A Covalently Attached Progesterone Molecule Outcompetes the Binding of Free Progesterone at an Allosteric Site of Cytochrome P450 3A4

Abstract

Because of its exceptional substrate promiscuity, human P450 3A4 (CYP3A4) is arguably the most important drug-metabolizing enzyme. CYP3A4 also has the particularity of binding multiple ligands simultaneously, which is associated with heterotropic or homotropic, positive or negative, cooperativity or allostery. Solving the kinetics of such complex systems remains challenging, and so is identifying the binding pockets involved. Progesterone (PRG) is a known allosteric activator of CYP3A4-catalyzed 7-benzyloxy-4-trifluoromethylcoumarin (BFC) debenzylation. We report herein the use of bioconjugation as a successful strategy to identify this PRG allosteric site. A progesterone analogue (PGM) was covalently attached, separately at several locations, near a peripheral binding pocket previously proposed to be an allosteric site. Studies of BFC debenzylation in the presence of free PRG revealed that two of the bioconjugates successfully positioned the covalently attached PGM moiety in a way that mimcs the allosteric activation observed with free PRG. Interestingly, the PGM bioconjugate with the better fit yielded a higher permanent activation of the enzyme.

Combining small-molecule bioconjugation and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to expose allostery: the case of human cytochrome P450 3A4

“…18,26,27 Above this concentration, progesterone was reported to compete with the substrate for oxidation. 18,28 If free progesterone is binding to the allosteric site at concentrations less than 25 m, we reasoned that the differential HDX profile of unlabeled CYP3A4 F215C (known to be activated by progesterone) in the presence and absence of progesterone (20 M) should resemble the HDX profiles of the allostery-mimicking bioconjugates. Surprisingly, progesterone binding to F215C resulted in H/D uptake behavior that contrasted with the F215C-PGM conjugate in several critical regions of the protein ( Figure 5, Figure S9).…”

“…We recently demonstrated that, as expected for a selective binding process, the impact of bioconjugation of a maleimide-containing progesterone derivative (PGM) on enzyme kinetics varies greatly depending on the location of the attachment point, which affects binding orientation. 17,18 The CYP3A4 F108C-PGM and F215C-PGM bioconjugates were found to effectively mimic progesterone allosteric activation towards 7-benzyloxy-4-trifluoromethylcoumarin (BFC) oxidation. 18 We also constructed a PGM conjugate for which allosteric activation was antagonized (G481C-PGM), and a conjugate (L482C-PGM) which emulated activation without the PGM moiety occupying the allosteric site (agonist-like).…”

“…17,18 The CYP3A4 F108C-PGM and F215C-PGM bioconjugates were found to effectively mimic progesterone allosteric activation towards 7-benzyloxy-4-trifluoromethylcoumarin (BFC) oxidation. 18 We also constructed a PGM conjugate for which allosteric activation was antagonized (G481C-PGM), and a conjugate (L482C-PGM) which emulated activation without the PGM moiety occupying the allosteric site (agonist-like). 18 Given the interesting differences in these functional effects, we reasoned that this collection of CYP3A4-PGM bioconjugates would enable a robust HDX-MS characterization of allosteric mechanisms in CYP3A4.…”

“…18 We also constructed a PGM conjugate for which allosteric activation was antagonized (G481C-PGM), and a conjugate (L482C-PGM) which emulated activation without the PGM moiety occupying the allosteric site (agonist-like). 18 Given the interesting differences in these functional effects, we reasoned that this collection of CYP3A4-PGM bioconjugates would enable a robust HDX-MS characterization of allosteric mechanisms in CYP3A4. The results presented herein reveal that, as expected, the conformational dynamics of CYP3A4 are significantly affected by the location of the covalently-tethered PGM ligand.…”

Alcohol-Induced Increase in the Content of CYP2E1 in Human Liver Microsomes Causes a Multifold Activation of CYP3A4 and Attenuates its Cooperativity

“…More recent studies compellingly demonstrated the presence of a separate allosteric effector binding site and revealed its role in the instances of heterotropic activation of CYP3A4 by various effectors, including ANF (Davydov et al, 2012;Davydov et al, 2013;Polic and Auclair, 2017;Denisov et al, 2018;Marsch et al, 2018;Denisov et al, 2019;Ducharme et al, 2019). This allosteric site is formed at the vicinity of the F' and G' helices and located at the interface between the two CYP3A4 molecules in the crystallographic dimer of CYP3A4 complex with peripherally-bound progesterone (PDB 1W0F (Williams et al, 2004)).…”