Novel rapidly diversifiable antimicrobial RNA polymerase switch region inhibitors with confirmed mode of action in Haemophilus influenzae

Journal of Bacteriology. 2012 Oct; 194(20):5504-12.

Buurman ET, Foulk MA, Gao N, Laganas VA, McKinney DC, Moustakas DT, Rose JA, Shapiro AB, Fleming PR

Departments of Bioscience and Chemistry, AstraZeneca R&D Boston, Infection Innovative Medicines Unit, Waltham, MA 02451, USA


A series of inhibitors with a squaramide core was synthesized following its discovery in a high-throughput screen for novel inhibitors of a transcription-coupled translation assay using Escherichia coli S30 extracts. The inhibitors were inactive when the plasmid substrate was replaced with mRNA, suggesting they interfered with transcription. This was confirmed by their inhibition of purified E. coli RNA polymerase. The series had antimicrobial activity against efflux-negative strains of E. coli and Haemophilus influenzae. Like rifampin, the squaramides preferentially inhibited synthesis of RNA and protein over fatty acids, peptidoglycan and DNA. However, squaramide-resistant mutants were not cross-resistant to rifampin. Nine different mutations were found in parts of rpoB or rpoC that together encode the so-called “switch region” of RNA polymerase. This is the binding site of natural antibiotics myxopyronin, corallopyronin, ripostatin and the drug fidaxomicin. Computational modeling using the X-ray crystal structure of the myxopyronin-bound RNA polymerase of Thermus thermophilus suggests a likely binding mode of these inhibitors that is consistent with the resistance mutations. The squaramides are the first reported non-natural-product-related, rapidly diversifiable antibacterial inhibitors acting via the switch region of RNA polymerase.


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