Protein Data Bank in Europe

Abstract

Structure-function relationships of nine rationally designed ritonavir-like compounds were investigated to better understand the ligand binding and inhibitory mechanism in human drug-metabolizing cytochrome P450 3A4 (CYP3A4). The analogs had a similar backbone and pyridine and tert-butyloxycarbonyl (Boc) as the heme-ligating and terminal groups, respectively. N-Isopropyl, N-cyclopentyl, or N-phenyl were the R₁-side group substituents alone (compounds 5a-c) or in combination with phenyl or indole at the R₂ position (8a-c and 8d-f subseries, respectively). Our experimental and structural data indicate that (i) for all analogs, a decrease in the dissociation constant (K_s) coincides with a decrease in IC₅₀, but no relation with other derived parameters is observed; (ii) an increase in the R₁ volume, hydrophobicity, and aromaticity markedly lowers K_s and IC₅₀, whereas the addition of aromatic R₂ has a more pronounced positive effect on the inhibitory potency than the binding strength; (iii) the ligands' association mode is strongly influenced by the mutually dependent R₁-R₂ interplay, but the R₁-mediated interactions are dominant and define the overall conformation in the active site; (iv) formation of a strong H-bond with Ser119 is a prerequisite for potent CYP3A4 inhibition; and (v) the strongest inhibitor in the series, the R₁-phenyl/R₂-indole containing 8f (K_s and IC₅₀ of 0.08 and 0.43 μM, respectively), is still less potent than ritonavir, even under conditions that prevent the mechanism based inactivation of CYP3A4. Crystallographic data were essential for better understanding and interpretation of the experimental results, and suggested how the inhibitor design could be further optimized.