Diamide Inhibitors of the Bacillus subtilis N-Acetylglucosaminidase LytG That Exhibit Antibacterial Activity

Abstract

N-Acetylglucosaminidases (GlcNAcases) play an important role in the remodeling and recycling of bacterial peptidoglycan by degrading the polysaccharide backbone. Genetic deletions of autolysins can impair cell division and growth, suggesting an opportunity for using small molecule autolysin inhibitors both as tools for studying the chemical biology of autolysins and also as antibacterial agents. We report here the synthesis and evaluation of a panel of diamides that inhibit the growth of Bacillus subtilis. Two compounds, fgkc (21) and fgka (5), were found to be potent inhibitors (MIC 3.8 ± 1.0 and 21.3 ± 0.1 μM, respectively). These compounds inhibit the B. subtilis family 73 glycosyl hydrolase LytG, an exo GlcNAcase. Phenotypic analysis of fgkc (21)-treated cells demonstrates a propensity for cells to form linked chains, suggesting impaired cell growth and division.

Keywords: N-acetylglucosaminidase; autolysin; diamide; inhibitor; peptidoglycan.

Figure 1

Structure of peptidoglycan showing the cleavage sites of the major characterized autolysins in B. subtilis. Inset - Structure of BI.fgba, an inhibitor of B. subtilis LytG.

Figure 2

Structures of diamides synthesized and tested against B. subtilis.

Figure 3

Crystal structure of the diamide fgka (5)

Figure 4

Characterization of diamide inhibitors fgkc (21) and fgka (5) against B. subtilis. (A) Plot of LytG residual activity against log[fgkc]. Assay performed using purified PG and enzyme activity monitored using a turbidometric assay at 600 nm. Non-linear regression was used to calculate the IC₅₀ of fgkc (21). Assays were run in biological and technical triplicate. (B) Promiscuous inhibitor screen for inhibitor aggregation – MIC values were determined in the presence or absence of 0.001% (v/v) Triton X-100. (C) Role of non-specific binding in fgkc (21) mode of action. MIC values were determined in the presence of varying [BSA]. (D) Microscopy of B. subtilis 11774 in the presence of sub-MIC (0.8×) concentration of fgkc (21). (E) MIC values for fgkc (21) against various B. subtilis autolysin mutants.