Phosphonopeptides as Antibacterial Agents: Mechanism of Action of Alaphosphin

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.