Identification of the Binding Site of Brucella VirB8 Interaction Inhibitors

• Published in: Chemistry & biology Vol. 19; no. 8; pp. 1041 - 1048
• Main Authors: Smith, Mark A., Coinçon, Mathieu, Paschos, Athanasios, Jolicoeur, Benoit, Lavallée, Pierre, Sygusch, Jurgen, Baron, Christian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 24.08.2012
• Subjects: Amino Acid Substitution; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Brucella - metabolism; Computer Simulation; Crystallography, X-Ray; Dimerization; High-Throughput Screening Assays; Kinetics; Protein Structure, Tertiary; Structure-Activity Relationship; Virulence Factors - antagonists & inhibitors; Virulence Factors - genetics; Virulence Factors - metabolism
• ISSN: 1074-5521; 1879-1301
• DOI: 10.1016/j.chembiol.2012.07.007

Secretion systems translocate virulence factors of many bacterial pathogens, enabling their survival inside the host organism. Consequently, inhibition strongly attenuates pathogenicity and can be considered a target for novel antimicrobial drugs. The type IV secretion system (T4SS) of the intracellular pathogen Brucella is a prerequisite for its virulence, and in this work we targeted the interactions of the essential assembly factor protein, VirB8, using small-molecule inhibitors. High-throughput screening identified several potent and specific inhibitors, and the target-binding site of these inhibitors was identified by X-ray crystallography, in silico docking, and analysis of the derivates of the inhibitor B8I-2. VirB8 interaction inhibitors bind to a surface groove opposite to the dimerization interface, and by varying the binding-site residues, we were able to determine which residues are required for inhibitor activity. E115 and K182 were found to be especially important, and changes at R114, Y229, and L151 also reduced inhibitor efficiency. ► Structural identification of the binding site for the VirB8 interaction inhibitor B8I-1 ► Confirmation of X-ray crystallographic analysis using in silico docking, and identification of the localized binding site and binding modes for other potent VirB8 interaction inhibitors ► Validation of the binding mode of VirB8 interaction inhibitor B8I-2 using synthesized B8I-2 inhibitor derivatives ► Verification of the VirB8 interaction inhibitor-binding site using protein variants to determine key interaction residues identified by in silico methods and B8I-2 inhibitor derivatives Anti-virulence drugs that disarm the pathogen, allowing host immune system to eliminate the infection might circumvent issue of bacterial resistance. Smith et al. develop such a molecule that targets an essential assembly factor of the Brucella type IV secretion system and presents an alternative to antibiotics.