Cell 169, 1240–1248.e23 (2017)

Bacterial RNA polymerase (RNAP) is an attractive antimicrobial target, as several drug classes potently inhibit this essential protein without targeting the polymerases of mammalian hosts. Yet, the emergence of drug resistance has prompted the search for agents that are less resistance prone. Maffioli et al. now report the identification of pseudouridimycin (PUM), a natural product that inhibits RNAP by a unique mode of action. The authors identified PUM, a pseudouridine nucleoside linked to a modified Gly-Gln dipeptide, from a target-based screen of actinobacterial and fungal culture extracts. PUM inhibits bacterial RNAP activity, specifically at sites of uridine incorporation, blocks growth of Gram-positive and Gram-negative bacteria and cures infections in a mouse model. PUM has no cross-resistance with the known RNAP inhibitor rifampin, and resistance to PUM develops only one-tenth as rapidly as resistance to rifampin. Sequencing of PUM-resistant mutants identified a unique binding site for PUM, which was confirmed by a crystal structure of a transcriptional initiation complex that reveals that the pseudouridine of PUM pairs with a deoxyadenosine in the DNA template whereas the dipeptide portion engages regions that explain PUM's specificity and potency. These structural data guided the synthesis of related analogs, showing that PUM offers a new chemical scaffold and defines a unique target site for further antimicrobial development.