Seq ID No: 1).
In still another embodiment of the present invention, the conformationally restricted cationic
peptides are designed by replacing some key positioned amino acid residues of lead peptide
template VS2 (Seq ID No: 1) with another amino acid residue having a high propensity of
occurrence in natural and synthetic antimicrobial peptides and containing the net charge and the
conformation constraining element (?Phe) as constant.
VS2 template- Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2 (Seq ID No: 1)
Set –I: - Ac-K-W-?F-X-K-?F-X-K-?F-X-K-NH2
Set –II: - Ac-K-W-?F-W-K- ?F-X-K-?F-X-K-NH2
Set- III: - Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2
Wherein, X= Substitution site, and Set-I-III are the conformationally restricted cationic
peptides
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Sequences of newly designed peptide analogues:
VS2: Ac – K – W – ?F – W – K – ?F – V –K – ?F – V – K – NH2 (Main template)
VS2-S: Ac – K – W – F – W – K – F – V –K – F – V – K – NH2 (Saturated analogue)
The peptide analogues along with the saturated analogue VS2-S, including VS2, VS2-1, VS2-
2, VS2-3, VS2-4, VS2-5, VS2-6, VS2-7, VS2-8, VS2-9, VS2-10, VS2-11, VS2-12, VS2-13, VS2-
14, VS2-15, VS2-16, VS2-31 and VS2-32 were synthesized and purified and characterized by
reverse phase- high performance liquid chromatography (RP-HPLC) (Figure- 1a to Figure-17a)
and mass spectrometry.
In yet another embodiment of the present invention, the conformationally restricted cationic
peptides are represented as VS2-1-VS2-32, and include new analogues as below:
VS2-1 Ac – K – W – ?F – W – K – ?F – G – K – ?F – G – K – NH2 (Seq ID No: 3)
VS2-2 Ac – K – W – ?F – W – K – ?F – L – K – ?F – L – K – NH2 (Seq ID No: 4)
VS2-3 Ac – K – W – ?F – W – K – ?F – I – K – ?F – I – K – NH2 (Seq ID No: 5)
VS2-4 Ac – K – V – ?F – W – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 6)
VS2-5 Ac – K – W – ?F – V – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 7)
VS2-6 Ac – K – L – ?F – L – K – ?F – V – K – ?F – V – K – NH2 (Seq ID No: 8)
VS2-8 Ac – K – L – ?F – W – K – ?F – V – K – ?F – V – K – NH2 (Seq ID No: 9)
VS2-12 Ac – K – W – ?F – K – I – ?F – K – I – ?F – I – K – NH2 (Seq ID No: 10)
VS2-11 Ac – K – W – ?F – K – W – ?F – K – L – ?F – L – K – NH2 (Seq ID No: 11)
VS2-13 Ac – K – W – ?F – K – L – ?F – K – L – ?F – L – K – NH2 (Seq ID No: 12)
VS2-14 Ac – K – V – ?F – L – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 13)
VS2-15 Ac – K – V – ?F – L – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 14)
VS2-16 Ac – R – V – ?F – W – R – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 15)
17
VS2-31 Ac – R – P – ?F – G – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 16)
VS2-32 Ac – R – G – ?F – P – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 17)
In an embodiment of the present invention, the conformationally restricted cationic peptides are
undecapeptides containing at least 11 amino acid residues.
In another embodiment of the present invention, the conformationally restricted cationic peptides
are acetylated at N-terminus and amidated at C-terminus.
In still another embodiment of the present invention, the conformationally restricted cationic
peptides exhibit a helical configuration.
In yet another embodiment of the present invention, the conformationally restricted cationic
peptides show activity against both gram-negative and gram-positive bacterial strains with high
activity and acceptable toxicity.
In still another embodiment of the present invention, the conformationally restricted cationic
peptides exhibit multiple modes of action including disruption of the bacterial cell wall,
penetration into the bacterial cells, interacting with negatively charged bacterial DNA and finally
leading to damage of super coiled DNA structure and its fragmentation.
In yet another embodiment of the present invention, the conformationally restricted cationic
peptides VS2-2-VS2-5 show activity against both gram-negative and gram-positive bacterial
strains with a growth inhibition up to 100%.
In another embodiment peptides of the present invention, the conformationally restricted cationic
peptides VS2-2 and VS2-3 exhibit stronger efficiency against both gram-negative and grampositive
sensitive bacterial strains and multi-drug resistant bacterial strains compared to the parent
peptide VS2.
18
In an embodiment of the present invention, the conformationally restricted cationic peptides along
with one or more pharmaceutically acceptable excipients can be used for preparing a
pharmaceutical composition.
In another embodiment of the present invention, the pharmaceutically acceptable excipients
include binders, diluents, disintegrants, suitable natural and synthetic polymers, preservatives,
solubilizers, surfactants, starch, lubricants, suitable colors, coating agents etc.
In another embodiment of the present invention, the pharmaceutical composition is in the form of
a solid, or a liquid.
In still another embodiment of the present invention, the pharmaceutical composition is
administrated via oral and injectable routes.
In still another embodiment of the present invention, the pharmaceutical composition can be used
for treating multi-drug resistant bacterial strains.
In yet another embodiment of the present invention, the multi-drug resistant bacterial strains
include a group consisting of Acinetobacter baumannii, Escherichia Coli, Staphylococcus aureus,
Streptococcus pneumonia, and Bacillus cereus.
ADVANTAGES OF THE INVENTION
1. Relatively short sequences
2. Easy to synthesize, purify and characterize
3. Comparatively higher stability against proteolytic degradation and longer half-life
4. Relatively high biocompatibility
5. Active against both gram-negative and gram-positive bacteria
6. Activity against bacterial strains having extensive drug resistance
7. Useful for preparation of pharmaceutical composition for treating multi-drug-resistant
bacterial strains
19
EXAMPLES
The following examples, which include preferred embodiments, will serve to illustrate the practice
of this invention, it being understood that the particulars shown are by way of example and for
purpose of illustrative discussion of preferred embodiments of the invention.
Example 1a:
Design, synthesis, purification, and characterization of new antimicrobial peptides:
17 new antimicrobial peptide analogues based on a known lead peptide
template of VS2, containing ?Phe (F), were designed and synthesizedusing Fmoc- solid phase
peptide synthesis.
Design of new analogues was based on the strategies containing replacement of some key
positioned amino acid residues with amino acid residues having high propensity of occurrence
in natural and synthetic antimicrobial peptides, containing the net positive charge and the
conformation constraining element (?Phe) as constant.
VS2 template- Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2
Set -I Ac-K-W-?F-W-K-?F-X-K-?F-X-K-NH2
Set -II Ac-K-W-?F-W-K- ?F-X-K-?F-X-K-NH2
Set- III Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2
X= Substitution site
The peptide analogues along with the saturated analogue VS2-S, including VS2, VS2-1, VS2-
2, VS2-3, VS2-4, VS2-5, VS2-6, VS2-7, VS2-8, VS2-9, VS2-10, VS2-11, VS2-12, VS2-13, VS2-
14, VS2-15, VS2-16, VS2-31 and VS2-32 were synthesized and purified and characterized by
reverse phase- high performance liquid chromatography (RP-HPLC) (Figure- 1a to Figure-11a, 12
to 17) and mass spectrometry.
Example 1b: Synthesis of cationic peptides (VS2-1- VS2-32)
Following four steps were involved during the synthesis of conformationally restricted cationic
peptides.
I. Preparation of Fmoc-X-dl-threo-ß-phenylserine:
20
Fmoc-X-dl-threo-ß-phenylserine (where Fmoc is 9-fluorenylmethoxy carbonyl and X is selected
from the group consisting of Lys [Boc], Trp [Boc], Val, Leu, Ile, Gly or Pro) was synthesized by
a method of salt coupling using mixed anhydride. Fmoc amino acid (15 mmol) dissolved in 15 ml
of distilled tetrahydrofuran (THF) was activated at 10-15°C for 20 min with isobutyl chloroformate
(IBCF) and n-methylmorpholine (NMM) (15 mmol each). A precooled solution of 15 mmol of dlthreo-
ß-phenylserine made in 1 equivalent of NaOH (15 ml) was added to the above-mentioned
mixed anhydride, and the reaction mixture was stirred at room temperature overnight at room
temperature. Following evaporation of THF, a solution of saturated citric acid was added to the
aqueous solution to reach a pH 2.0. The precipitate obtained was dissolved in 100 ml ethyl acetate
and transferred to a separating funnel. Following the removal of the lower aqueous layer, the ethyl
acetate layer was washed extensively with water (3-4 times). The complete removal of citric acid
was confirmed by measuring the pH and the final solution had neutral pH. The ethyl acetate layer
was further washed with saturated NaCl brine and allowed to pass through a bed of anhydrous
sodium sulfate. Evaporation of ethyl acetate on a rotary evaporator resulted in solid dipeptide acids
i.e., Fmoc-X-dl-threo-ß-phenylserine., wherein X is selected from the group consisting of Lys
[Boc], Trp [Boc], Val, Leu, Ile, Gly or Pro).
II. Preparation of Fmoc-X-?Phe azalactone:
Fmoc-X-dl-threo-ß-phenylserine was mixed with anhydrous sodium acetate in distilled acetic
anhydride and stirred overnight. The thick slurry obtained was mixed with ice and stirred at 8 to
10°C. Following trituration, the respective yellow dipeptide azalactone were filtered on a sinter
funnel and dried to constant weight. The authenticity and purity of the azalactones were assessed
by TLC and RP-HPLC.
III. Peptide synthesis:
Peptides were synthesized as C-terminal amides using standard Fmoc chemistry on rink amide
MBHA (4-methylbenzhydrylamine hydrochloride salt) resin in the manual mode, with DIPCDI
and oxymapure as coupling agents. Couplings were carried out using DMF at a 3-fold molar
excess. Removal of Fmoc was carried out using 20% piperidine in DMF. Both the coupling of
amino acids and the Fmoc deprotection were monitored by the Kaiser test. ?Phe or ?F was
introduced into peptides as an Fmoc-X-?Phe azalactone (wherein, X is Lys [Boc], Trp [Boc], Val,
21
Leu, Ile, Gly or Pro) dipeptide block, which was allowed to couple overnight in DMF. At the
completion of assembly of the peptides, following Fmoc removal, the amino termini were
acetylated using 20% acetic anhydride in DCM. After acetylation of the peptides, the resin was
washed extensively with DMF, DCM, and methanol (3 times each) and dried in a desiccator under
vacuum.
IV. Cleavage of the peptides from resin:
The peptides were cleaved by stirring the resin in a cleavage mixture (95% TFA, 2.5% water, and
2.5% TIS) for 2 h at room temperature. The suspension was filtered using a sinter funnel, TFA
was rotary evaporated, and the peptides were precipitated by adding cold dry ether. Ether was
filtered through a sinter funnel, and the peptides on the funnel were dissolved in 10% acetic acid
and lyophilized for further characterization and use.
Example 2: Reverse phase- high performance liquid chromatography
The synthesized cationic peptides namely VS2-1, VS2-2, VS2-3, VS2-4, VS2-5, VS2-6, VS2-7,
VS2-8, VS2-9, VS2-10, VS2-11, VS2-12, VS2-13, VS2-14, VS2-15, VS2-31, and VS2-32 were
purified along with the parent VS2, using the reverse-phase-high-performance liquid
chromatography (RP-HPLC). The purified cationic peptides characterized were further screened
to determine the respective antimicrobial activity, hemolytic activity, and in-vitro efficacies.
Example 3: Antimicrobial activity screening
The antimicrobial activities of cationic peptides (VS2- VS2-32) were carried out using the broth
dilution method. The peptide analogs were screened against both gram-negative Escherichia coli
bacteria (E. coli) (ATCC-25922), as well as the gram-positive Staphylococcus aureus bacteria (S.
aureus) (ATCC-25923).
Bacterial cells grown overnight were diluted in Mueller-Hinton (MH) broth to a density of 105
CFU/ml. One hundred microliters of this culture was aliquoted into the wells of a 96-well flatbottom
microtiter plate (Costar), 90 µl of MH broth, and 10 µl of stocks of each peptide (in PBS)
was added. This mixture was incubated at 37°C in a rotary shaker incubator set at 200 RPM. After
18 h of incubation, the optical density at 600 nm (OD600) was measured using a microtiter plate
22
reader. The MIC90 values (the concentration at which 90% of bacterial growth inhibition was
observed) were calculated.
The peptides shortlisted from the preliminary antimicrobial screening were further screened for
their antimicrobial activity against a panel of gram-negative and gram-positive bacterial strains
including gram-positive-Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-3615),
Streptococcus pneumoniae (ATCC 700902), Staphylococcus haemolyticus (MTCC-3383), and
gram-negative-Klebsiella pneumoniae (MTCC-432), Acinetobacter baumannii (MTCC-9829),
Pseudomonas aerginosa (MTCC-3542), and Proteus vulgaris (MTCC-1771).
Results:
All the cationic peptides (VS2- VS2-32) were active against both gram-negative and gram-positive
bacteria which are further presented in Figure-1[A-G] andFigure-2[A-H]. From the preliminary
screening, 4 peptide analogs were taken. All the four peptide analogs (VS2-VS5) were found active
and more efficient than the parent peptide VS2. Peptide analogs (VS2-2-VS2-5,) show activity
against both gram-negative and gram-positive bacterial strains with a growth inhibition percentage
of up to 100%, MIC90 values for the same are presented in Figures-3-9. These results have
indicated a broad-spectrum antimicrobial behavior of these new peptide analogs.
MIC determination: The minimum inhibitory concentrations (MIC values) were determined for
all the peptide analogs and are shown in Figure-1[A-G] (for E. coli) and Figure -2[A-H] (for S.
aureus).
Example 4: Haemolysis activity screening
The hemolysis activity assays were performed with all the cationic peptides (VS2-2-VS2-5) in
various concentrations ranging from 5 µM-150 µM to determine the respective in-vitro toxicity.
Human blood in 10% citrate phosphate dextrose was obtained from the Rotary Blood Bank, New
Delhi, India. Red blood cells (RBCs) were harvested by centrifugation at 1,000 × g for 5 min.
RBCs were washed with phosphate-buffered saline (PBS) (3-5 times). The packed cell volume
obtained was used to make a 0.8% (vol/vol) suspension in PBS; 100 µl of the RBC suspension was
transferred to each well of a 96-well microtiter plate and mixed with 100 µl of peptide solution in
23
PBS at different concentrations. The microtiter plate was incubated at 37°C for 60 min and after
incubation, the plates were centrifuged at 1,000 × g for 5 min. OD at 540nm was measured using
a microtiter plate reader to determine RBC lysis. Cells incubated with PBS alone were used as the
negative control, and RBCs lysed using 0.1% Triton X-100 were used as a positive control with
100% lysis.
Results:
The results showed that all the cationic peptides (VS2-2-VS2-5) exhibit an acceptable hemolytic
activity. The results further depict that the newly designed and synthesized peptides exhibit lesser
hemolytic activity than the parent peptide i.e., VS2 at their 5X MIC concentration, which are
further presented in Figure-10.
Example 5: Proteolytic activity screening
The proteolytic activity assays were performed with antimicrobial peptides including VS2-2 and
VS2-3 with VS2-S (Seq ID No: 2) which is the saturated analogue of VS2 having phenylalanine
instead of a,ß-dehydrophenylalanine were treated with 10% serum containing a cocktail of
proteases for 24hr and stability was checked using RP-HPLC on C18 column.
Results:
RP-HPLC spectra of VS2-S after 0hr of treatment showed a single peak which implicated that the
peptide was in its intact form and pure. Treatment of VS2-S with serum for 24hr showed an intense
dip in the intensity of the peptide peak with the appearance of different other peaks which clearly
indicted degradation of the peptide which are further presented in Figure-11 (A-B). While in the
case of VS2-2 and VS2-3, no change in the peptide peak indicated their high stability towards
proteases, which are further presented in Figure-11 (C-F).
Example 6: In-vitro efficacy study
The peptide analogs (VS2-2-VS2-5) were tested to determine the respective in-vitro efficacies
against both drug-sensitive and extensive drug-resistant bacterial strains. The peptide analogs were
tested against various drug-sensitive bacterial strains of gram-negative character including
Acinetobacter baumannii (BAA747), Escherichia coli (ATCC25922), and gram-positive character
24
including Staphylococcus aureus (BAA1709), and Bacillus cereus (NCIM2106), and with
extensive drug-resistant bacterial strains of gram-negative character including Acinetobacter
baumannii (BAA 1605), and Escherichia Coli (Clinical isolate), and gram-positive character
including Staphylococcus aureus (ATCC 43300), and Streptococcus pneumonia (ATCC 700902).
Results:
The results showed that the newly designed and synthesized peptide analogs (VS2-2-VS2-5) are
much more active than the parent peptide VS2. The analog peptides VS2-2 and VS2-3 also exhibit
greater efficiency against both gram-negative and gram-positive sensitive bacterial strains as well
as multi-drug resistant bacterial strains compared to that of the parent peptide VS2. (Table 1-3).
S.
No
Organism MIC (µM)
Peptide-
A
(VS2)
Peptide
-
B
(VS2-
2)
Peptid
e-
C
(VS2-
3)
Peptid
e-
D
(VS2-
4)
Peptide
-
E
(VS2-
5)
Vanco
mycin
Levoflox
acin
1. Acinetobacter
baumannii
20 20 10 30 30 >21.5 0.34
2. Escherichia
coli
20 20 10 20 30 >21.5 0.08
3. Staphylococcu
s
aureus
15 5 10 20 30 0.67 0.69
4. Bacillus cereus 5 5 5 5 5 0.33 0.17
Table-1: Antimicrobial activity against drug sensitive bacterial strains
Sl. No Organism MIC (µM)
Peptide
-
A
(VS2)
Peptide
-
B
(VS2-
2)
Peptide
-
C
(VS2-
3)
Peptide
-
D
(VS2-
4)
Peptide
-
E
(VS2-
5)
Vancomyci
n
Levofloxaci
n
1. Acinetoba
cter
baumanni
i
15 10 10 30 30 0.19 50
25
2. Escherich
ia
coli
(Clinical
isolate)
15 10 10 30 40 0.19 =0.39
Table-2: Antimicrobial activity against gram-negative bacterial strains having multi-drug
resistance.
Sl.
N
o
Organism MIC (µM)
Peptid
e-
A
(VS2)
Peptid
e-
B
(VS2-
2)
Peptid
e-
C
(VS2-
3)
Peptid
e-
D
(VS2-
4)
Peptid
e-
E
(VS2-
5)
Vancomyc
in
Levofloxac
in
1. Staphylococc
us
aureus
20 =5 10 30 40 0.78 6.25
2. Streptococcu
s
peumoniae
40 20 20 40 70 0.19 1.56
Table-3: Antimicrobial activity against gram-positive bacterial strains having extensive drug
resistance.
Example 7:
Screening for in-vitro antimicrobial activity and haemolysis of peptide-based antimicrobials:
Evaluation of antimicrobial activity of peptide analogues was carried out using broth dilution
method against both gram-negative (Escherichia coli, E. coli) as well as gram-positive
(Staphylococcus aureus, S. aureus) bacteria. Minimum inhibitory concentrations (MIC values)
were determined for all the peptide analogues and are shown in Figure-1a to Figure-7a (for E. coli)
and Figure-18 to Figure-33 (for S. Aureus). Results of in-vitro antimicrobial activity experiments
showed that all the new helical cationic peptide analogues synthesized were active against both
gram-negative and gram-positive bacteria.
Next, the haemolysis activity assays were performed for all the peptide analogues to determine
their in-vitro toxicity and results are presented in Figure-34.
Example 8:
Screening for in-vitro toxicity and antimicrobial activity of 4 selected peptide-based
antimicrobials against a large panel of gram-positive and gram-negative bacteria:
26
Based on in-vitro antimicrobial activity screening and haemolysis studies, 4 analogues were down
selected for further studies and cellular toxicity studies were carried out using HEK293T cells for
these down selected analogues. Results of cellular toxicity studies are presented in Figure-35. To
further confirm antimicrobial activities of the four shortlisted peptide analogues, these peptides
were again screened for their antimicrobial activity against a panel of gram-negative and grampositive
bacterial strains including gram positive- Bacillus Cereus, Staphyllococcus Epidermis,
Streptococcus Pneumoniae, Staphyllococcus Haemolyticus and gram-negative- Klebsiella
Pneumoniae, Acinobacter Baumannii, Pseudomonas Aerginosa, Proteus Vulgaris.
All the four peptide analogues were found active and more efficient than the parent peptide i.e.VS2
against both gram-negative and gram-positive bacterial strains and the results are presented in the
Figure-36 to Figure-42. These results have clearly indicated a broad-spectrum antimicrobial
behaviour of these new peptide analogues.
Example 9:
Screening for antimicrobial activity for down selected peptide analogues at a contract
research organisation’s (CRO) site (M/s Anthem Biosciences Pvt Ltd):
Next, the four down-selected peptide analogues, VS2-2, VS2-3, VS2-4 and VS2-5 along with
the parent template VS2 for comparison were screened for their antimicrobial activities against
drug sensitive as well as multi-drug resistant and extensively drug resistant gram-negative and
gram-positive bacterial strains in an industrial setup at M/s Anthem Biosciences Pvt Ltd,
Bangalore as per the CLSI guidelines by Micro broth dilution method. Results of these studies
are provided in Table-3 to Table-8.
Table-3 Compiled MIC (minimum inhibitory concentration) results for VS2 and its 4 down
selected analogues, VS2-2, VS2-3, VS2-4 and VS2-5 against drug sensitive gram-negative and
gram-positive bacterial strains.
S. No. Organism Type MIC (µM)
VS2 VS2-
2
VS2-3 VS2-4 VS2-5
27
1 Acinetobacter
baumannii
BAA747
Gramnegative,
Drug
sensitive
20 10 30 30 30
2 Escherichia
coli
ATCC25922
Gramnegative,
Drug
sensitive
20 10 10 20 30
3 Staphylococcus
aureus
BAA1709
Grampositive,
Drug
sensitive
15 5 10 20 30
4 Bacillus cereus
NCIM2106
Grampositive,
Drug
sensitive
5 5 5 5 5
Table-4 Compiled MBC (minimum bactericidal concentration) results for VS2 and its 4
down selected analogues, VS2-2, VS2-3, VS2-4 and VS2-5 against drug sensitive gramnegative
and gram-positive bacterial strains.
Table-5 Compiled MIC (minimum inhibitory concentration) results for VS2 and its 4 down
selected analogues, VS2-2, VS2-3, VS2-4 and VS2-5 against multi-drug resistant gram
negative and gram-positive bacterial strains.
28
Table-6 Compiled MBC (minimum bactericidal concentration) results for VS2 and its 4
down selected analogues, VS2-2, VS2-3, VS2-4 and VS2-5 against multi-drug resistant gram
negative and gram-positive bacterial strains.
S. No. Organism Type
MBC (µM)
VS2 VS2-2 VS2-3 VS2-4 VS2-5
1.
Acinetobacter
baumannii
BAA 1605
Gram-negative, Multidrug
resistance
20 10 10 30 30
2. Escherichia coli,
Clinical
Gram-negative, Multidrug
resistance
30 20 15 40 50
3.
Staphylococcus
aureus
ATCC 43300
Gram-positive, Multidrug
resistance
20 < 5 10 30 50
4.
Streptococcus
pneumoniae
ATCC 700902
Gram-positive, Multidrug
resistance
50 20 30 70 100
Table-7 Compiled MIC (minimum inhibitory concentration) results for VS2 and its 4 down
selected analogues, VS2-2, VS2-3, VS2-4 and VS2-5 against extensive-drug resistant gram
negative and gram-positive bacterial strains.
29
S.
No
Organism Type
MIC (µM)
Meropenem
Vancomycin
Colistin
VS2 VS2-2 VS2-3 VS2-4 VS2-5
1.
Klebsiella
pneumoniae
(NDM)
Gramnegative,
Extensivedrug
resistance
15 5 5 20 50 25 1.563 >25
2.
Staphylococ
cus aureus
(Reduced
vancomycin
susceptibilit
y)
Grampositive,
Extensivedrug
resistance
30 15 20 30 70 > 200 > 200 0.098
Table-8 Compiled MBC (minimum bactericidal concentration) results for VS2 and its 4
down selected analogues, VS2-2, VS2-3, VS2-4 and VS2-5 against extensive-drug resistant
gram negative and gram-positive bacterial strains.
S.
No
Organism Type
MIC (µM)
Meropenem
Vancomycin
Colistin
VS2 VS2-2 VS2-3 VS2-4 VS2-5
1.
Klebsiella
pneumoniae
(NDM)
Gramnegative,
Extensivedrug
resistance
15 5 10 20 50 50 1.563 >25
2.
Staphylococ
cus aureus
(Reduced
vancomycin
susceptibilit
y)
Grampositive,
Extensivedrug
resistance
30 15 20 50 70 >200 >200 0.195
Results of these studies have clearly have indicated that all the four shortlisted VS2 analogues
are active against both gram-positive as well as gram-negative bacterial strains in industrial
setup as well. The peptides were found active against both drug-sensitive, drug resistant and
extensive drug resistant gram-positive and gram-negative bacterial strains.
30
Example 10:
Proteolytic stability of peptide analogues:
The proteolytic activity assays were performed with antimicrobial peptides including VS2-2
and VS2-3 with VS2-S which is the saturated analogue of VS2 having phenylalanine instead
of a,ß-dehydrophenylalanine were treated with 10% serum containing a cocktail of proteases
for 24hr and stability was checked using RP-HPLC on C18 column.
RP-HPLC spectra of VS2-S after 0hr of treatment showed a single peak which implicated that
the peptide was in its intact form and pure. Treatment of VS2-S with serum for 24hr showed
an intense dip in the intensity of the peptide peak with the appearance of different other peaks
which clearly indicted degradation of the peptide which are further presented in Figure-43 (AB).
While in the case of VS2-2 and VS2-3, no change in the peptide peak indicated their high
stability towards proteases, which are further presented in Figure-43 (C-F).
Example 11:
Mechanism of action studies for the antimicrobial peptides:
In this, cell wall disruption, peptide induced cell membrane permeation and DNA binding and
fragmentation assays were carried out. VS2-5 was taken as a representative peptide for the
mechanism of action studies. Transmission electron microscopy (TEM) images of bacterial cells
(both S. aureus and E. coli) treated with VS2-5 showed disruption of bacterial cell wall indicating
high cell wall disruption capability of antimicrobial peptide VS2-5 (Figure-44 to Figure-45).
Membrane permeabilizing activity of VS2-5 was determined using propidium iodide (PI) assay.
Confocal images of cells treated with cationic antimicrobial peptide VS2-5 showed permeation of
cell impermeable dye PI into the cells both gram-positive as well as gram negative cells indicating
high membrane permeabilization activity of VS2-5 peptide analogues (Figure-46 to Figure-47).
Next, the effect of VS2-5 peptide on DNA inside the bacterial cells was investigated by staining
of nucleic acids using 4',6-diamidino-2-phenylindole dye (DAPI) which binds strongly to adenine–
thymine rich regions in DNA and give fluorescence. Confocal images of bacterial cells, both gramnegative
as well as gram-negative cells, treated with VS-5 peptide showed fragmentation of the
DNA inside the cells representing the interaction of VS2-5 peptide with the DNA and causing
DNA damage (Figure-48 to Figure-49). These results clearly showed that these novel antimicrobial
31
peptides act through multiple mode of mechanism of actions and this will be very difficult for
bacteria to recover from these changes and gain resistant against these compounds.
Example 12:
In-vivo toxicity and maximum tolerated dose (MTD) determination studies:
The acute in-vivo toxicity studies were carried out in accordance to OECD Guidelines-423. For
this, animals were fasted prior to dosing (with the mouse, food but not water was withheld for
3-4 hours). Following the period of fasting, the animals were weighed and the test peptides
namely VS2-2, VS2-3 and VS2-5 were be administered intravenously at predetermined doses.
After the test peptides were administered, food was withheld for a further 1-2 hours. The dose
level used as the starting dose was 50 mg/kg body weight (according to literature e.g., maximum
tolerated dose for vancomycin, a glycopeptide) for intravenous route and 300 mg/kg for
subcutaneous route. Dose escalation study was carried out as per the flow charts given below in
Figure-50.
The results of in-vivo acute toxicity and maximum tolerated dose (MTD) studies on 3
shortlisted VS2 analogues namely VS2-2, VS2-3 and VS2-5 for intravenous route (i.v.) are
presented in the table below in Table-9:
These results clearly underscore that the new shortlisted VS2 analogues i.e., VS2-2, VS2-3 and
VS2-5 are much safer than the parent peptide i.e., VS2 whose MTD was 2mg/kg.
32
Results of in-vivo toxicity and maximum tolerated dose (MTD) studies on 3 shortlisted VS2
analogues namely VS2-2, VS2-3 and VS2-5 for subcutaneous route (s.c.) are presented in the table
below in Table-10:
These results indicate that the new VS2 analogues can also be injected through s.c. route at
higher concentrations as depot formulation.
In-vivo safety and toxicity studies were also carried out for topical use in eyes with an industrial
partner i.e., SIPRA Laboratories, Hyderabad. In-vivo acute eye irritation studies for the one
most active peptides i.e., VS2-2 were carried out in New Zealand white rabbits in compliance
with OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November
26th, 1997 by decision of the OECD Council [C(97)186/Final] at SIPRA laboratories. Results
showed that both VS2-2 was completely safe at 0.4% peptide concentration. No eye irritation or
any other observable toxicity was observed at this concentration for VS2-2 (Table-11 to Table-18)
33
34
35
Example 13:
In-vivo efficacy study using drug sensitive bacterial strain in mouse thigh infection
model:
As proof of principle, in-vivo efficacy studies were carried out using gram-positive bacterial (drugsensitive)
thigh infection model in Balb/c mice. Both the administration routes i.e., i.v. and s.c.
were explored for the screening of in-vivo efficacy of peptide analogs. Based on in-vitro
antimicrobial activity and in-vivo toxicity studies two VS2 analogs namely VS2-2 and VS2-3 were
shortlisted for in-vivo efficacy studies using the bacterial thigh infection model.
For this, staphylococcus aureus bacterial (1X106) cells were injected in the thigh (i.m. injection)
of Balb/c neutropenic mice to create thigh infection. Post 2hr of bacterial inoculation, treatment
was initiated using both i.v. and s.c. routes for PBS (negative control), VS2-2, VS2- 3 and
vancomycin (positive control). After 24hr of treatment thigh tissues were excised, homogenized
in PBS, and plated on an agar plate. The next day, colonies grown on agar plates in different groups
were counted. Results of in-vivo efficacy study showed that both VS2-2 and VS2-3 were
completely active and controlled bacterial growth in the thigh as good as vancomycin. The new
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analogues, VS2-2 and VS2-3 were found active upon treatment through i.v. as well as s.c. route of
administration.
Example 14:
In-vivo efficacy study using extensively drug resistant bacterial strain in mouse thigh infection
model:
We investigated in-vivo efficacy of these peptides using mouse thigh infection model (infected
with extensively-drug resistant bacterial strains, Staphylococcus aureus, ATCC 700699, grampositive,
reduced susceptibility towards last resort of antibiotic available for multi-drug resistant
gram-negative bacteria i.e., vancomycin and Klebsiella pneumoniae, NDMBAA2146, gramnegative,
resistant to almost all antibiotics in clinic). Thigh infection in neutropenic mice were
created and treated with antibacterial peptides VS2-2 and VS2-3. The Inventors observed up to
0.52 log growth reduction in the case of Staphylococcus aureus and 0.12 log reduction in
Klebsiella pneumoniae. These results clearly underscored the potential of these newly designed
peptide based antimicrobials for their further development.
Example 15:
In-vitro toxicity and efficacy, and in-vivo efficacy studies using clinically isolated bacterial
strains:
We also have screened for antimicrobial activity of shortlisted peptides against clinically isolated
drug-resistant bacterial strains. All the four shortlisted peptides including VS2-2, VS2- 3, VS2-4,
and VS2-5 along with the parent peptide, VS2, were tested for their antimicrobial efficacy. The
results of antimicrobial activity against a panel of clinically isolated bacterial strains are presented
below in Table-19.
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These results clearly indicated that the newly designed analogues were active against these
clinically isolated drug resistant bacterial strains. Out of four shortlisted VS2 analogous, two
namely VS2-2 and VS2-3 had much higher antibacterial activity against these microbial strains
than the other two analogues i.e., VS2-4, VS2-5, and the parent peptide VS2.
Example 16: Assessing the efficacy of peptides in in-vitro on retinal cells (ARPE- 19) and invivo
in C57BL/6 mice in Bacterial Endophthalmitis model by intravitreal route
(i) Bacterial Strains and Culture condition: The bacterial strains used in the study were clinical
isolates from the vitreous of patients diagnosed clinically with infectious endophthalmitis after
routine microbiological work-up and antibiotic susceptibility testing by ViTEK 2 and E-test. The
chosen strains were Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus
(Gram-positive). Following 12-to-18- hour incubation, 1-2 colonies were transferred to 1 mL of
BHI broth to achieve 0.5 Mcfarland (1.5×108 cells/ml). MOI and CFU were calculated
accordingly. For in-vitro experiments, retinal cells (ARPE-19) were infected with 10:1 MOI of
bacteria and for in-vivo experiment, 5000 CFU/µL was resuspended in sterile BHI broth.
(ii) In-vitro experiments: ARPE-19 cells were seeded onto 6-well culture dish and was grown to
confluency (90%). The growth media was replaced by incomplete media and the cells were
infected with 10:1 MOI of Staphylococcus aureus and Pseudomonas aeruginosa independently
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and incubated for an hour to establish infection in the cells. After an hour, 1µg of the most active
peptide, VS2-2, was added to the appropriate wells and was continuously monitored for bacterial
reduction. After 6 hours of post-infection, the supernatant was collected and stored for further use.
Cells were lysed in RLT and betamercaptoethanol for q-PCR analysis of proinflammatory
cytokines (IL6, IL8 and TNFa) level (Figure-51).
(iii) In-vivo experimental endophthalmitis model: Both male and female C57BL/6J mice (8
weeks of age; Sipra Labs, Hyderabad) were used in these studies. All animals were maintained
according to institutional guidelines and the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. Mice were anesthetized with a mixture of ketamine and
xylazine. Eyes were cleaned with betadine 10% solution (Win- Medicare Pvt Ltd, India) and
topical anaesthetic (0.5% proparacaine HCl (Ophthetic Allergan, Hormigueros, Puerto Rico) was
instilled in each eye before injection. Intravitreal injections were performed under surgical
microscope (ZEISS Stemi 508 Stereo Microscope, Germany). Needle was inserted just posterior
to the superior limbus, and 1µl of bacterial suspension containing 5000 CFU was injected directly
into the mid-vitreous. Contralateral eyes were injected with 1X sterile phosphate-buffered saline
(PBS; surgical control) or left undisturbed (absolute control). Peptide B was injected intravitreally
6-hour post-infection in one group and 24-hour postinfection in the other group. Eyes were
clinically assessed by an ophthalmologist throughout the course of infection by hand-held slit
lamp, immunohistochemical and histologic analysis, and whole eye quantification of bacterial
growth.
(iv) Clinical Evaluation, Histopathology, and Intraocular bacterial growth: The clinical
changes occurring during experimental endophthalmitis were scored independently by masked
ophthalmologist with the aid of a hand-held slit-lamp biomicroscope (PSLAIA-11, Appasamy
associates, Chennai, India). Ocular inflammation in infected and peptide-treated eyes was scored
by a blind-folded ophthalmologist based on the scoring of anterior segment inflammation,
presence/absence of red reflex, vitreous inflammation, and retinal clarity.
Clinical changes were graded on a scale from 0 (No disease) to 4+ (Highest disease severity) based
on the criteria. Photographs of mouse eyes were taken for visualization of progression and
alleviation of disease severity. Infected eyes and peptide treated eyes were harvested 24- hours
post peptide treatment.
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The eyes were enucleated and stored in 500µl of sterile phosphate-buffered saline and
homogenized by bead beating (EzLyser Genetix Biotech Asia) with 3.2-mm stainless steel beads
for 90 seconds at maximum speed. The homogenates were serially diluted and plated on BHI agar
plates. Rest of the volume was used for multiplex ELISA to analyse cytokine (IFN?, IL1ß, IL6 and
TNFa) levels in infected and treated eyes. The total protein concentration was calculated by BCA
method and samples were diluted accordingly and 10µg of protein was taken for ELISA. For
histopathological scoring of endophthalmitis, at indicated time points, eyes were enucleated and
fixed in Davidson fixative, then embedded in paraffin for sectioning. Tissue sections (7 µm) were
cut through the pupillary-optic nerve axis at four different depths and three H&E-stained sections
from each depth were prepared. Disease severity was scored by a masked observer using defined
grading criteria ranging from 0 (no disease) to 4 (maximum disease) based on the extent of
inflammation in the cornea, anterior chamber, vitreous, and integrity of retinal architecture. H&E,
MPO (neutrophil marker) and GFAP (retinal stress marker) staining were performed. All the
processed sections were examined with an Olympus light microscope (BX51) by masked
pathologist. Sections of MPO were analysed by counting the total positive cells in the posterior
chamber of ten 400× random microscope fields per case and averaging the number.
(a) Establishing mice model endophthalmitis: We found that intravitreal injections of 5000
CFU/eye resulted in reproducible endophthalmitis as clinical and histological features of induced
endophthalmitis in mice resembles with multiple characteristics of human endophthalmitis such
as corneal haze, vitreous haze and intraocular inflammation. Disease severity measured by clinical
score, showed a time-dependent progression. Microscopic examination of the infected eyes
coincided with the clinical scores.
(b) Clinical scoring: Disease progression in infected eyes and alleviation in peptide treated eyes
was monitored, and images were captured with slit lamp and eyes were clinically scored for
corneal, vitreous and retinal clarity (Figure-52). Clinical scoring showed the drastic reduction of
inflammation and improved retinal clarity after peptide treatment (Green) in comparison to 30-
hour and 48-hour infected eyes. Infected eyes had mean clinical scoring of around 2+ to 3+ while
the peptide treated eyes had 0 to 1+ scoring where the vitreous and retinal clarity were close to
normal eyes (Figure-53).
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Quantification of bacterial colonies for mice infected with Pseudomonas aeruginosa (Gramnegative)
and Staphylococcus aureus (Gram-positive) and treated with VS2-2 intravitreally
clearly showed significant reduction in bacterial load (Figure-54). Histopathological analysis of
eye balls excised from the infected and VS2-2 treated groups clearly showed significant reduction
in inflammatory cells with bacterial load which signifies its high efficacy with no toxicological
effects (Figure-55). The eye inflammation may also be reduced by experiment using lower
concentrations of peptide with repeated treatments to completely clear bacterial infection.
We observed significant increases in the inflammatory cells in the infected eyes when compared
to the control group, however VS2-2 treated eyes showed significant reduction in inflammatory
cells with no bacteria load assed microbiologically (Figure-56).
Results of glial fibrillary acidic protein (GFAP) stained eyeball sections infected and treated with
VS2-2 clearly indicated significant reduction in brown retinal layer and therefore reduction in
retinal stress in peptide treated groups post 6hr infection (Figure-57). ,CLAIMS:We Claim:
1. A conformationally restricted cationic antimicrobial peptide/s for treating multi-drug
resistant bacterial strains comprising cationic peptides, which are undecapeptides
containing at least 11 amino acid residues with a, ß dehydrophenylalanine, and positively
charged residues, acetylated at N-terminus and amidated at C-terminus.
2. The peptide as claimed in claim 1, wherein cationic antimicrobial peptide/s are designed
by replacing key positioned amino acid residues of lead peptide template VS2 (Seq ID No:
1) with another amino acid residue having a high propensity of occurrence in natural and
synthetic antimicrobial peptides and containing the net charge and the conformation
constraining element (?Phe) as constant, where:
VS2 template- Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2 (Seq ID No: 1), wherein
Set –I: - Ac-K-W-?F-X-K-?F-X-K-?F-X-K-NH2
Set –II: - Ac-K-W-?F-W-K- ?F-X-K-?F-X-K-NH2
Set- III: - Ac-K-W-?F-W-K-?F-V-K-?F-V-K-NH2
Wherein, X= Substitution site, and Set-I-III are the conformationally restricted cationic
peptides.
3. The peptide as claimed in claim 1, wherein cationic antimicrobial peptide/s comprises
following sequence/s:
VS2-1 Ac – K – W – ?F – W – K – ?F – G – K – ?F – G – K – NH2 (Seq ID No: 3)
VS2-2 Ac – K – W – ?F – W – K – ?F – L – K – ?F – L – K – NH2 (Seq ID No: 4)
VS2-3 Ac – K – W – ?F – W – K – ?F – I – K – ?F – I – K – NH2 (Seq ID No: 5)
VS2-4 Ac – K – V – ?F – W – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 6)
VS2-5 Ac – K – W – ?F – V – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 7)
VS2-6 Ac – K – L – ?F – L – K – ?F – V – K – ?F – V – K – NH2 (Seq ID No: 8)
VS2-8 Ac – K – L – ?F – W – K – ?F – V – K – ?F – V – K – NH2 (Seq ID No: 9)
VS2-12 Ac – K – W – ?F – K – I – ?F – K – I – ?F – I – K – NH2 (Seq ID No: 10)
42
VS2-11 Ac – K – W – ?F – K – W – ?F – K – L – ?F – L – K – NH2 (Seq ID No: 11)
VS2-13 Ac – K – W – ?F – K – L – ?F – K – L – ?F – L – K – NH2 (Seq ID No: 12)
VS2-14 Ac – K – V – ?F – L – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 13)
VS2-15 Ac – K – V – ?F – L – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 14)
VS2-16 Ac – R – V – ?F – W – R – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 15)
VS2-31 Ac – R – P – ?F – G – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 16)
VS2-32 Ac – R – G – ?F – P – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 17)
4. The peptide as claimed in claim 1, wherein cationic antimicrobial peptide/s comprises
following sequence/s:
VS2-2 Ac – K – W – ?F – W – K – ?F – L – K – ?F – L – K – NH2 (Seq ID No: 4)
VS2-3 Ac – K – W – ?F – W – K – ?F – I – K – ?F – I – K – NH2 (Seq ID No: 5)
VS2-4 Ac – K – V – ?F – W – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 6)
VS2-5 Ac – K – W – ?F – V – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 7)
5. The peptide as claimed in claim 1, wherein the bacterial strains comprises gram positive
and gram negative bacteria.
6. A method of synthesising the conformationally restricted cationic antimicrobial peptides,
as claimed in claim 1 comprises:
a) synthesising the peptides as C-terminal amides based on Fmoc chemistry on rink amide
MBHA (4-methylbenzhydrylamine hydrochloride salt) resin, with DIPCDI and oxymapure
as coupling agents wherein the coupling is carried out using DMF at a 3-fold molar excess,
followed by the removal of Fmoc using 20% piperidine in DMF;
b) introduction of ?Phe or ?F into peptides as an Fmoc-X-?Phe azalactone wherein, X is
Lys [Boc], Trp [Boc], Val, Leu, Ile, Gly or Pro dipeptide block, which was allowed to
couple overnight in DMF, followed by Fmoc removal at the completion of assembly of the
peptides and acetylating the amino termini by approx. 20% acetic anhydride in DCM;
43
c) washing the resin extensively with DMF, DCM, and methanol, for approx. 3 times each
followed by drying in a desiccator under vacuum.
7. The method as claimed in claim 6, wherein Fmoc-X-dl-threo-ß-phenylserine, where Fmoc
is 9-fluorenylmethoxy carbonyl and X is selected from the group consisting of Lys [Boc], Trp
[Boc], Val, Leu, Ile, Gly or Pro, is prepared by the steps comprising:
a) dissolving approx. 15 mmol of Fmoc amino acid in 15 ml of distilled tetrahydrofuran
(THF) followed by activating at 10-15°C for 20 min with isobutyl chloroformate (IBCF)
and n-methylmorpholine (NMM) (15 mmol each);
b) adding a precooled solution of 15 mmol of dl-threo-ß-phenylserine made in 1 equivalent
of NaOH (15 ml), to the mixed anhydride obtained in step (a), followed by stirring the
reaction mixture at room temperature overnight;
c) evaporating the THF followed by addition of a solution of saturated citric acid to the
aqueous solution to achieve a pH of 2.0, to obtain a precipitate;
d) dissolving the precipitate in 100 ml ethyl acetate and transferring to a separating funnel
for the removal of the lower aqueous layer, where the ethyl acetate layer was washed
extensively with water for approx.3-4 times for the complete removal of citric acid;
e) washing the ethyl acetate layer with saturated NaCl brine and allowed to pass through a
bed of anhydrous sodium sulfate followed by evaporation of ethyl acetate on a rotary
evaporator to obtain the solid dipeptide acids comprising Fmoc-X-dl-threo-ß-
phenylserine., wherein X is selected from the group consisting of Lys [Boc], Trp [Boc],
Val, Leu, Ile, Gly or Pro).
8. The method as claimed in claim 6, wherein Fmoc-X-?Phe azalactone is prepared by the
steps of:
a) mixing Fmoc-X-dl-threo-ß-phenylserine with anhydrous sodium acetate in distilled
acetic anhydride and stirring overnight to obtain the thick slurry;
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b) mixing the slurry obtained in step (a) with ice and stirring at 8 to 10°C for titration to
obtain yellow dipeptide azalactone which was filtered on a sinter funnel and dried to
constant weight.
9. The method as claimed in claim 6, wherein the peptide is cleaved from resin by the cleavage
mixture comprising 95% TFA, 2.5% water, and 2.5% TIS, for 2 h at room temperature
followed by filtering the suspension, rotary evaporating the TFA, precipitating the peptides
by adding cold dry ether, filtering the ether, and dissolving the peptides on the funnel in 10%
acetic acid followed by lyophilization of the peptides.
10. A pharmaceutical composition comprising peptide/s, as claimed in claim 1, selected from
a group comprising of:
VS2-2 Ac – K – W – ?F – W – K – ?F – L – K – ?F – L – K – NH2 (Seq ID No: 4)
VS2-3 Ac – K – W – ?F – W – K – ?F – I – K – ?F – I – K – NH2 (Seq ID No: 5)
VS2-4 Ac – K – V – ?F – W – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 6)
VS2-5 Ac – K – W – ?F – V – K – ?F – V – K – ?F – W – K – NH2 (Seq ID No: 7)
wherein the peptide is present in an amount ranging from 5µm to 150 µm.