Heterotropic Cooperativity for CYP3A4‐Mediated 1′‐Hydroxylation of Midazolam by Berberine: An In Silico Modeling and Simulation Study
Authors: Jeevan GC B. , McDonald Matthew G. , Tian Dan‐Dan , Thummel Kenneth E. , Rettie Allan E. , Paine Mary F. , Natesan Senthil 
Published: June 2021

ObjectiveCytochrome P450 (CYP) 3A4, a membrane‐associated enzyme located primarily in the liver and small intestine, is involved in the oxidation of >50% of marketed drugs. Due to its broad substrate specificity and voluminous binding pocket, CYP3A4 can bind two or more drugs simultaneously, leading to non‐hyperbolic kinetics and heterotropic cooperativity, resulting in activation or inhibition of substrate metabolism. In vitro experiments involving human liver and intestinal microsomes suggested that a major constituent of goldenseal, berberine, both activates and inhibits CYP3A‐mediated 1′‐hydroxylation of midazolam at low (≤5 μM) and high (≥100 μM) concentrations, respectively. Molecular dynamics (MD) simulations were conducted to investigate the structural basis for the observed dual cooperativity effects of berberine on midazolam 1′‐hydroxylation. Similarities in structure and membrane partitioning between berberine and progesterone suggested the progesterone site as a potential allosteric site.MethodsAll‐atom MD simulations using explicit water were conducted using a midazolam‐bound crystal structure of CYP3A4 (PDB ID 5TE8) anchored in a 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) bilayer with its modeled transmembrane helix. Two additional systems were modeled: (1) berberine docked (using MOE software) in the progesterone binding allosteric site, topologically distinct from the midazolam binding site, and (2) berberine placed at its favorable bilayer depth, near the progesterone binding site. Each all‐atom MD simulation was conducted for 250 ns using NAMD2.12 software and CHARMM36 and CGenFF force fields.ResultsAnalyses of trajectories from all three MD simulations indicated that the conformational changes associated with the dynamics of the CYP3A4‐midazolam complex in which berberine was docked to the peripheral progesterone‐binding site were distinct from that shown in the other two simulations. Berberine caused notable conformational changes in the F‐F′ loop, increasing immobilization of midazolam (decrease in root‐mean‐square‐deviation) within the binding site, as well as decreasing the distance between the heme iron and C1 of midazolam, possibly resulting in activation (positive allosteric modulation). Additionally, a berberine molecule docked in the vicinity of the progesterone site entered the substrate binding site, altering the interaction of the midazolam molecule with the binding site residues to modulate metabolism. Berberine may also block the dissociation of 1′‐hydroxymidazolam from the binding site, causing inhibition (negative cooperativity).ConclusionComputational results provide a structural basis for the concentration‐dependent heterotropic cooperativity of CYP3A4 consistent with both activation and inhibition of midazolam 1′‐hydroxylation.Support or Funding InformationNational Institutes of Health ‐ R15GM13129 and U54AT008909This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.