Identification of structurally diverse menaquinone-binding antibiotics with in vivo activity against multidrug-resistant pathogens

The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action (MOA). Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, MOA due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs). Their predicted products (MBA1–6) were rapidly accessed using a synthetic-bioinformatic natural product (syn-BNP) approach, which relies on bioinformatic structure prediction followed by chemical synthesis. Among these six structurally diverse MBAs, four makeup two new MBA structural families. The most potent member of each new family (MBA3, MBA6) proved effective at treating methicillin-resistant Staphylococcus aureus infection in a murine peritonitis-sepsis model. The only conserved feature present in all MBAs is the sequence “GXLXXXW”, which we propose represents a minimum MK-binding motif. Notably, we found a subset of MBAs were active against Mycobacterium tuberculosis both in vitro and in macrophages. Our findings suggest that naturally occurring MBAs are a structurally diverse and untapped class of mechanistically interesting, in vivo active, antibiotics.