Structure-Based Inhibitor Design for Evaluation of a CYP3A4 Pharmacophore Model

Abstract

Human cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme that oxidizes and clears the majority of drugs. CYP3A4 inhibition may lead to drug–drug interactions, toxicity, and other adverse effects but, in some cases, could be beneficial and enhance therapeutic efficiency of coadministered pharmaceuticals that are metabolized by CYP3A4. On the basis of our investigations of analogs of ritonavir, a potent CYP3A4 inactivator and pharmacoenhancer, we have built a pharmacophore model for a CYP3A4-specific inhibitor. This study is the first attempt to test this model using a set of rationally designed compounds. The functional and structural data presented here agree well with the proposed pharmacophore. In particular, we confirmed the importance of a flexible backbone, the H-bond donor/acceptor moiety, and aromaticity of the side group analogous to Phe-2 of ritonavir and demonstrated the leading role of hydrophobic interactions at the sites adjacent to the heme and phenylalanine cluster in the ligand binding process. The X-ray structures of CYP3A4 bound to the rationally designed inhibitors provide deeper insights into the mechanism of the CYP3A4–ligand interaction. Most importantly, two of our compounds (15a and 15b) that are less complex than ritonavir have comparable submicromolar affinity and inhibitory potency for CYP3A4 and, thus, could serve as templates for synthesis of second generation inhibitors for further evaluation and optimization of the pharmacophore model.

Membrane Fluidity Modulates Thermal Stability and Ligand Binding of Cytochrome P4503A4 in Lipid Nanodiscs

“…Available crystal structures of CYP3A4 indicate that it is approximately 50% α-helical and 8% β-sheet in secondary structure. 11,25–28 This is in good agreement with our experimental results, and the minor discrepancies in secondary structure content may likely be explained, in part, by the fact that the available crystal structures do not include the crystallographically disordered N-terminal helix in the calculation of secondary structure.…”

The Cytochrome P450 3A4 has three Major Conformations: New Clues to Drug Recognition by this Promiscuous Enzyme

“…Aromaticity and hydrophobicity of the ligand substituents stabilize the protein-ligand complex and drastically improves affinity and inhibitory potency. [18,19] The importance of compound lipophilicity in substrate binding and in metabolic rate was outlined. [22] That explains why the parameter Log(P) has a central role in the flowchart of Figure 2.…”

N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

“…In cases when the thiazole has been replaced with another azaheterocycle, coordination is generally maintained. Additionally, in examples wherein the azaheterocycle is absent, the orientation of the remaining scaffold remains consistent with that of ritonavir Poulos, 2012, 2013;Kaur et al, 2015). Moreover, the observation that deaza-ritonavir (the heme coordinating nitrogen is replaced by C-H) produces a partial type I spectral change is similarly consistent with "thiazole-first" binding.…”

“…Structural modifications abolishing the phenyl groups substantially lower the affinity of these molecules for CYP3A4 and result in the observation of multiple ligand-binding orientations in their crystal structures (Sevrioukova and Poulos, 2013;Kaur et al, 2015). Nonetheless, thiazole coordination to the heme iron and orientation of the isopropyl thiazole unit near the protein surface remain consistent features of these structures.…”