Unraveling the Structural Basis of Selective Inhibition of Human Cytochrome P450 3A5

Abstract

The human cytochrome P450 (CYP) CYP3A4 and CYP3A5 enzymes metabolize more than one-half of marketed drugs. They share high structural and substrate similarity and are often studied together as CYP3A4/5. However, CYP3A5 preferentially metabolizes several clinically prescribed drugs, such as tacrolimus. Genetic polymorphism in CYP3A5 makes race-based dosing adjustment of tacrolimus necessary to minimize acute rejection after organ transplantation. Moreover, the differential tissue distribution and expression levels of CYP3A4 and CYP3A5 can aggravate toxicity during treatment. Therefore, selective inhibitors of CYP3A5 are needed to distinguish the role of CYP3A5 from that of CYP3A4 and serve as starting points for potential therapeutic development. To this end, we report the crystal structure of CYP3A5 in complex with a previously reported selective inhibitor, clobetasol propionate (CBZ). This is the first CYP3A5 structure with a type I inhibitor, which along with the previously reported substrate-free and type II inhibitor-bound structures, constitute the main CYP3A5 structural modalities. Supported by structure-guided mutagenesis analyses, the CYP3A5–CBZ structure showed that a unique conformation of the F–F′ loop in CYP3A5 enables selective binding of CBZ to CYP3A5. Several polar interactions, including hydrogen bonds, stabilize the position of CBZ to interact with this unique F–F′ loop conformation. In addition, functional and biophysical assays using CBZ analogs highlight the importance of heme-adjacent moieties for selective CYP3A5 inhibition. Our findings can be used to guide further development of more potent and selective CYP3A5 inhibitors.

Differential Effects of Clotrimazole on X-Ray Crystal Structures of Human Cytochromes P450 3A5 and 3A4

“…Significant induced fits are not seen in the structure of CYP3A4 with the antifungal fluconazole (Figure 1) and for other small substrates, and these structures are very similar to the ligand free structure of CYP3A4 (Sevrioukova, 2019a). Clotrimazole is smaller than other compounds crystallized with CYP3A5, clobetasol propionate (Wang et al, 2021), azamulin (Hsu and Johnson, 2022) and ritonavir (Hsu et al, 2018), and the clotrimazole complex may not exhibit an induced fit when it is bound to the heme iron in the proximal active site.…”

Identification of Potential Cytochrome P450 3A5 Inhibitors: An Extensive Virtual Screening through Molecular Docking, Negative Image-Based Screening, Machine Learning and Molecular Dynamics Simulation Studies

“…Several studies have already used CYP3A5 as a potential target for the in silico assessment of potential molecules [ 31 , 32 , 33 , 34 ]. Hence, the above data and research findings substantiated that CYP3A5 is a crucial and validated drug target for cardiovascular and other diseases [ 35 ]. Overall, the main objective of the current study was to screen the large chemical database against the CYP3A5 target for cardiovascular diseases through molecular docking, NIB screening, ML-based screening followed by pharmacokinetics and a toxicity assessment, molecular dynamics (MD) simulation, and molecular mechanics generalized Born surface area- (MM-GBSA) based binding free energy calculation.…”

Changes in Alprazolam Metabolism by CYP3A43 Mutants

“…The coordinates of the heme moiety were transferred from the CYP3A4 template to the CYP3A43 model. The CYP3A5 structure (PDB code: 7LAD [ 28 ]) was selected for modeling, as it is the only structure available in the Protein Data Bank (PDB) [ 29 ] with one co-crystallized ligand, thus indicating high comparability to the CYP3A43 model. The resulting model for CYP3A43 was curated and carefully checked in the MOE software package (Molecular Operating Environment 2020.0901, Chemical Computing Group ULC, Montreal, QC, Canada), including the setting of protonation states at pH 7.4 [ 30 ] in the OPLS-AA force field [ 31 ].…”