Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism

Abstract

Cytochrome P450 3A4 (CYP3A4) is the main P450 enzyme for drug metabolism and drug–drug interactions (DDIs), as it is involved in the metabolic process of approximately 50% of drugs. A detailed mechanistic elucidation of DDIs mediated by CYP3A4 is commonly believed to be critical for drug optimization and rational use. Here, two typical probes, midazolam (MDZ, substrate) and testosterone (TST, allosteric effector), are used to investigate the molecular mechanism of CYP3A4-mediated heterotropic allosteric interactions, through conventional molecular dynamics (cMD) and well-tempered metadynamics (WT-MTD) simulations. Distance monitoring shows that TST can stably bind in two potential peripheral sites (Site 1 and Site 2) of CYP3A4. The binding of TST at these two sites can induce conformational changes in CYP3A4 flexible loops on the basis of conformational analysis, thereby promoting the transition of the MDZ binding mode and affecting the ratio of MDZ metabolites. According to the results of the residue interaction network, multiple allosteric communication pathways are identified that can provide vivid and applicable insights into the heterotropic allostery of TST on MDZ metabolism. Comparing the regulatory effects and the communication pathways, the allosteric effect caused by TST binding in Site 2 seems to be more pronounced than in Site 1. Our findings could provide a deeper understanding of CYP3A4-mediated heterotropic allostery at the atomic level and would be helpful for rational drug use as well as the design of new allosteric modulators.

Assessing the role of residue Phe108 of cytochrome P450 3A4 in allosteric effects of midazolam metabolism

“…Based on the full-length structural model, 13 the substrate MDZ (S-MDZ) was deposited in the CYP3A4 active site in a pose similar to the MDZ co-crystallized structure (PDB ID: 5TE8 11 ). Subsequently, effector MDZ (E-MDZ) and TST were introduced into the two peripheral allosteric sites (Site 1 and Site 2), as we reported in our previous work, 12,13 generating two wild-type complexes CYP3A4-MDZ-MDZ (homotropic allostery complex) and CYP3A4-MDZ-TST (heterotropic allostery complex) (Fig. S3, ESI †).…”

“…[2][3][4][5] CYP3A4-mediated drug metabolism often exhibits regiospecificity and is primarily caused by the allosteric effects of substrate concentration (homotropic allostery) and other drugs (heterotropic allostery). [6][7][8][9][10][11][12][13][14][15][16] Deeper insight into the microscopic process and potential key factors in CYP3A4-mediated drug metabolism can help us understand the allosteric effects, drug-drug interactions (DDIs), and offer detoxification response to toxic xenobiotics. 17,18 Many studies have used midazolam (MDZ) as a probe to investigate the complicated process of CYP3A4-mediated drug metabolism.…”

“…17,18 Many studies have used midazolam (MDZ) as a probe to investigate the complicated process of CYP3A4-mediated drug metabolism. [6][7][8][9][10][11][12][13] MDZ can be hydroxylated by CYP3A4 to two metabolites (1 0 -OH-MDZ and 4-OH-MDZ), and the ratio of the two metabolites can be allosterically modulated by substrate concentration (MDZ), testosterone (TST), progesterone (PGS), and other drugs (Fig. S1, ESI †).…”

“…S1, ESI †). [6][7][8][9][10][11][12][13][14][15] Effectors (MDZ, TST, and PGS) may bind in peripheral allosteric sites of CYP3A4, resulting in the accumulation of 4-OH-MDZ. 9,10,15 In our previous studies, 12,13 the binding of effectors MDZ and TST in peripheral allosteric sites of CYP3A4 (Site 1 and Site 2 in Fig.…”

“…[6][7][8][9][10][11][12][13][14][15] Effectors (MDZ, TST, and PGS) may bind in peripheral allosteric sites of CYP3A4, resulting in the accumulation of 4-OH-MDZ. 9,10,15 In our previous studies, 12,13 the binding of effectors MDZ and TST in peripheral allosteric sites of CYP3A4 (Site 1 and Site 2 in Fig. S2, ESI †) regulates the regioselectivity of MDZ metabolism primarily by inducing conformational changes in the flexible F 0 -F and B-C loops of CYP3A4.…”