title: Novel rapidly diversifiable antimicrobial RNA polymerase switch region inhibitors with confirmed mode of action in Haemophilus influenzae.
abstract: 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 the natural antibiotics myxopyronin, corallopyronin, and ripostatin and the drug fidaxomicin. Computational modeling using the X-ray crystal structure of the myxopyronin-bound RNA polymerase of Thermus thermophilus suggests a 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.
authors: Departments of Bioscience, AstraZeneca R&D Boston, Infection Innovative Medicines Unit, Waltham, Massachusetts, USA. Ed.Buurman@astrazeneca.com
authors: Ed T
authors: ET
authors: Buurman
authors: Melinda A
authors: MA
authors: Foulk
authors: Ning
authors: N
authors: Gao
authors: Valerie A
authors: VA
authors: Laganas
authors: David C
authors: DC
authors: McKinney
authors: Demetri T
authors: DT
authors: Moustakas
authors: Jonathan A
authors: JA
authors: Rose
authors: Adam B
authors: AB
authors: Shapiro
authors: Paul R
authors: PR
authors: Fleming
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000201:enzymology*; D057166:High-Throughput Screening Assays / Q000379:methods; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012293:Rifampin / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin