Computational Discovery of Picomolar Q_o Site Inhibitors of Cytochrome bc₁ Complex

Abstract

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 Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20 507-fold improved binding affinity (K(i) = 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 (K(i) = 83.00 pM) bound to the chicken bc(1) 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.

Computational and Experimental Insights into the Mechanism of Substrate Recognition and Feedback Inhibition of Protoporphyrinogen Oxidase

“…This kind of kinetic time-course demonstrated that the enzymatic activity decreased gradually along with the product formation and finally the enzyme became inhibited. The curvilinear functions displayed by the curves were consistent with the presence of a slow, tight-binding inhibitor [24]. This type of kinetic behavior is usually due to a process characterized by the rapid formation of reactant-enzyme complex, followed by a slower dissociation of the product-enzyme complex [25].…”

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway

“…Kanamycin at a final concentration of 30 g ml Ϫ1 was added into the broth to prevent loss of pWDX plasmid in the complement trials, and 0.03 mM isopropyl-␤-D-thiogalactopyranoside was added to the cultures of FS14⌬pqqF containing pqqF-pWDX to induce the expression of PqqF protein. Three parallel controls were prepared for every strain, and 3-ml cultures were taken out for measurements of the prodigiosin concentration and pH at 6,12,24,36,48,60, and 72 h. Prodigiosin concentration was measured at 534 nm with an extinction coefficient of 7.07 ϫ 10 4 from liquid cultures as previously described (60). The pH was determined from the supernatant using Sartorius pH analyzer (model PB-10).…”

Structural Basis of Resistance to Anti-Cytochrome bc₁ Complex Inhibitors: Implication for Drug Improvement

“…Since no adjustment is made by Phe275, trifloxystrobin may just miss this energetically favorable interaction. By consequently taking advantage of π-π stacking interactions with Phe275 combined with the stabilizing effect of filling cavities, the first pico molar methoxy-acrylate based inhibitors were developed and structurally characterized[47]. …”

“…In the field of drug design for bc 1 , a recent example where a combination of traditional and modern ideas named pharmacophore-like fragment virtual screening (PFVS) resulted in the first pico molar methoxyacrylate-based inhibitors was recently introduced by the Yang group[47, 110]. …”