Subnanomolar inhibitor of cytochrome bc1 complex designed by optimizing interaction with conformationally flexible residues

Article abstract of DOI:10.1021/ja905756c

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 K_i 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 K_i 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 K_i 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.