New solutions for antibiotic resistance are urgently needed. Drug-resistant Streptococcus pneumoniae alone is estimated to cause 1.2 million infections with an excess of 96 million USD in medical costs per annum. Previous studies identified new diamide small molecules as growth inhibitors of non-pathogenic, Gram-positive Bacillus subtilis affecting the cell wall autolysin LytG. This is the first report of small molecule inhibitors of a bacterial autolysin that demonstrated antimicrobial activity in whole cell assays. A panel of 65 diamides were screened for antimicrobial activity against S. pneumoniae using the resazurin microtiter assay. Screening identified three compounds with low micromolar activity against S. pneumoniae. Mode-of-action studies using genetic screening of autolysin mutants lytB, lytC, pmp23, and dacA/B indicated the diamides targeted the cell wall. Desensitization of the diamides was observed in the lytB mutant, prompting in vitro screening against LytB, an endo-N-acetylglucosaminidase. In vitro results with LytB contradicted the genetic screen, underscoring the complexity in autolysin homeostasis. While LytB is not the molecular target, the genetic screening suggests the cell wall is the target of the diamides. Synergy of our diamides with antibiotics of known mode-of-action corroborate the cell wall as a target. The compounds were dismissed as promiscuous ligands of common targets, including DNAse and intercalation, and eukaryotic assays indicated specificity to the bacteria. Toxicity of the lead compounds were assessed using the established Drosphilia melanogaster survival assay. Our findings suggest cell wall homeostatic enzymes as a class of drug targets with great therapeutic potential and diamide inhibitors as good lead compounds.

One strep at a time: Chemogenomic screening of antimicrobial diamides in Streptococcus pneumoniae

256th National Meeting (2018)

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