Phosphonopeptides as Antibacterial Agents: Mechanism of Action of Alaphosphin

TLDR

Alaphosphin is actively transported into bacterial cells via peptide permeases, cleaved by intracellular aminopeptidases to release l‑1‑aminoethylphosphonic acid, which then inhibits alanine racemase. Alaphosphin selectively blocks peptidoglycan synthesis by accumulating inside cells and competitively or irreversibly inhibiting alanine racemase (and possibly UDP‑N‑acetylmuramyl‑L‑alanine synthetase), leading to reduced d‑alanine pools and accumulation of UDP‑N‑acetyl‑muramyl intermediates.

Abstract

The novel antibacterial peptide mimetic alaphosphin ( l -alanyl- l -1-aminoethylphosphonic acid) selectively inhibited peptidoglycan biosynthesis in both gram-negative and gram-positive bacteria. It induced accumulation of uridine diphosphate- N -acetyl-muramyl-tripeptide in gram-positive organisms and significantly reduced the intracellular pool levels of d -alanine. Alaphosphin was actively transported into bacterial cells by stereospecific peptide permeases and was subsequently hydrolyzed by intracellular aminopeptidases to yield l -1-aminoethylphosphonic acid. This alanine mimetic rapidly accumulated inside susceptible cells to yield a concentration which was 100- to 1,000-fold in excess of that of the precursor peptide in the surrounding medium. In the case of susceptible gram-negative organisms, it was shown that 1-aminoethylphosphonic acid was incorporated into a metabolite which was tentatively identified as uridine diphosphate- N -acetylmuramyl-aminoethylphosphonate. The primary intracellular target site of 1-aminoethylphosphonic acid was alanine racemase (EC 5.1.1.1), which was reversibly and competitively inhibited in the gram-negative organisms Escherichia coli and Pseudomonas aeruginosa and irreversibly inhibited in a time-dependent manner in the gram-positive organisms Staphylococcus aureus and Streptococcus faecalis . A secondary target site could be uridine diphosphate- N -acetylmuramyl- l -alanine synthetase [EC 6.3.2.8(b)]. The mechanism of action of alaphosphin may be regarded as involving at least three stages: (i) active transport by peptide permeases; (ii) intracellular peptidase cleavage; and (iii) action of l -1-aminoethylphosphonate on alanine racemase.