ANTIMICROBIAL

PEPTIDES

- Shveta Jaishankar
Int. Ph. D
MBGU
Sketch
• Introduction
• Structure
• Classification
• Biosynthesis
• Selectivity
• Mode of Action
• Disadvantages
• Bacterial Resistance
• Clinics & Commercialization
 INTRODUCTION
• Host Defense Peptides
• Evolutionarily conserved
• Extremely Diverse
• Produced in all classes of life
• Broad Spectrum antibiotics ( Therapeutic
Agents)
• Immunomodulators
• Resists Resistance development in microbes
 STRUCTURE
• Around 12- 50 amino acids long
• Rich in positively charged
residues
• Secondary structures are active
and they can be-
-α Helical
-β stranded
-β hair pin
-Extended
• Have both hydrophilic and
hydrophobic groups
(Amphipathic)
• Contain mostly L - isomers
Nature,Vol 415,Jan 2002,389-395
 CLASSIFICATION
• Multiple ways to classify – mostly based on structure, amino acid
sequence and net charge.

AMPs

Linear Unique Amino Lipo Macro

Anionic Cyclic Acid Containing
Cationic peptides peptides

Biochimica et Biophysica Acta 1462 (1999) 11-28,

Nat. Rev. Microbiol, Vol 3,march 2005,238-250
ANIONIC PEPTIDES
• Small peptides (750 Da),
occur in mM concentration.
• Requires Zn+2 as cofactor.
• Active against Gram +ve and
gram -ve bacteria
• Usually rich in Glu & Asp
amino acids
E.g. Maximin H5 (Amphibians),
Dermcidin ( Humans)
Dermcidin
LINEAR CATIONIC PEPTIDES
• Small ( <40 residues).
• Cationic amphipathic α helices
[In lipid environment].
• Increased helical content
correlates with increased
antimicrobial activities.
• No Cys residues
E.g. Gramicidin D(Bacteria)
Cecropins ( insects)
Magainins , Buforins
(Amphibians)
CAP18 ( Hare), LL37 (Humans)
Magainins
 PEPTIDES WITH UNUSUAL AMINO ACID LIPOPEPTIDES
COMPOSITION
• Also called peptaibols.
• Cationic peptides
• α helical structure, high
• Rich for a particular amino acid . proportion of α-amino-
• No Cys residues hence mostly isobutyric acid.
linear, others are extended • Acylated at N-terminals.
forms.
• 1,2 amino alcohol at C- Terminal.
E.g. Histatins (His; Humans)
E.g. Trichogin ( Mushroom)
Prophenins (Phe, Pro; Pigs)
Polymixin B ( Bacillus sps.)
Indolicidin ( Trp; Cattle)
Alamethicin
Drosocin (Pro, Arg; fruit fly)

Drosocin
Alamethicin
H - Gly - Lys - Pro - Arg - Pro - Tyr - Ser - Pro - Arg - Pro - Thr - Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-
Ser - His - Pro - Arg - Pro - Ile - Arg - Val - OH Val-Aib-Aib-Glu-Gln-Phl
 CYCLIC PEPTIDES
E.g.
Disulphide bond containing
peptides
-Brevinins 1,tachyplesins 2,,
Defensins - 3 (α,β,θ;Humans), Nisin
Drosomysin>3, protegrins 4
Backbone cyclised peptides • Ring formed by disulphide bonds or
-Gramicidin S, Tyrocidines by cyclisation of peptide backbone
Lantibiotics
• Can be anionic or cationic, usually
Nisin ( Lactococcus sps),
Cinnamycin (Streptomyces) have anti-parallel β sheet structure
• Maintain hydrophilic – hydrophobic
balance for antimicrobial action.
• Includes peptides with polycyclic
ring formed by thio ester bond.
(Lantibiotics)

Tyrocidines
 MACROPEPTIDES
• Can be large peptide structures (containing huge rings in
them) or can be fragments of large peptides.
• In cyclic compounds cystine knots motifs are present;
gives high rigidity to backbone.
E.g. Circulin A & B (antiviral),Kalata, cyclopsychotride.
(Rubiaceae family)
Lactoferricin (lactoferrin), Casocidin (caesin).

Lactoferricin B

Circulin A
 BIOSYNTHESIS
• In response to infections.
• Encoded by genome
-Gene product processed by
proteolysis
-Post translational modification
• Non ribosomal synthesis
• Cleavage of intact proteins
-Lactoferrin (pepsin)
-Heat denatured Egg white
Lysozyme.
- Derivatives of H2A histone’s
N-Terminal; Buforins and
parasin Microbiology and Molecular Biology Reviews, March
2006, p. 121-146, Vol. 70, No. 1
Biochimica et Biophysica Acta 1462 (1999) 11-28
 3 Important Human AMPs
CATHELICIDINS
• Produced by skin keratinocytes,
mast cells and Neutrophils.
• Has cathelin domain of about 100
residues at C – Terminal
Cathelin ( Cathepsin L (protease)
inhibitor)
• Called hCAP-18 in humans
(processed product is LL- 37)
• Part of Wound healing and
inflammation components,
induces intracellular matrix
proteoglycans.
• Has both antibacterial and
immunomodulatory functions.
J Am• Acad
Binds to LPS2005,
Dermatol, of bacteria.
52,3,381-390 LL - 37
DEFENSINS
• Produced by keratinocytes, epithelial cells.
• β sheet structure, 29 – 40 residues long.
• Have 6 – 8 Cys residues forming disulphide bonds.
• Divided into 2 subfamilies (α and β defensins) based on alignment of the
disulphide bonds.
α defensins β defensins
•S-S bonds at C 1-6, 2-5, 3-5. •S-S bonds at 1-5, 2-4, 3-6.
•Neutrophil granules, paneth cells •Skin, lung and gut epithelial cells,
neutrophils
•Increases TNF- α and IL – 1 •Chemotaxis of memory T- Cells,
expression in activated immature dendritic cells
monocytes •Induces histamine release and
•Antiviral activity against prostaglandin production
adenoviruses, HIV

•θ defensin are produced in non human primate’s leukocytes and are

cyclic octapeptides that have antibacterial and antifungal activity.
Nat. Revi. Immunol. 3, 710-720 (Sep 2003)
GRANULYSIN • Present in granules of Tc cells
and NK cells
• Belong to saposin protein
family, are cationic and
amphipathic.
• Is initially a 15kDa protein
processed to a 9 kDa product
• Acts against bacteria,
mycobacteria, fungi and
entamoebas
• Lyses variety of tumour cells
and induces apoptosis.
J. Biol Chem. April 23, 2004, 279,17834-17841
 SELECTIVITY
Act specifically only on microorganism
• Cationic property ( more positively charged)
• Presence of cholesterol and zwitterionic lipid head groups in
animal cell membranes.
• Presence of acidic phosphotidyl lipids in bacterial cell
membranes
• Transmembrane potential difference

Nature ,415 , Jan 2002,389-395

 MODE OF ACTION
• 3 main steps involved
-Attraction
-Attachment
-Permeability
-Intracellular killing
Models
-Barrel Stave Model
-Carpet Model
-Toroidal pore Model
PLoS Pathogens, 3 October 2010 , Vol.6, Issue
10, e1001067
Nature reviews microbiology, Vol 3,march 2005,238-
250
BARREL STAVE MODEL

Nature reviews microbiology, Vol 3,march

2005,238-250
CARPET MODEL

Nature reviews microbiology, Vol 3,March 2005,238-

250
TOROIDAL MODEL

Nature reviews microbiology, Vol 3,march 2005,238-

250
Intracellular Killing

Nature reviews microbiology, Vol 3,march 2005,238-

250
AMPs and DISEASES

Clinical Immunology (2010) 135, 1–11

 DISADVANTAGES
• Only 3 AMPs are commercially available that are
used as topical creams and solutions.
• This is because
- High toxicity
- Access to location
- Slow process due to tissue infiltration action
- Proteases action
- Not same in in vivo and in vitro
- Expensive
- Activity depends on serum salt conc. and pH
 BACTERIAL RESISTANCE
• By reduction in net negative
charge on cell surface (addition of
D- alanine to surface teichoic
acids).
• By increase in net positive charge
by addition of L – Lysine.
• Changes in membrane proteins
• ATP binding cassette transporters
in efflux of AMPs
• Lipid A moiety alteration (amino
arabinose, myristate, palmate
group addition.)
• Presence of capsule.
• Proteolysis by enzymes.
http://nizetlab.ucsd.edu/Research/AntimicrobialP
eptides/
 CLINICS
• Drug properties
-Good activity
-Appropriate function
-Low toxicity
-In vivo stability
-Inexpensive production
•Using the current knowledge available in the field of AMPs biotechnology firms
have began developing AMPs with improved activity, less toxicity and less resistance
profiles.
•Still at an early stage of technologic maturation, and many hurdles have yet to be
overcome

•Other than drugs activity, AMPs are being used in other biotech application such as
development of
-Transgenic plants ( fights phytopathogens)(defensin, thionins)
-Transgenic fish ( in aquaculture)(lactoferricin)
-Potential sensors and biomarkers of diseases
-Food preservation (nisin,bacteriocins)
Central
European
Journal of
Biology 2(1)
2007 1–33
 Some Drugs
Pexiganan ( MSI -78) ----- Genaera [ LOCILEX®]
- 1st antimicrobial peptide to undergo
commercial development
- 22 amino acid analogue of magainin 2
(cationic peptide)
-Broad spectrum antibacterial activity
- Topical treatment of diabetic foot ulcers
- Phase III trial involved combination therapy
- Disapproved by FDA since it was not more
affective then other drugs used to treat foot
Iseganan ( IB-367 ) ---- Intrabiotics
ulcer.
-Derived from protegrin (cationic;pig
leukocytes)
-Broad spectrum anti bacterial &
antifungal activity
-Phase II trials involved its usage as
aerosolized isogenan HCl to reduce
bacterial burden in lungs during
pulmonary infections in cyctic fibrosis
patients.
-Phase III trials failed –used as oral mouth
rinse to prevent stomatitis, ulcerative
mucositis and ventilator associated
Curr Eye Res. 2005 July ; 30(7): 505–515. pneumonia.
 OMIGANAN ( MBI – 226) ---- (Migenix)
-Analogue of indolicidin (bovine
neutrophils; cationic)
-Anti bacterial and anti fungal activity
-Prevents catheter related infections
-Phase III trials showed reduced
catheter colonization
-FDA consultation in process
MBI - 594AN ---- (Microbiologix)
-Cathelicidin based indolicidin like
novel peptide
-Treatment of acne ( P. acnes )
-Phase II trials successful, non
irritating and non toxic.
-Reduced inflammation and
lesions

Curr Eye Res. 2005 July ; 30(7): 505–515.

 XMP 629 ---- (XOMA)
-9 amino acid derivative of BPI
-antimicrobial activity against P.
acnes with minimal MBC
-Phase II trials failed with synthetic
peptide
-NEUPREX® is an injectable
formulation of rBPI .
P113 ( Dermegen) --- (Periodontix)
-12 amino acid long cationic
peptide derived from histatins.
-activity against gram positive and
negative bacteria and C. albicans.
-Used as mouthrinse to prevent
oral candidasis in HIV patients
-P113D used to prevent lung
infections by P. aeroginosa in cystic
fibrosis.
-Phase III trials
Curr Eye Res. 2005 July ; 30(7): 505–515.
DAPTOMYCIN -(Eli lilly & Co) ---(CUBICIN®)
-Cyclic anionic lipopeptide
-Treatment of complicated skin and skin
structure infections.
-Activity against gram +ve bacteria only,
GRE and MRSA.
-Activity depends on calcium.

POLYMIXNS ( B & E)
-Old cyclic cationic lipopeptides
-Anti gram negative bacterial activity
-Topical application for wounds, burns
etc
-Usually combined with broad spectrum
antibiotics ( neomycin sulphate and
bacitracin).
-Neuro and nephro toxic hence cannnot
be given systemically
Central European Journal of Biology 2(1) 2007 1–33
 GRAMICIDIN S
-Cyclic penta decapeptides
-Has alternating D and L
amino acids
-Produced by Bacillus brevis
-Anti gram +ve bacterial
activity.
-Administered topically since
it causes haemolysis in low conc.

Central European Journal of Biology 2(1) 2007 1–33

 Preclinical staged
Drugs
PLECTASIN (Novozymes)
-Fungal origin defensin
-Treats pneumonia and systemic
infections ( S. pneumonia)
-Tolerated at high doses
-It binds to Lipid II

MERSACIDIN (Novacta)
-Lantibiotic from Bacillus sps
- Has rare amino acid
methyllanthionine
-anti gram positive bacterial
activity
-Optimization of its
analogues

J Mol Med (2007) 85:317–329

 CONCLUSION
• Structurally diverse molecules exhibiting antimicrobial
activity (Broad spectrum and high potency)
• Considered part of innate immune system. Also have
immunomodulatory activities(in inflammation,
angiogenesis and tissue repair)
• Has evaded microbial resistance to some extent.
• Potential alternatives to antibiotics to treat or prevent
infections.
• Therapeutic activities include antimicrobial, antiviral,
antiparasitic and anticancer.
 FUTURE ASPECTS
• Need for effective and safe treatments for diseases
• Much yet to be learned about the physiology and
their multifunctional characteristics
• Many AMPs yet to be discovered
• Lot of optimism for future clinical practice of AMPs
• Hence, the present foci would be to identify more
of such novel peptides, re-design the existing
peptides to get rid of their toxicity and develop
novel recombinant protocols to obtain greater
yield of peptides at a lower cost