Midazolam as a Probe for Drug–Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation

Abstract

We developed an efficient and sensitive probe for drug–drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F–F′ loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug–drug interactions in human P450 CYP3A4.

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

“…Very recently, Sligar et al . reported the allosteric pathway in midazolam (MDZ) hydroxylation by CYP3A4, showing that the allostery could also alter the product pattern (1OH vs. 4OH of MDZ) [40] . Hackett, Atkins and the co‐workers described the allosteric interactions at the molecular level with the self‐assembled CYP3A4 nanodiscs.…”

“…[38,39] Very recently, Sligar et al reported the allosteric pathway in midazolam (MDZ) hydroxylation by CYP3A4, showing that the allostery could also alter the product pattern (1OH vs. 4OH of MDZ). [40] Hackett, Atkins and the co-workers described the allosteric interactions at the molecular level with the self-assembled CYP3A4 nanodiscs. They found that the allosteric ligand remained dynamic at the Phe-cluster binding site in the FÀ G region, which located at the interface of enzyme and membrane.…”

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

“…Two of these molecules are accommodated inside the substrate-binding pocket, while the third interacts with CYP3A4 at the protein–membrane interface at the pocket formed between the F-F’ and G-G’ loops on one side and lipid head groups on another. This was confirmed by the effect of mutations at the residues 212–215 [ 25 , 26 ], and by the binding of covalently linked dimers of TST at the substrate-binding pocket and the allosteric effect caused by TST monomer [ 28 ]. The binding of TST and PGS at this peripheral site with high affinity is also corroborated by two X-ray structures [ 29 , 30 ].…”

“…Importantly, experimental protocols for in vitro studies should provide quantitative and reproducible measurements of essential biochemical parameters, such as catalytic rates and binding constants. The incorporation of heterologously expressed CYP3A4 in lipid nanodiscs allows one to obtain an array of data supporting the general mechanistic understanding of homotropic and heterotropic cooperativity in CYP3A4 [ 10 , 11 , 24 , 25 , 26 , 27 ] by defining the inherent properties of states with differing number of molecules bound. Based on previous results obtained with substrates and effectors such as (i) testosterone (TST) and alpha-naphthoflavone (ANF) [ 27 ]; (ii) progesterone (PGS) and carbamazepine [ 11 , 25 ]; and (iii) atorvastatin and dronedarone [ 10 ], the general picture of homotropic and heterotropic interactions of substrates and effectors in CYP3A4 is available.…”

“…Such a protocol should distinguish between the direct competitive inhibition of the metabolism of the probe substrate, and various effects (inhibition, activation, or a change in the site of metabolism) caused by interactions with the allosteric site. It should be also relatively simple and easy to scale up to high throughput screening, unlike our own global analysis approach, described above, which provides a detailed mechanistic understanding of heterotropic interactions but requires multiple experiments performed with highly purified structurally homogeneous preparations of CYP3A4 [ 10 , 11 , 24 , 25 , 26 ].…”