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

N

H

NH2

O

R H

n

Amino Acid

Peptoid Monomer

• 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

MICHD

ColiE

SR.

10Selectivity Ratio:

Antibacterial Activity

• 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.

Peptoid 1 H-(NLys-Nspe-Nspe)-NH2

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-to-length 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

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

1block = H-(NLys)4-(Nspe)8-NH2

2scramble = H-NLys-Nssb-Nspe-Nssb-Nspe-NLys-Nspe-NLys-Nssb-Nssb-Nspe-NLys-NH2

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. Antimicrobial peptoids (amptoids) can function like antimicrobial peptides.

2. They are selective for bacteria if they posses a cationic charge and are moderately hydrophobic.

3. A defined helical structure is not required for antimicrobial activity, rather hemolytic activity.

4. 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