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While <jats:italic>E. coli<\/jats:italic> RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of <jats:italic>E. coli <\/jats:italic>and <jats:italic>K. pneumoniae <\/jats:italic>RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP \u03b2 subunit gate loop. We focused on high\u2010affinity RfaH interactions with the RNAP \u03b2\u2019 subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by <jats:italic>in silico<\/jats:italic> docking using ZINC database, 3 ligands were able to inhibit <jats:italic>E. coli<\/jats:italic> RfaH recruitment <jats:italic>in vitro<\/jats:italic>. The most potent lead was active against both <jats:italic>E. coli <\/jats:italic>and <jats:italic>K. pneumoniae<\/jats:italic> RfaHs <jats:italic>in vitro<\/jats:italic>. Our results demonstrate the feasibility of identifying RfaH inhibitors using <jats:italic>in silico<\/jats:italic> docking and pave the way for rational design of antivirulence therapeutics against antibiotic\u2010resistant pathogens.<\/jats:p>","DOI":"10.1111\/mmi.14093","type":"journal-article","created":{"date-parts":[[2018,8,3]],"date-time":"2018-08-03T13:01:37Z","timestamp":1533301297000},"page":"128-142","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["In silico discovery of small molecules that inhibit RfaH recruitment to RNA polymerase"],"prefix":"10.1111","volume":"110","author":[{"given":"Dmitri","family":"Svetlov","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry The Ohio State University  Columbus OH 43210 USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Da","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of Chemistry and 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