Computational Discovery of Picomolar <i>Q</i>_o Site Inhibitors of Cytochrome <i>bc</i>₁ Complex

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Qo site inhibitors of the cytochrome bc1 complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (Ki = 881.80 nM, porcine bc1), the most potent compound 4f displayed 20 507-fold improved binding affinity (Ki = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (Ki = 83.00 pM) bound to the chicken bc1 at 2.70 Å resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.