Subnanomolar Inhibitor of Cytochrome bc1 Complex Designed by Optimizing Interaction with Conformationally Flexible Residues

Cytochrome bc1 complex (EC 1.10.2.2, bc1), an essential component of the cellular respiratory chain and the photosynthetic apparatus in photosynthetic bacteria, has been identified as a promising target for new drugs and agricultural fungicides. X-ray diffraction structures of the free bc1 complex and its complexes with various inhibitors revealed that the phenyl group of Phe274 in the binding pocket exhibited significant conformational flexibility upon different inhibitors binding to optimize respective π−π interactions, whereas the side chains of other hydrophobic residues showed conformational stability. Therefore, in the present study, a strategy of optimizing the π−π interaction with conformationally flexible residues was proposed to design and discover new bc1 inhibitors with a higher potency. Eight new compounds were designed and synthesized, among which compound 5c, with a Ki value of 570 pM, was identified as the most promising drug or fungicide candidate, significantly more potent than the commercially available bc1 inhibitors, including azoxystrobin (AZ), kresoxim-methyl (KM), and pyraclostrobin (PY). To our knowledge, this is the first bc1 inhibitor discovered from structure-based design with a potency of subnanomolar Ki value. For all of the compounds synthesized and assayed, the calculated binding free energies correlated reasonably well with the binding free energies derived from the experimental Ki values, with a correlation coefficient of r2 = 0.89. The further inhibitory kinetics studies revealed that 5c is a noncompetitive inhibitor with respect to substrate cytochrome c, but it is a competitive inhibitor with respect to substrate ubiquinol. Due to its subnanomolar Ki potency and slow dissociation rate constant (k−0 = 0.00358 s−1), 5c could be used as a specific probe for further elucidation of the mechanism of bc1 function and as a new lead compound for future drug discovery.