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Peptoids that Mimic the
Structure, Function, and
Mechanism of Helical
Antimicrobial Peptides
PNAS. 2008. Vol. 105(8). 2794-2799
Antimicrobial Peptides
Are natural peptides that defend the host
organism against bacterial infection.
 They typical contain both positively
charged and hydrophobic residues.
 Interact with the cellular membrane of the
respective pathogens in a selective
manner.

Mechanisms of Action of
Antimicrobial Peptides
Carpeting
Integration
Phase
Transition
Nature 415: 389-395
Proteolysis of CAMPs

Cationic antimicrobial
peptides are very
susceptible to
proteolytic
degradation by
bacterial and host
proteases.
Nature Reviews Microbiology 4, 529-536 (July 2006)
Peptoids
R
H
NH2
Peptoid Monomer
N
n
H
O
Amino Acid
• Peptoids are resistant to
proteolysis.
• Can form stable helical
structures.
• Are biocompatible.
• Easy to synthesize.
JACS. 125(44), 13525. 2003
Peptoid Secondary Structure
Magainin-2
Peptoid 1
Peptoid Library
Peptoid Library
Selectivity Ratio:
SR 
HD
MIC
10
E .Coli
Antibacterial Activity
Peptoid 1  H-(NLys-Nspe-Nspe)-NH2
• The antimicrobial activity of Peptoid 1 on biosafety-level 2 bacterial
strains was assessed.
• The minimum inhibitory concentration (MIC) of Peptoid 1 against
each respective strain was compared to Pexiganan.
Cytotoxicity Analysis for Selected
Peptoids Against A549 Cells
Peptoid
E.Coli
MIC, μM
B. Subtilis
MIC, μM
1
3.5
0.88
1-Pro6
3.1
1.6
1-NHis6,12
3.5
6.9
115mer
5.5
1.4
Pexiganan
3.1
1.6
Melittin
1.6
0.78
• The ID10 and ID50 are the minimum inhibitory dose of the
peptoid on A549 carcinoma lung epithelial cells.
Structural-Activity Analysis:
Chirality
1 = H-(NLys-Nspe-Nspe)4-NH2
1enantiomer = H-(NLys-Nrpe-Nrpe)4-NH2
Peptoid
E.Coli
MIC, μM
B. Subtilis
MIC, μM
SR
1
3.5
0.88
6.0
1enantimomer
3.5
0.88
4.6
Conclusion: the formation of the left handed helix does affect the
mechanism of interaction of the peptoid with that bacterial target.
Structural-Activity Analysis:
Length
H-(NLys-Nspe-Nspe)X-NH2
X = 2, 3, 4, 5
• NOTE: The charge-tolength ratio remains the
same, 1:3.
Peptoid
E.Coli
MIC, μM
B. Subtilis
MIC, μM
SR
1
3.5
0.88
6.0
16mer
27
27
>8.1
19mer
9.1
1.2
>16
115mer
5.5
1.4
0.55
Conclusion: Although they all form
helical structures, their respective
antimicrobial activity varies such that
there is an optimum length of peptoid.
Although this is true, the two shorter
peptoids illustrated a selectivity ratio
higher than peptoid 1.
Structural-Activity Analysis:
Hydrophobicity
2 = H-(NLys-Nssb-Nspe)4-NH2
2-Nsmb = H-(NLys-Nsmb-Nspe)4-NH2
2-Nsna = H-(NLys-Nssb-Nspe-NLys-Nspe-Nsna)2-NH2
Peptoid
Std. B*, %
SR
2
39
>3.9
2-Nsmbr
48
>16
2-Nsna
47
7.6
*Percent Acetonitrile in H20, 0.1% v/v TFA
Structural-Activity Analysis:
Hydrophobicity
1 = H-(NLys-Nspe-Nspe)4-NH2
1-Nsna = H-(NLys-Nspe-Nspe-NLys-Nspe-Nsna)2-NH2
1-NHis = H-(NLys-Nspe-Nspe-NLys-Nspe-NHis)2-NH2
1-Pro = H-NLys-Nssb-Nspe-NLys-Nspe-LPro-(NLys-Nspe-Nspe)2-NH2
Peptoid
% Std. B
E.Coli
MIC, μM
B. Subtilis
MIC, μM
SR
1
48
3.5
0.88
6.0
1-Nsna
53
3.3
1.6
1.2
1-NHis
37
3.5
6.9
>31
1-Pro
40
3.1
1.6
20
Conclusion: the moderately hydrophobic peptide demonstrate greater
selectivity than peptoids of increased hydrophobicity, due to the similar
antimicrobial activity and diminished hemolytic activity.
Structural-Activity Analysis:
Charge
1-NGlu1,4,7,10 = H-(NGlu-Nspe-Nspe)4-NH2
1-NGlu4,10 = H-(NLys-Nspe-Nspe-NGlu-Nspe-Nspe)2-NH2
Peptoid
E.Coli
MIC, μM
B. Subtilis
MIC, μM
SR
1
3.5
0.88
6.0
1-NGlu4,10
>110
6.9
0.17
1-NGlu1,4,7,10
>219
>219
N/A
Conclusion: due to the reduced charge or negative charge on the peptoids the
inherent antimicrobial activity was diminished or nonexistent, while there was
either minimal or no selectivity for bacteria over mammalian cells.
Structural-Activity Analysis:
Amphipathicity
1block = H-(NLys)4-(Nspe)8-NH2
2scramble = H-NLys-Nssb-Nspe-Nssb-Nspe-NLys-Nspe-NLys-NssbNssb-Nspe-NLys-NH2
Peptoid
E.Coli
MIC, μM
B. Subtilis
MIC, μM
SR
1
3.5
0.88
6.0
1block
6.9
1.7
2.6
2
31
3.9
>3.9
2scramble
31
15
>3.9
Conclusion: the antimicrobial activity of the 1block peptoid was diminished and
illustrated more hemolytic activity (does it form an α-helix?), demonstrating that
terminal organization is less selective than facial. The 2scramble peptide was used
to illustrate global amphipathicity, and it had similar antimicrobial activity with
respect to 2 and no hemolysis was demonstrated; suggesting low global
amphipathicity.
Assessment of helical Structure in
Artificial Lipid SUVs
POPC
CD Spectra analysis was used to assess the respective helical structure of
peptoids in the presence of both anionic bacterial like and zwitterionic
erythrocyte like membranes.
Nature 444, 775-779(7 December 2006)
Surface Analysis


X-ray reflectivity is an
analytical technique
utilized to assess the
surface of a material.
It can assess the
electron density, film
thickness and surface
roughness.
http://www.hlphys.uni-linz.ac.at/hl/lva/xray_lecture_WS200708/ReflectivityTutorial_schreiber.pdf
Orientation of Peptoid in DPPG
Lipid Monolayer
The X-ray reflectivity studies illustrated
that the peptoids orients itself within the
monolayer at a 56o angle.
Conclusions
1.
2.
3.
4.
Antimicrobial peptoids (amptoids) can function like
antimicrobial peptides.
They are selective for bacteria if they posses a cationic
charge and are moderately hydrophobic.
A defined helical structure is not required for
antimicrobial activity, rather hemolytic activity.
The peptoids interact with the membrane by inserting
itself into the lipid layer – as the xray data suggests.
However, the mechanism of action cannot be deduced
at this time, but is similar to that of anitmicrobial
peptides.
Supporting Material
Hemolysis curves of respective peptoids. Note that peptoids 16mer, 1-NGlu1,4,7,10,
1-NHis6,12, and 2scrambled did not illustrate hemolytic activity at the concentrations
examined.
Supporting Material
CD Spectra of peptoid 1 and its length variants.
Supporting Material