Structure and mechanism of the complex between cytochrome P4503A4 and ritonavir

Abstract

Ritonavir is a HIV protease inhibitor routinely prescribed to HIV patients that also potently inactivates cytochrome P4503A4 (CYP3A4), the major human drug-metabolizing enzyme. By inhibiting CYP3A4, ritonavir increases plasma concentrations of other anti-HIV drugs oxidized by CYP3A4 thereby improving clinical efficacy. Despite the importance and wide use of ritonavir in anti-HIV therapy, the precise mechanism of CYP3A4 inhibition remains unclear. The available data are inconsistent and suggest that ritonavir acts as a mechanism-based, competitive or mixed competitivenoncompetitive CYP3A4 inactivator. To resolve this controversy and gain functional and structural insights into the mechanism of CYP3A4 inhibition, we investigated the ritonavir binding reaction by kinetic and equilibrium analysis, elucidated how the drug affects redox properties of the hemoprotein, and determined the 2.0 Å X-ray structure of the CYP3A4-ritonavir complex. Our results show that ritonavir is a type II ligand that perfectly fits into the CYP3A4 active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen, which decreases the redox potential of the protein and precludes its reduction with the redox partner, cytochrome P450 reductase.crystal structure | cytochrome P450 | inhibitor

CYP3A4 Mediates Growth of Estrogen Receptor-positive Breast Cancer Cells in Part by Inducing Nuclear Translocation of Phospho-Stat3 through Biosynthesis of (±)-14,15-Epoxyeicosatrienoic Acid (EET)

“…Indeed, ritonavir has recently been demonstrated by us to inhibit the growth of the MCF7, T47D, and MDA-MB-231 lines studied in this report as well as growth of the MDA-MB-231 xenograft (22). The inhibitory effect of ritonavir on CYP3A4 has recently been confirmed in a co-crystal structure demonstrating that ritonavir covalently and irreversibly binds by its thiazole nitrogen to the CYP3A4 heme iron, thereby reducing the redox potential of the enzyme and preventing reduction by cytochrome P450 reductase (35). Although ritonavir inhibits CYP3A5 (K I ϭ 0.12 M) as well as 3A4 (K I ϭ 0.10 M) (36), a potential limitation of ritonavir may be an increase of CYP2C8 epoxygenase activity, suggested by the observed increase in CYP2C8 mRNA when CYP3A4 is silenced.…”

2-Aminobenzothiazole derivatives: Search for new antifungal agents

“…3. As a figure of merit, the same binding topology has also been experimentally observed in the X-ray structures of other thiazoles in complex with cytochrome different from the CA-CYP51, but functionally related to our target [28]. At the same time, the amino group at position 2 reinforces the binding engaging Thr311 and Gly307 with hydrogen-bonding mediated polar interactions.…”

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

“…In 3A4, the "roof" above the heme is formed by a cluster of phenylalanine side chains. These phenylalanine side chains expand outward relative to the ligand-free structure (67,68) to accommodate erythromycin (734 Da) (69), ritonavir (721 Da) (70), desthiazolylmethyloxycarbonyl ritonavir (580 Da) (71), or two molecules of ketoconazole (531 Da) stacked antiparallel and vertically above the heme (69). In contrast, bromoergocryptine (656 Da) elicits only small changes relative to ligandfree structures when bound (72).…”