Phosphinic acid inhibitors of D-alanyl-D-alanine ligase

Abstract

We report the synthesis of a series of phosphinic acid dipeptide analogues, NH2CH(R1)PO(OH)CH2CH(R2)CO2H, related to DAla-DAla. The best of these compounds are potent, essentially irreversible inhibitors of DAla-DAla ligase, and their preferred stereochemistry was shown by chiral synthesis of (1(S)-aminoethyl)(2(R)-carboxy-1-n-propyl)phosphinic acid, 12b, and by X-ray crystallography of its derivative benzyl [1(S)-[(benzyloxycarbonyl)-amino]ethyl](2(R)-carbomethoxy-1-propyl) phosphinate, 13, to correspond to the stereochemical configuration of DAla-DAla at both centers. A mechanism for the inhibition of DAla-DAla ligase by these compounds is proposed to involve an ATP-dependent formation of phosphorylated inhibitor within the enzyme's active site. The antibacterial activities of these compounds are modest although their spectra include both Gram-positive and Gram-negative susceptible organisms. The best antibacterial activity was shown by (1(S)-aminoethyl) [2-carboxy-2(R)-(methylthio)-1-ethyl]phosphinic acid, 3e, whose MIC's range from 4-128 micrograms/mL on nine of a panel of 11 bacterial organisms. Combination of one of the more active phosphinic acids 12b with the alanine racemase inhibitor fluoro-D-alanine enhances the antibacterial spectrum of the latter on several strains of bacteria and inhibits fluoro-D-alanine's self-reversal, which normally occurs at concentrations several fold higher than its MIC level. This inhibition of fluoro-D-alanine self-reversal is consistent with an involvement of DAla-DAla ligase inhibition in the antibacterial activity of these compounds.

Structure of the Mycobacterium tuberculosis d -Alanine: d -Alanine Ligase, a Target of the Antituberculosis Drug d -Cycloserine

“…Ligation is completed upon the nucleophilic attack of the amine at the D-Ala 2 site on the phosphorylated D-Ala at the D-Ala 1 site to yield D-Ala:D-Ala (13,39). Inhibitors mimicking D-Ala or D-Ala:D-Ala have been discovered, including D-cycloserine (DCS), cis-and trans-DL-cyclothreonine, and compounds related to reaction transition state intermediates, such as phosphinate and phosphonate derivatives (32,34). DCS has been used as an anti-TB agent and is the only Ddl inhibitor currently used in the treatment of bacterial infections (46).…”

Allosteric inhibition of Staphylococcus aureus d -alanine: d -alanine ligase revealed by crystallographic studies

“…The tight binding of D-cycloserine to DDl suggests that the antibiotic binds to the high-affinity D-alanine site (10). Compounds designed to mimic the Dalanine:D-alanine dipeptide or the reaction transition-state intermediates led to the developments of phosphinate and phosphonate dipeptide analogs (11)(12). These phosphinates or phosphonates, after being phosphorylated by DDl, bind to the protein tightly and inhibit the reaction (11)(12).…”

“…Compounds designed to mimic the Dalanine:D-alanine dipeptide or the reaction transition-state intermediates led to the developments of phosphinate and phosphonate dipeptide analogs (11)(12). These phosphinates or phosphonates, after being phosphorylated by DDl, bind to the protein tightly and inhibit the reaction (11)(12). The best in vitro IC 50 of these compounds are Ϸ4 M on Streptococcus faecalis DDl, and their antibacterial activities are not significant (11).…”

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

“…Assuming a direct hydrolysis route, the mechanism for D-Ala-D-Ala hydrolysis by VanX would begin with attack of a Zn-activated H20 on the carbonyl group of the first D-Ala residue to produce the tetrahedral adduct V that then eliminates to yield the two D-Ala products (Scheme I). Phosphoruscontaining amino acid analogs have been successfully used to mimic unstable tetrahedral intermediates involved in both peptide hydrolysis and formation and have proved to be an effective strategy to design potent inhibitors (16,17 To date, nearly all reported examples of slow-binding inhibition exhibit biphasic inhibition kinetics due to rapid formation of an initial E-I complex followed by a slow equilibrium conversion to a tighter-binding E.I* complex (13,18 Given this Ddl precedent, studies of proteolysis and phosphinate protection were examined in VanX. Limited proteolysis of VanX by chymotrypsin, trypsin, and endoproteinase Lys-C showed similar results.…”