In Situ Biofilm Affinity-Based\nProtein Profiling Identifies\nthe Streptococcal Hydrolase GbpB as the Target of a Carolacton-Inspired\nChemical Probe

Natural products are important precursors for antibiotic\ndrug design.\nThese chemical scaffolds serve as synthetic inspiration for chemists\nwho leverage their structures to develop novel antibacterials and\nchemical probes. We have previously studied carolacton, a natural\nproduct macrolactone fromSorangium cellulosum, and discovered a simplified derivative, <b>A2</b>, that maintained\napparent biofilm inhibitory activity, although the biological target\nwas unknown. Herein, we utilize affinity-based protein profiling (AfBPP) <i>in situ</i> during biofilm formation to identify the protein\ntarget using a photoexcitable cross-linking derivative of <b>A2</b>. From these studies, we identified glucan binding protein B (GbpB),\na peptidoglycan hydrolase, as the primary target of <b>A2</b>. Further characterization of the interaction between <b>A2</b> and GbpB, as well as PcsB, a closely related homologue from the\nmore pathogenic S. pneumoniae, revealed\nbinding to the catalytic CHAP (cysteine, histidine, aminopeptidase)\ndomain. To the best of our knowledge, this is the first report of\na small-molecule binder of a conserved and essential bacterial CHAP\nhydrolase, revealing its potential as an antibiotic target. This work\nalso highlights <b>A2</b> as a useful tool compound for streptococci\nand as an initial scaffold for the design of more potent CHAP binders.