Identification of a small-molecule inhibitor of bacterial AraC family activators.
Protein members of the AraC family of bacterial transcriptional activators have great promise as targets for the development of novel antibacterial agents. Here, we describe an in vivo high-throughput screen to identify inhibitors of the AraC family activator protein RhaS. The screen used two Escherichia coli reporter fusions: one to identify potential RhaS inhibitors and a second to eliminate nonspecific inhibitors from consideration. One compound with excellent selectivity, OSSL_051168, was chosen for further study. OSSL_051168 inhibited in vivo transcription activation by the RhaS DNA-binding domain to the same extent as the full-length protein, indicating that this domain was the target of its inhibition. Growth curves showed that OSSL_051168 did not affect bacterial cell growth at the concentrations used in this study. In vitro DNA-binding assays with purified protein suggest that OSSL_051168 inhibits DNA binding by RhaS. In addition, we found that it inhibits DNA binding by a second AraC family protein, RhaR, which shares 30% amino acid identity with RhaS. OSSL_051168 did not have a significant impact on DNA binding by the non-AraC family proteins CRP and LacI, suggesting that the inhibition is likely specific for RhaS, RhaR, and possibly additional AraC family activator proteins.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.
Jeff M
JM
Skredenske
Veerendra
V
Koppolu
Ana
A
Kolin
James
J
Deng
Bria
B
Kettle
Byron
B
Taylor
Susan M
SM
Egan
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D051777:AraC Transcription Factor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D008826:Microbial Sensitivity Tests; D008954:Models, Biological; D005810:Multigene Family; D011485:Protein Binding / Q000187:drug effects; D011804:Quinolines / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000032:analysis; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000378:metabolism
C584457:1-butyl-4-nitromethyl-3-quinolin-2-yl-4H-quinoline; D000900:Anti-Bacterial Agents; D051777:AraC Transcription Factor; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011804:Quinolines; C084095:RhaS protein, E coli; D054852:Small Molecule Libraries; D015534:Trans-Activators
AraC family activators; antibacterial agents; cell-based assays; high-throughput screening; inhibitor