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Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (K D 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)