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 competitive-noncompetitive 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.

Fig. 1.

Structure of ritonavir.

Fig. 2.

Spectral changes induced by ritonavir in CYP3A4. Absorbance spectra of ferric ligand-free (__), ferric ritonavir-bound (-..-..), ferrous ritonavir-bound (- - -), and ferrous-CO adduct (….) of 3 μM CYP3A4 were recoded in buffer A.

Fig. 3.

Equilibrium titration of CYP3A4 with ritonavir. Spectral changes in 8 μM ferric (A), 1.5 μM ferrous (B), and 2 μM ferric BEC-bound CYP3A4 (C) were recorded as described in Materials and Methods. Absorbance changes in ferric, ferrous, and ferric BEC-bound CYP3A4 were plotted against the ritonavir concentration (D, E, and F, respectively). The derived K_s values are listed in Table 1.

Fig. 4.

Kinetics of ritonavir binding to CYP3A4. Ligation of 1, 2, 4, 8, and 16 μM ritonavir (traces a - e, respectively) to 1.5 μM ferric (A), ferrous (C) or BEC-bound ferric CYP3A4 (E) was monitored at 426, 442, and 423 nm, respectively. The observed rate constants (k_obs) for the fast and slow phases were calculated from the biexponential fits (solid lines) to the kinetic traces. The limiting rate constants and kinetic K_d’s derived from the plots of k_obs for the fast phase vs. ritonavir concentration (B, D, and F) are listed in Table 1.

Fig. 5.

CPR cannot reduce ritonavir-bound CYP3A4. Spectra were recorded under anaerobic conditions in buffer A before (___) and after addition of NADPH and catalytic amounts of human CPR to ligand-free (A), androstenedione- (B), or ritonavir-bound CYP3A4 (C) in the absence (- - - -) or presence of CO (…).

Fig. 6.

Crystal structure of the CYP3A4-ritonavir complex. (A), The active site cavity of ritonavir-bound CYP3A4. Ritonavir is green and in CPK representation; the heme is red. (B), Aromatic residues surrounding ritonavir. 2Fo-Fc (blue) and Fo-Fc (green) electron density maps around the heme and ritonavir are contoured at 1 and 3 σ, respectively. (C), An umbrella-like charge-charge/H-bonding network connected to the isopropyl-thiazole moiety of ritonavir via a highly ordered water molecule (w1).

Fig. 7.

Comparison of ritonavir- and ketoconazole-bound CYP3A4. The structures are rendered in white and light blue, respectively. (A), Relative orientation of ritonavir (green), ketoconazole molecules (yellow and orange) and the interacting aromatic residues. (B), Conformational differences around the polar “umbrella.” Blue and red dotted lines represent electrostatic/H-bonding interactions in the ritonavir- and ketoconazole-ligated CYP3A4, respectively.