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. 2016 Jun 13;18(6):287-91.
doi: 10.1021/acscombsci.6b00039. Epub 2016 May 26.

Peptoid Library Agar Diffusion (PLAD) Assay for the High-Throughput Identification of Antimicrobial Peptoids

Kevin J Fisher  1 , Jeremy A Turkett  1 , Ashley E Corson  1 , Kevin L Bicker  1
Affiliations

Peptoid Library Agar Diffusion (PLAD) Assay for the High-Throughput Identification of Antimicrobial Peptoids

Kevin J Fisher et al. ACS Comb Sci. .

Abstract

Rapid emergence of antimicrobial resistant organisms necessitates equally rapid methods for the development of new antimicrobial compounds. Of recent interest have been mimics of antimicrobial peptides known as antimicrobial peptoids, which exhibit similar potency to the former but with improved proteolytic stability. Presented herein is a high-throughput method to screen libraries of antimicrobial peptoids immobilized on beads embedded into solid media. Termed the peptoid library agar diffusion (PLAD) assay, this assay allows for individual chemical manipulation of two identical peptoid strands. One strand can be released to diffuse out from a solid support bead and interact with the microorganism during screening. The other strand can be cleaved after screening from beads showing strong antimicrobial activity and analyzed by mass spectrometry to deconvolute the structure of the peptoid. This method was applied to a small library of peptoids to identify an antimicrobial peptoid with modest efficacy against the ESKAPE pathogens.

Keywords: antimicrobial; combinatorial library; high-throughput; peptoids.

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Conflict of interest statement

Notes The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
General PLAD Assay screening schematic. (Stage 1) PLAD linked peptoids immobilized on beads are imbedded into soft agar containing a small amount of reducing reagent and inoculated with the microorganism of interest. (Stage 2) Overnight incubation results in growth of a microorganismal lawn, reduction of the disulfide, and release of the beta-strand peptoid from the bead. Peptoids with antimicrobial properties generate a zone of inhibition around the bead. (Stage 3) Beads showing zones of inhibition are removed from the plate, alpha-strand peptoids are cleaved from the bead, and the peptoid structure is determined by MS/MS.
Figure 2
Figure 2
Synthesis of the PLAD linker, resulting in identical peptoid strands that can be chemically manipulated orthogonally. The beta-strand peptoid can be released using a reducing reagent to cleave the disulfide, followed by release of the alpha-strand using cyanogen bromide to cleave at the C-terminal methionine.
Figure 3
Figure 3
(A) A proof-of-concept known antimicrobial peptoid, C134mer immobilized on the PLAD linker. (B) Evaluation of the zone of inhibition around a bead in response to varying concentrations of three reducing reagents; β-mercaptoethanol (BME), dithiothreitol (DTT), and tris(2-carboxyethyl)phosphine (TCEP).
Figure 4
Figure 4
(A) Amines incorporated into a low diversity, proof-of-concept library on the PLAD linker. (B) Average zones of inhibition for the antimicrobial peptoids identified from screening the proof-of-concept library. Minimum inhibitory concentration (MIC) values for peptoid K15 tested against the ESKAPE pathogens.
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