Investigation of the molecular and mechanistic basis for the regioselective metabolism of midazolam by cytochrome P450 3A4

Fu, Tingting; Zheng, Qing‐Chuan; Zhang, Hongxing

doi:10.1039/d2cp00232a

Cited by 5 publications

(24 citation statements)

References 59 publications

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“…However, a minor product, 4OH-MDZ, is also generated by CYP3A4, and its fraction reaches ~35% at high MDZ concentrations. Therefore, the metabolism of MDZ is allosterically regulated, and the mechanism of the homotropic cooperative interactions of MDZ has been studied previously by many authors [ 38 , 44 , 45 , 46 , 47 , 48 , 49 ]. As described in our recent study [ 26 ], we performed the global analysis of the MDZ metabolism by CYP3A4 in nanodiscs and resolved the binding constants and fractions of 1OH and 4OH products formed when one or two MDZ molecules are bound.…”

Section: Introductionmentioning

confidence: 99%

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4

et al. 2022

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Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F’ loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

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Section: Introductionmentioning

confidence: 99%

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4

et al. 2022

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“…16,18 It was also found in our previous work that homotropic allostery can affect the ratio of MDZ metabolites by modulating the conformation of the "sandwich" structure. 16 On the other hand, CYP3A4-mediated MDZ metabolism also exhibits heterotropic allostery. 12,13 TST is another commonly used probe that regulates MDZ metabolism, as TST can promote the generation of the 4-OH-MDZ metabolite.…”

Section: Introductionmentioning

confidence: 57%

“…In this pose, the C1′ and C4 groups are almost parallel to heme (Fe−C1′ and Fe−C4 is about 5.0 Å), and the hydroxylation seems to be possible at both oxidation sites (1′-OH-MDZ and 4-OH-MDZ). According to our previous experience with homotropic allostery, 16 to further explore the binding diversity of MDZ in the CYP3A4 active site, we performed a 200 ns WT-MTD simulation and constructed the free energy landscape (Figure 5A). As illustrated in Figure 5A, three low-energy wells (a, b, and c) can be observed along the distance parameters (Fe−C1′ and Fe−C4), which indicates there are three types of MDZ- and it is clear that the hydroxylation at the C4 group is favorable.…”

Section: Mechanism Of Heterotropic Allostery In Mdz Metabolism 321 Re...mentioning

confidence: 99%

“…Midazolam (MDZ, shown in Figure S1) is considered as a typical probe for the estimation of CYP3A4-mediated DDIs, due to its special and complex metabolic profile as well as the high fraction (90%) metabolized by CYP3A4. − MDZ is metabolized by CYP3A4 to two metabolites (Figure S1): 1′-hydroxy (1′-OH-MDZ) and 4-hydroxy (4-OH-MDZ). − The ratio of metabolites (1′-OH-MDZ/4-OH-MDZ) can be modulated by substrate concentration and other drugs, which are known as homotropic and heterotropic allostery. − …”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Zheng

2022

J. Chem. Inf. Model.

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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.

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“…This implies that the MM/GBSA method may overestimate the charge-charge interactions and thus cause a large deviation between the calculated and experimental binding free energies. Additionally, the binding of any substrate to proteins should consider two aspects, i.e., thermodynamic and kinetic factors ( Camilloni and Pietrucci, 2018 ; c; 2020; Lyu et al, 2020 ; Zhang et al, 2020 ; Fu et al, 2022 ). The MM/GBSA method can only reflect the effects from the thermodynamic perspective, which might also result in some deviations in describing the substrate binding.…”

Section: Resultsmentioning

confidence: 99%

Exploring the Binding Mechanism of a Supramolecular Tweezer CLR01 to 14-3-3σ Protein via Well-Tempered Metadynamics

et al. 2022

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Using supramolecules for protein function regulation is an effective strategy in chemical biology and drug discovery. However, due to the presence of multiple binding sites on protein surfaces, protein function regulation via selective binding of supramolecules is challenging. Recently, the functions of 14-3-3 proteins, which play an important role in regulating intracellular signaling pathways via protein–protein interactions, have been modulated using a supramolecular tweezer, CLR01. However, the binding mechanisms of the tweezer molecule to 14-3-3 proteins are still unclear, which has hindered the development of novel supramolecules targeting the 14-3-3 proteins. Herein, the binding mechanisms of the tweezer to the lysine residues on 14-3-3σ (an isoform in 14-3-3 protein family) were explored by well-tempered metadynamics. The results indicated that the inclusion complex formed between the protein and supramolecule is affected by both kinetic and thermodynamic factors. In particular, simulations confirmed that K214 could form a strong binding complex with the tweezer; the binding free energy was calculated to be −10.5 kcal·mol−1 with an association barrier height of 3.7 kcal·mol−1. In addition, several other lysine residues on 14-3-3σ were identified as being well-recognized by the tweezer, which agrees with experimental results, although only K214/tweezer was co-crystallized. Additionally, the binding mechanisms of the tweezer to all lysine residues were analyzed by exploring the representative conformations during the formation of the inclusion complex. This could be helpful for the development of new inhibitors based on tweezers with more functions against 14-3-3 proteins via modifications of CLR01. We also believe that the proposed computational strategies can be extended to understand the binding mechanism of multi-binding sites proteins with supramolecules and will, thus, be useful toward drug design.

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Investigation of the molecular and mechanistic basis for the regioselective metabolism of midazolam by cytochrome P450 3A4

Abstract: Cytochrome P450 3A4 (CYP3A4) is the most important P450 enzyme for drug metabolism and drug-drug interaction, due to it is responsible for the biotransformation of approximately 50% of clinically used...

Cited by 5 publications

References 59 publications

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4

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

Exploring the Binding Mechanism of a Supramolecular Tweezer CLR01 to 14-3-3σ Protein via Well-Tempered Metadynamics

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