An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates

Abstract

Peptide-Peptide Nucleic Acid (PNA) conjugates targeting essential bacterial genes have shown significant potential in developing novel antisense antimicrobials. The majority of efforts in this area are focused on identifying different PNA targets and the selection of peptides to deliver the peptide-PNA conjugates to Gram-negative bacteria. Notably, the selection of a linkage strategy to form peptide-PNA conjugate plays an important role in the effective delivery of PNAs. Recently, a unique Cysteine- 2-Cyanoisonicotinamide (Cys-CINA) click chemistry has been employed for the synthesis of cyclic peptides. Considering the high selectivity of this chemistry, we investigated the efficiency of Cys-CINA conjugation to synthesize novel antimicrobial peptide-PNA conjugates. The PNA targeting acyl carrier protein gene (acpP), when conjugated to the membrane-active antimicrobial peptides (polymyxin), showed improvement in antimicrobial activity against multidrug-resistant Gram-negative Acinetobacter baumannii. Thus, indicating that the Cys-CINA conjugation is an effective strategy to link the antisense oligonucleotides with antimicrobial peptides. Therefore, the Cys-CINA conjugation opens an exciting prospect for antimicrobial drug development.

Keywords: antimicrobial agents; antisense oligonucleotides; cell-penetrating peptides; conjugation; peptide nucleic acids.

FIGURE 1

CINA mediated conjugation to generate peptide-PNA antisense conjugates.

FIGURE 2

The benzhydryloxyearbonyl (Bhoc) adduct during the Trifluoroacetic acid (TFA) cleavage of PNA and optimisation of cleavage protocol.

FIGURE 3

Synthetic strategies and efficiency of Cys-CINA in the synthesis of complex peptide-PNA conjugates. (A) Synthesis of cyclic peptide-PNA conjugate: CINA-OctGly-Dab-Thr-Dab-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr* (1. 2 equivalents), PNA: Cys-PEG-Cyt-Thy-Cyt-Ade-Thy-Ade-Cyt-Thy-Cyt-Thy-Thy-Gua-Lys-NH2 (1 equivalent), 10 M TCEP, PBS, pH 7.4. (B) Analytical HPLC spectra for polymyxin, PNA and crude polymyxin-PNA conjugate. (C) “One-pot” synthesis of thioether cross-linked cathelicidin-PNA conjugate: CINA-PEG-Val-Cys-Lys-Arg-Trp-Lys-Lys-Trp-Lys-Arg-Lys-Trp-Lys-Lys-Trp-Cys-Val-NH2 (1. 2 equivalents), PNA: Cys-PEG-Cyt-Thy-Cyt-Ade-Thy-Ade-Cyt-Thy-Cyt-Thy-Thy-Gua-Lys-NH2 (1 equivalent) 10 M TCEP, PBS, pH 7.4, 1,2-dibromobutene. (D) Analytical HPLC spectra for linear cathelicidin, PNA, crude cathelicidin-PNA conjugate, and in situ side-chain cross-linking. [TCEP: tris(2-carboxyethyl) phosphine, PEG: 8-Amino-3,6-Dioxaoctanoic Acid, PBS: phosphate buffer saline].

FIGURE 4

The inhibition zone of peptides, PNA, and peptide-PNA conjugates against A. baumannii 5075D (A) polymyxin and PNA No zones of inhibition observed, (B) polymyxin-acpP PNA conjugate zone of inhibition observed.

FIGURE 5

Bacterial flow cytometry analysis. Early log phase of A. baumannii 5075, 5075D and 5075R were treated with LASP-072, LASP-097 and LASP-132 (0.04 mM, 1 and 24 h). Bacterial cells were stained with PI and analyzed by fluorescence-activated cell sorting (FACS) flow cytometry. The histograms represent the percentage of PI-positive cells after each treatment.

FIGURE 6

Cell viability (%) of the A549 cells treated with LASP-072, LASP-097 and LASP-132 (0.04 mM, 24 h) using XTT assay (mean ± SD; n = 4). Tukey’s multiple comparisons test was performed and p > 0.05.