FIELDOF INVENTION:
The present invention relates to a antimicrobial peptide of high therapeutic value having
antj-endotoxin activity. In another aspect, the present invention is related to the
development of therapeutic approaches for treating diseases resulting from microbial
infections caused by Gram negative bacteria, Gram positive bacteria, and fungi. Also
encompassed within the scope of the invention is the development of polypeptides with
endotoxin-neutralizing capability. The present invention also articulates to the design of
a novel antimicrobial peptide on the basis of a heptad repeat sequence having Leucine,
arginine, lysine, tryptophan and a proline residue substituted in place of a single leucine
residue at an 'a' or 'd' position with high therapeutic index. Since the designer peptide
starts with leucine and arginine at the N-terminus and proline at the eighth place it is
named as LR8P (SEQ ID NO. 1)
Designer peptide: SEQ ID NO. 1
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Leu Arg Arg Leu Leu Lys Trp Pro Arg Arg Leu~Leu—Lys~Leu—Leu*
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Leu • Leucine
Arg •' Arginine
- Lys-——• Lysine
'ST Trp —• Tryptophan
Q- Pro • Proline
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BACKGROUND OF THE INVENTION:
Worldwide for last several years there have been reports of an increase in the number
of microbes that are resistant to conventional antibiotic drugs [Giedraitiene et a/,.
Medicina (Kaunas), 2011, 47, 137-46]. This has given an impetus to the research for the
discovery of new drugs and searches for alternative chemical entities that can fight
against resistant microorganisms. Antimicrobial peptides (AMPs) that have been
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1dentified~from~all1iv^^ microorganisms are
now being seen as lead molecules. These peptides are generally produced in response
to infection and injury. Being the tools of innate immunity, the AMPs are generally
produced by the stimuli provided by Gram positive, Gram negative bacteria and some
viruses. Two major classes of the cationic antimicrobial peptides are the a-helical and
the p-sheet peptides. The (3-sheet class includes cyclic peptides constrained in this
conformation either by intramolecular disulfide bonds, e.g., defensins and protegrins or
by an N-terminal to C-terminal covalent bond, e.g., gramicidin S and tyrocidines [ Papo
et a/,. Peptides, 2003, 24, 1693-703]. Unlike the p-sheet peptides, a-helical peptides
e.g., cecropins, magainins and melittins are more linear molecules that mainly exist as
disordered structures in aqueous media and become amphipathic helices upon
interaction with the hydrophobic membranes.
Not only the live bacteria but also the dead bacteria pose a serious threat to human
health. Even after the bacteria are degraded, it sheds off lipopolysaccharide which has
the ability to induce a very strong pro-inflammatory response [Mogensen et a/,. Clin
Microbiol f?ei/,2009, 22, 240-73] in the host. Most conventional drugs fail to inhibit this
response which when uncontrolled may lead to the clinical condition of sepsis: Sepsis
has very high mortality rate and the leading cause of deaths in ICUs[Martin et a/,. Expert
Rev Anti Infect Ther ,2012, 10, 701-6] against which still no proper treatment exists.
Therefore, there have been worldwide efforts to identify and develop new molecules
that can neutralize LPS-induced pro-inflammatory responses with in vivo efficacy. A
- - number of naturally occurring proteins and polypeptides have been reported to bind and
*T- neutralize LPS. These include polymyxin B (PmB), Limulus-anti-LPS factor (LALF),the
human neutrophil-derived CAP18 and bactericidal/permeability-increasing protein (BPI).
PniB and LALF detoxify LPS in vitro and provide protection against endotoxin-mediated
E lethaNty in experimental animals. However, toxicity precludes their clinical use against
Gram-negative bacterial infections. The CAP18 protein inhibits LPS responses in vitro
but currently available data are not sufficient for assessment as a potential therapeutic
agent in the treatment of sepsis. Limitations to the therapeutic utility of the BPI protein
or the truncated recombinant derivatives rBPI23 or rBPI21 in clinical endotoxic shock,
are the rapid clearance from the circulation and lowered efficacy against complex
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"2)~A"yet"an"oth"er"dbjective^f"the presenrinventioh"is~toliesign non-toxic peptides so
that they are well tolerated in the mammalian host and qualify as lead molecules
for drug development.
3) A still another objective of the present invention is to make available a novel set
of antimicrobial peptides with strong activity against both Gram positive and
Gram negative bacteria as well as with negligible toxic activity against normal
mammalian cells.
4) A yet another objective of the present invention is to develop a peptide that
efficiently neutralizes endotoxin-mediated cytokine production both in human
monocytes and mouse macrophages.
5) A still another objective of the present invention is to design.peptides with
efficacy to prevent septic shock in mice challenged with lethal doses of LPS.
6) A yet another objective of the study was to introduce novel methodology for the
design of cell-selective antimicrobial peptides with in vivo antibacterial property.
SUMMARY OF THE INVENTION:
-Accord jnglyrthe~present~m^
peptide of SEQ ID No. 1. The peptide comprises of 15 amino acid residues including,
amino acids, Leu, Arg, Lys, Trp and Pro from N terminus to C terminus, wherein the
peptides are cationic, amphipathic, alpha-helical including the respective hydrophobic
residues at the "a" and "d" positions of the heptad repeat sequence of the corresponding
designed peptide of or any functional derivative, fragment, salt or ester thereof.
It js an objective of the present invention to provide a method for preparation of
ro bioactive peptides with peptide of SEQ ID No. 1 as the active component.
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S •• .. A yet another objective of the present invention is to develop a peptide for the
?T preparation of pharmaceutical composition that efficiently inhibits bacterial and fungal
growth. '§ A still another objective of the present invention is to develop a peptide which inhibits
endotoxemia or sepsis jn therapeutically effective dosage.
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BRIErDESCRIPTIONOF THEDIAGRAMS:
Figure 1: Study of haemolysis induced by LR8P in hRBCs in dose-dependent manner.
Percentage haemolysis and concentrations of peptides (as indicated in the figure) are
given in V and X axis respectively. LR8P demonstrated -6 % haemolytic activity
respectively at a concentration of 100 pM indicative of its less cytotoxicity.
Figure 2; Estimation of cell viability by MTT assay in 3T3 murine fibroblast cells after
treatment of LR8P . The cells showed ~ 75% viability at 100 pM peptide concentrations.
Figure 3: Effect of LR8P on the production of Nitric Oxide in LPS-stimulated
macrophages. The peptides showed dose-dependent Nitric oxide inhibition in response
to 100 ng/ml of LPS in RAW 264.7 cell line.
Figure 4: Effect of LR8P on the production of TNF-a in LPS-stimulated macrophages.
The peptide showed dose-dependent TNF-a inhibition in response to 100 ng/ml of LPS
in RAW 264.7 cell line.
Figure 5 : Effect of LR8P on the production of TNF-a in LPS-stimulated macrophages.
T-he-peptide~showed-dose=dependent"TNF-a"inhibition:in"response"totOOng/mLofLPS^
in Bone Marrow-derived Macrophages (BMDMs)
Figure 6; Effect of LR8P on the production of TNF-a in LPS-stimulated human
monocytes. The peptide showed dose-dependent TNF-a inhibition in response to 100
ng/ml of LPS in human leukemia cell line THP-1.
Figure 7: Detection of binding of LR8P to LPS. Binding of LR8P to LPS was
S? determined by quantitative chromogenic Limulus amoebocyte lysate (LAL) assay. The
.2 efficiency of LR8P to bind to LPS was analyzed by estimating their ability to inhibit the
LPS mediated activation of LAL enzyme. Inhibition in substrate colour production as a
function of inhibition of LAL enzyme's activity was estimated arid the percentage LPS
2 . binding was calculated.
S: : Figure 8: Percentage inhibition of TNF-a in BALB/c mouse serum when treated with
LPS 0111;B4 (10mg/kg) iand LR8P when administered simultaneously with LPS in mice
at a concentration of 2.5 mg/kg and 5 mg/kg.
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of LPS mediated pro-inflammatory response in vivo. Survival of LR8P treated mice
when they were administered a lethal dose of LPS 0111 :B4 at a concentration of 12
mg/kg. Two different concentrations of peptides were used i.e. 5 mg/kg and 2.5 mg/kg.
Figure 10: Inhibition of CFU when mice were administered with S. aureus 25923 and
LR8P. Mice were injected intraperitoneally with mid-log phase grown S.aureus (2X 106)
and then LR8P after 30 minutes. Percentage Inhibition of CFU was determined in the
spleen, liver and kidney by plating them on MH plates at 37°C 24 hours post infection.
Each group contained 5 mice.
Figure 11: Survival assay of Mice administered intraperitoneally with P. aeruginosa
ATCC BAA 427 (2x107cfu/ml) and LR8P (7 mg/kg) The control untreated mice
received an equalvolume of PBS. Each group contained five mice.
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Abbreviations
AMP
BMDMs
BPI
CD
CFU
DCM
DIC
DIPEA
DMF
DMSO
FBS
Frhoc
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Antimicrobial Peptide
^Pl?©_JI]arfQ.VI!Lderiy.ed macrophages .
bactericidal/permeability-increasing protein
Circular dichroism
Colony forming units
Dichloromethane
Di-isoproyl carbodiimide, Differential interference contrast
Diisopropylethylamine
Dimethyl formamide
Dimethyl sulfoxide
Fetal Bovine Serum
N-(9-fluprenyl) methoxycarbonyl
2-(1H-benzotriazoie-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
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IL-6
LAL
LALF
LPS
MDR
mg
MIC .
min
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1H-6-Chlordbenzotriazole-1»yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
N' hydroxybenzotriazole
High performance liquid chromatography
Human red blood cells
Intensive Care Unit
lnterleukin-6
Limulus amebocyte lysate
Lirhulus antilipopolysaccharide (anti-LPS) factor
Lipopolysaccharides
Multi Drug Resistant
Milligram
Minimum inhibitory concentration
Minutes
Milliliters
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DETAItED DESCRIPTION OF THE INVENTION:
The present invention provides a methodology by using leucine zipper motif as a
template to design cell selective anti-bacterial and anti-endotoxin peptides. While
designing the peptides a single proline residue was inserted at the specific position of
the peptide sequence so that its self-assembly is disturbed and cytotoxicity is reduced.
Of many such designer peptides, LR8P (SEQ ID No. 1) was significantly non-haemolytic
against human red blood cells, human blood was taken from healthy volunteer and for
this purpose, we have an approval from the Institutional ethics committee no. 131
CDRI/IEC/2014/A5 regarding this experiment with human blood. The MTT [3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was performed to examine
the toxic activity of the designed peptides against murine fibroblast cell line 3T3 by a
standard procedure [Nordahl et ai} J Biol Chem, 2005, 280, 34832-9]. 3T3 cells line
was procured from the cell line repository, Tissue Culture unit, CSIR-CDRI. LR8P was
highly active against both Gram-positive and Gram negative bacteria which were
manifested in the form of its lower MIC values against these bacteria which was also
appreciably retained in the presence of serum. The antibacterial activity of the peptide
LR8P (SEQ ID No. 1) was assayed in sterile 96 well plate in 100pl final volume under
aerobic conditions [Ahmad et a/,. J.Biol. Chem, 2006,281, 22029-38]. All the bacteria
used in the antibacterial assay viz. E. co//(ATCC 9637), E. coll (ATCC 25922), B.
subtilis (ATCC 6633), B.megaterium (NRRL B-4272), Pseudomonas aeruginosa (ATCC
BAA-427), Staphylococcus aureus (ATCC 25923), Staphylococcus aureus (ATCC
700699 MRSA), Staphylococcus aureus (ATCC BAA-44 MDR Strain), Staphylococcus
aureus (ATCC 700698 MRSA), Staphylococcus aureus (ATCC 29213), Staphylococcus
• - — « • » - - « - ;
ro aureus (ATCC 33592 Gentamycin Resistant) and Klebsiella pneumoniae (ATCC
27736) were purchased from ATCC and cultured as per their recommendations.
Moreover, it also exhibited very significant activity against a number of fungi and MRSA,
•MDR and gentamycin-resistant strains of S. aureus. The in vitro antifungal activity of
^ - LR8P (SEQ ID NO, 1) was evaluated against Candida albicans ATCC 10231, Candida
parapsilosis ATCC 22019 and Cryptococcus neoformans. Candida albicans ATCC
10231 and Candida parapsilosis ATCC 22019 were purchased from ATCC and cultured
CNI as per their recommendations while Cryptococcus neoformans was provided by CSIR-
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mg/kg) showed very significant clearance of the bacteria from the key organs like
spleen, liver and kidney. Treatment of LR8P (7mg/kg) to mice administered with P.
aeruginosa (ATCC BAA-427) showed 60% survival indicating appreciable efficacy of
this peptide in rescuing it against this gram-negative bacteria. LR8P also showed the
ability to neutralize LPS-induced pro-inflammatory responses in murine macrophage cell
line, RAW 264.7, human monocytic cell line, THP-1 and rat bone marrow-derived
macrophages. Further, LR8P inhibited the production of pro-inflammatory cytokines in
LPS-treated BALB/c mice. A single dose of 2.5mg/Kg of LR8P showed 80% survival of
mice treated with a lethal dose of LPS (12 mg/kg), while 5.0 mg/Kg single dose of this
peptide showed 100% survival of the mice group treated with the same lethal dose of
LPS. RAW 264.7 were procured from the cell line repository of the tissue culture Unit,
CSIR-CDRI and bone marrow-derived macrophages (BMDMs) were isolated from
BALB/C mice. The BALB/C mice for experiments were provided by National Laboratory
Animal Center (N.L.A.C), CSIR-CDRI (Lucknow). This centre takes care of humane
•handling of animals and follows Indian Nationalscience Academy guidelines for this
purposer NLAG is registered with theCommittee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA) for breeding and experiments on
animals. All animal procedures were carried out according to the protocols, approved by
the Institutional Animal Ethics Committee No. IAEC/2010/79 and National Laboratory
Animal Centre (Lucknow). Animal protocols were adhered to the guidelines of CPCSEA
(Committee for the Purpose of Control and Supervision of Experiments on Animals;
Registration No. 34/GO/ReBi/S/99CPCSEA dated 12.3.15), Govt, of India. THP-1 cells
ro were procured from the cell line repository, tissue culture unit; CSIR-CDRI to check the
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inhibition of peptide LR8P dependent LPS mediated TNF-a production. TNF-a promotes
?T the inflammatory response, which, in turn, causes many of the clinical problems
o associated with several autoimmune disorders such as rheumatoid arthritis, ankylosing
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spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa and
refractory asthma [Popa et a/,. Lipid Res, 2007, 48, 751-62].
Altogether, the results suggest that LR8P possesses significant properties for its
development as an antimicrobial and anti-endotoxin agent.
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: j) The presence of leucine zipper-like sequence confers self-assembling
property in an AMP which often results in the enhancement of the cytotoxicity
of the peptide. Taking Leucine zipper motif as a template and inserting single
proline residue at specific 'a' or 'd' position of the sequence so that selfassembly
property of the peptide is disturbed and new molecules are
produced with low cytotoxicity.
ii) The positively charged residues were positioned in the peptide in such a way
that the peptide is amphipathic in nature. Further, the position of the charged
residues remained the same in both the peptides.
iii) Proline residue was introduced at 'a' or 'd' position of a leucine zipper
sequence since it is known as a helix breaker as well as it is an imino acid
with a secondary amino group instead of a primary amino group. Thus,
proline residue provides stability in a peptide against the proteolytic enzyme.
Proline-containing AMPs are considered as promising lead molecules in
antimicrobial drug development program.
iv) The~peptide~was~synthesize~d~by~s~olid^
purity.
Design, synthesis and purification of antimicrobial peptides:
Heptad repeat sequence is the repetition of seven amino acid residues (a to g) in which
, each "a" and "d" positions are occupied by hydrophobic residues[Yadav et al}. J Biol
ST : Chem, 2003, 278, 51023-34]. Leucine zipper sequence, first identified in DNA binding
,P- proteins, is a special case of heptad repeat sequence in which every seventh residue
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:jH ("a" position) is a leucine residue. Often the "d" positions of a leucine zipper sequence
E are also occupied by leucine/isoleucine residues. We identified and characterized a
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•;:g melittin which seems to play a crucial role in maintaining cytotoxicity of the peptide [
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Asthana et a/,. J Biol Chem, 2004, 279, 55042-50]. The substitution of leucine
residue(s) at "a" and /or "d" position(s) can alter the assembly as well as the stability of
protein/peptide containing this motif. Thus, there is ample opportunity to modulate the
11
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•^assembly of a peptideby placing different amino acids at "a" and "d" pdsitions^of the
heptad repeat sequence, Therefore, the two 15-residue designer peptides with all the
"a" and "d" positions occupied by Leucine residues except a single 'a* or 'd* position with
a single proline residue were designed. Proline is an important amino acid which
appears in the segment of protein/peptide of biological importance. It possesses a
unique structural property to create bend/nick in the secondary structure of a
peptide/protein which many times turn out to be important for its biological properties. A
bactencin analogue having proline residues showed very strong microbicidal activity
[Podda et al,.Biochim BiophysActa, 2006, 1760, 1732-40].The designer peptide, LR8P
(SEQ ID No. 1) was synthesised on solid phase (Merrifield J. Am. Chem.
Soc, 1963, 85 (14), 2149-54 ) employing glass sintered assembly and applying
nitrogen gas for continuous shaking.
Rjnk amide MBHA resin (loading capacity, 0.63 mmol/g) all the N-a Fmoc and sidechain
protected amino acids were procured from Nova biochem Switzerland. 1-
hydroxybenzotriazole (HOBT), N, N'-di-isopropylcarbodiimide (DIG), 1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU) and N, N'-diisopropylethylamine (DIPEA)
were purchased from Sigma, India. Dichloromethane, N, N' dimethylformamide (DMF)
and piperidine were purchased from Spectrochem . The peptide was purified by HPLC
using C18 reverse phase column by running a linear gradient of acetonitrile (0.1%TFA)
in water (0.1%TFA) starting from 20% and ending at 80% in 40 minutes.
EXAMPLES
'3T The following examples are given by a way of illustration only and should not construe
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g The peptide LR8P was synthesized by Solid phase peptide synthesis using manual
glass sintered assembly using RINK Amide MBHA Resin. 0.5 mg of resin was preswollen
in Dicholoromethane (DCM).Afterwards, DCM was removed and the Fmoc of
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to~rock in the20o/d"piperidine solutionfor 30-minutes. When there was 15 minutes left,-
the first amino acid from the C terminus i.e. Leucine (Leu) was activated by weighing
4.5 equivalents of amino acid combining with 0.5 M DIPEA in DMF and
HCTU/HOBt(both 0.5 M in DMF) in a glass scintillation vial. The activation was allowed
to occur for 15 minutes. After the Fmoc cleavage of resin is complete which is called
deprotection, the residual piperidine was washed out from resin with DMF, three times.
The deprotection was confirmed by doing Kaiser test which showed blue colour. The
activated amino acid/HCTU/HOBt/DIPEA solution was dumped onto the resin and rock
at room temperature for 2 hours. The resins were steadily shaken using a constant
supply of N2 gas. The attachment of first amino acid to the resin called as coupling was
again confirmed by doing Kaiser test showing colourless resins indicating that the first
amino acid was coupled. The Fmoc of the first attached amino acid was again removed
by 20 % piperidine (v/v) and next amino acid was coupled as described above.
Example 2:
Toxicity studies-Haemolytic activity and Cell viability assay.
Haemolytic activity
The haemolytic activity profile is an important parameter for any drug development
programme. Higher haemolytic activities lead to a large number of rejections of
bioactive compounds in clinical trials. Hence, the lyses of human red blood cells
(hRBCs) by the peptides were measured to determine the cytotoxicity of these peptides.
Fresh human blood was taken in a vial in the presence of an anticoagulant, ACD
(Acetate citrate dextrose), washed thrice in PBS. Peptides, dissolved in water were
ST added to the suspension of red blood corpuscle (4% final in v/v) in PBS (50mM
phosphate buffer and 0.15M NaCI, pH 7.4) to the final volume of 200 pL and incubated
at 37°C for 30 min. Then treated samples of hRBCs were centrifuged for 10 min at 800
g and monitored the released haemoglobin by measuring the absorbance of the
supernatant at 540 nm. For negative and positive controls hRBC in PBS and in 0.2%
-5 (final concentration) of Triton-X 100 were used respectively. The percentage of
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leucine zipper peptide, LR8P with a proline residue at 8th position exerted the least
damage to hRBCs membrane as evidenced by the insignificant release of haemoglobin
-even up to 100|jM peptide concentration (Figure 1).
Cell viability assay: The MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromjde] assay was performed to examine the toxic activity of the designed peptides
against murine fibroblast cell line 3T3 by a standard procedure. Fully confluent t25 flask
of 3T3 cell line was suspended in complete media and 10,000 cells were seeded in
,each well of 96 well plates and incubated for overnight in CO2 incubator for adherence.
Then complete media were substituted with incomplete media. After that, the varying
concentration of water-dissolved peptide was added and incubated for 2 hrs. ThenIO |jl
of MTT (cone. 5mg/ml) solution were added to each well and again incubated for 2 hrs.
After that incomplete media were discarded from 96 well plates and 200|jl of DMSO
.were added to each well to dissolve the crystals. The well in which no peptide was
added was considered as the control for 100% viability of cells. Readings of these
""samples werelaken at 550 nm by an ELISA reader. The viability of the peptide-treated
cells was calculated with respect to the control cells of 100% viability. The experiments
were performed in triplicates and the representative data were reported.
The results of cellviability / cytotoxicity showed a similar trend of haemolytic activity. In
brief, the peptide LR8P did not demonstrate any significant cytotoxicity against tested
mammalian cells namely hRBCs and 3T3 cells even upto 100 pM concentrations
^ (Figure:2).
a Example 3: Antibacterial assay
5 The antibacterial activity of the peptide LR8P (SEQ ID No. 1) was assayed in sterile 96
?T we|l plate in 100pl final volume under aerobic conditions. Bacteria were grown at 37°C
o with shaking at 150 rpm in appropriate growth medium to the mid-log phase as
^ determined by the optical density of 0.4-0.5 at 600 nm, diluted in media in order to attain
~105-106cfu/ml. Then 50 pi of diluted bacterial culture was added to 50 pi of water
containing 2-fold serially diluted different peptides in each well and incubated for 18-20
^ h at 37°C. The antibacterial activity of peptides as expressed by their MIC values (the
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peptide concentration that causes 100% inhibition of microbial growth), were assessed
by measuring the absorbance at 600 nm (Table: 1).
Minimum inhibitory concentration (MIC) in uM
Microorganism
E. coli (ATCC 9637)
E. coli (ATCC 25922)
B. Subtilis (ATCC 6633)
B.megaterium (NRRL B-4272)
Pseudomonas aeruginosa (ATCC BAA-427)
Staphylococcus aureus (ATCC 25923)
Staphylococcus aureus(ATCC 700699 MRSA)
Staphylococcus aureus(ATCC BAA-44 MDR
Strain*)
Staphylococcus aureus(ATCC 700698 MRSA)
Staphylococcus aureus{ATCC 29213)
Staphylococcus aureus(ATCC 33592 Gentamycin
Resistant)
Klebsiella pneumoniae (ATCC 27736)
LR8P
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Table 1
This MDR strain is resistant to ampicillin, amoxicillin/clavulanic acid,
ciprofloxacin, cephalothin, doxycycline, gentamicin, erythromycin, imipenem,
methicillin, penicillin, tetracycline, oxacillin azithromycin, clindamycin,
ceftriaxone, rifampin, amikacin and tobramycin
Example 4:
Antibacterial assay in presence of Fetal Bovine Serum (FBS):
The inactivation of antimicrobial peptides in serum has hindered efforts to develop
antimicrobial peptides for systemic use. As a consequence, less challenging conditions
15
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(e.g., phosphate buffer) are commonly used to examine antibacterial activity. To
evaluate antibacterial selectivity more appropriately, we performed the antimicrobial
assay of these two peptides against E.coli and S.aureus in the presence of 25%.FBS
and their MIC values are shown (Table: 2). Despite the decrease in their activity in the
presence of serum, LR8P (SEQ ID No. 1) appreciably retained its antibacterial
properties against the tested bacteria. The activity of peptides in serum is a
manifestation of their possible activity in in vivo.
Minimum inhibitory concentration (MIC) in uM in 25% serum
LR8P
E. coli. ATCC 25922
9±0..6
S. aureus ATCC 25923
21±0.9
Table: 2
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Antifungal Activity Assay
In this methodology, the minimum inhibitory concentration (MIC) of the peptides was
determined according to the standard microbroth dilution technique as per NCCLS
guidelines. Briefly, testing was performed in flat-bottomed 96-well tissue culture plates
in RPMI 1640 medjum buffered with MOPS (3-[N-morpholino] propanesulfonic acid) for
fungal strains. Initial inocula of fungal were maintained at 1-5 x 103 cells/mL These
plates were incubated in a moist chamber at 35 °C, and an absorbance at 492 nm was
recorded on a VersaMax microplate reader after 48 h for C. albicans ATCC 10231 and
C. parapsilosis ATCC 22019 and 72 h for C. neoformans. The MIC values are given in
Tables.
Minimum inhibitory concentration (MIC) in uM
Fungi
Candida albicans ATCC 10231
Cryptococcus neoformans
Candida parapsilosis ATCC-22019
LR8P
6.3
6.3
12.6
Table 3
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Anti-endotoxin Properties of LR8P
Inhibition of LPS mediated Nitric Oxide production: Bone marrow derived
macrophages were plated at 2.5x 105cells/well in 24-well plates and then incubated with
LPS in the presence of various concentrations of peptides. The cells with and without
LPS addition were taken as positive and negative controls for LPS-induced and basal
level of nitric oxide production respectively. The nitric oxide production was measured
by using Griess reagent, and nitric oxide in micromolar concentration was estimated
with the help of standard nitrite curve. 100pl of Griess reagent was added with 100pl of
culture supernatant to observe nitrate accumulation after respective treatment (Figure:
3). Absorbance was taken at 548 nm.
Inhibition of LPS mediated TNF-a Production: TNF-q promotes the inflammatory
response, which, in turn, causes many of the clinical problems associated with several
autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, inflammatory
bowel disease, psoriasis, hidradenitis suppurativa and refractory asthma. We examined
the ability of LR8P to inhibit the LPS mediated production of TNF-a. We observed that
both the peptides were able to inhibit the production of TNF-a in RAW 264.7 cell
line(Figure 4), bone marrow derived macrophage cells (BMDMs) (Figure 5) and human
monocytic cell line THP-1 (Figure 6).
a) Endotoxin neutralization assay (LAL assay) - The ability of LR8P (SEQ ID No. 1)
to bind LPS was assessed using a quantitative chromogenic Limulus amoebocyte
S1 lysate (LAL) with QCL-1000 (LONZA 50-647U) kit. Endotoxin neutralization
Q.
experiments were carried out following the protocols recommended by the *
?T manufacturer. Stock solutions of peptides were prepared in pyrogen-free water
'•£&£- •..,:. „ provided with the kit. Peptides at various concentrations were incubated with 1EU or
% - 0.5EU endotoxin units (EU) of LPS in a flat bottom nonpyrogenic 96-well tissue
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this mixture was then added to equal volume of LAL reagent (50pl), and the mixture
ex; was further incubated for 10 min followed by the addition of 100 |jl of LAL
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by the addition of 25% acetic acid, and the yellow color that developed due to
cleavage of the substrate was measured spectrophotometrically at 410nm. The
reduction of absorbance at 410 nm as a function of peptide concentrations is directly
proportional to the inhibition of LPS by the peptide (Figure: 7).
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Example 7
a) in vivo Anti-bacterial activity of LR8P; To check the ability of LR8P to clear the
bacterial load in vivo, BALB/c mice of 4-6 weeks were divided into four (Himedia)
was subcultured in fresh MH broth to log phase. All mice were inoculated i.p. with
Staphylococcus aureus ATCC 25923 (106 CFU/mouse) in PBS. Peptide LR8P
(7mg/kg) was administered i.p. at 1 hour post-inoculation of bacteria. The
positive and.negative controls were injected with only bacteria and PBS
respectively. After 24 hours of injection organs like spleen, liver and kidney were
taken out, homogenized in sterile PBS(500 pi). Samples were diluted
approximately 100-fold and plated on MH agar for scoring the colony numbers
(Figure 10).
b) Anti-bacterial Survival assay of mice: The survival rate among 6-week-old
BALB/c mice intraperitoneally injected with P. aeruginosa ATCC BAA 427(2x 107
cfu/ml) was examined. Following intraperitoneal injection of LR8P (single dose of
7 mg/kg), or PBS (negative control), and mortality were recorded daily over a 7-
day period (Figure: 11 ).The organs of the dead mice were taken out and
examined for their integrity to elude the chances of death due to injury.
Advantages of the Invention
As an outcome of the distinctive properties of LR8P (SEQ ID No. 1) applications
include:
1) The developed peptide of SEQ ID No. 1 possesses antibacterial, anti-fungal
and anti-endotoxin properties.
2) The peptide is useful in the treatment of bacterial infections either alone or in
combination with conventional antibiotics.
3) Since these peptides show minimal cytotoxicity, they could be potential lead
molecules for the development of antibacterial drugs.
. 4) Considering the fact that there is no preventive drug for sepsis, LR8P can be
a lead molecule in this field as it has shown excellent potential to neutralize
and inhibit the pro-inflammatory responses elicited by LPS both in in vitro and
in in vivo

We Claim:
1. An antimicrobial peptide of SEQ ID No. 1 comprising of 15 amino acid
residues including, Leucine, Arginine, Lysine, Tryptophane and Proline from
N terminus to C terminus, wherein the peptides are cationic, amphipathic,
alpha-helical including the respective hydrophobic residues at the "a" and "d"
positions of the heptad repeat sequence of the corresponding designed
peptide of or any functional derivative, fragment, salt or ester thereof.
2. The antimicrobial peptide of SEQ ID No. 1 as claimed in claim 1 inhibits the
growth of both Gram-positive and Gram negative bacteria, wherein said
Gram-negative bacteria are selected from the group consisting of E. coli and
P. aeruginosa and Gram positive bacteria selected from group containing
Staphylococcus aureus and Klebsiella pneumoniae of different ATCC strains
and their resistant versions.
3. The antimicrobial peptide of SEQ ID NO. 1 as claimed in claim 1 inhibits the
growth of fungi selected from the group consisting of Candida albicans,
Cryptococcus neoformans and Candida parapsilosis ATCC 22019.
4. A method for preparation of peptide of SEQ ID No. 1 as claimed in claim 1
by utilizing heptad repeat sequence of leucine comprises of the following
steps:
i. pre-swelling the resin (0.5 mg) in Dicholoromethane (DCM),
ii. removing the DCM from the resin,
iii. deprotecting the resin by cleaving the N-(9-fluorenyl)
methoxycarbonyl (Fmoc) from resin by adding 5 ml of 20% (v/v)
piperidine in Dimethyl formamide (DMF),
iv. allowing resin to rock in the 20% piperidine solution for nearly 30
minutes,
v. activating the first amino acid leucine (Leu) on the C terminus when
15 minutes left, in a glass scintillation vial for 15 minutes, wherein, the
leucine is 4.5 equivalents combined with 0.5 Molar
Diisopropylethylamine (DIPEA) in DMF and HCTU/HOBt (Both 0.5
Molar in DMF),
vi: -washing-off the piperidinefrom resinwithDMF for three-times after
deprotecting the resin,
vii. Dumping the activated amino acid/HCTU/HOBt/DIPEA solution as
obtained in step (v) onto the resin and allowing it to rock at room
temperature for nearly 2 hours,
viii. shaking the resins using a constant supply of nitrogen (N2) gas,
ix. coupling the first amino acid to the resin,
x. again removing the Fmoc of the first attached amino acid by 20 %
piperidine (v/v),
xi. coupling the next amino acid as described in steps (i) to (xi).
5. The method of preparation of peptide of SEQ ID No. 1 as claimed in claim 2
involves insertion of single D or L-isomers of any amino acids at key
positions of heptad repeat sequence of leucine wherein the amino acid
comprises of:
Ala, Cys ,Asp, Glu, Phe, Gjy, His, lie, Lys, Leu, Met, Asri, Pro, Gin, Arg,
Ser, Thr, Val, Trp, Tyr, Asn, Asp or Gin.
6. The antimicrobial peptide of SEQ ID No. 1 as claimed in claim 1 is useful in
the preparation of a pharmaceutical composition for inhibiting the growth of
bacteria and fungi, neutralizing lipopolysaccharide mediated proinflammatory
response both in mouse and human cell lines in vitro, inhibiting
an endotoxemia or sepsis wherein the peptide as claimed in claim 1 is an
active component.