Combining Small-Molecule Bioconjugation and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to Expose Allostery: the Case of Human Cytochrome P450 3A4.

We report a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). This strategy avoids issues related to weak substrate binding and ligand relocalization. The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4), the most important drug-metabolizing enzyme. Allosteric activation and inhibition of CYP3A4 by pharmaceuticals is an important mechanism of drug interactions. We performed HDX-MS analysis on several CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic) or do not show activation while still blocking the allosteric site (antagonistic). This allowed us to better define the position of the allosteric site, the protein structural dynamics associated with allosteric activation, and the presence of coexisting conformers.

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

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 mimics the allosteric activation observed with free PRG. Interestingly, the PGM bioconjugate with the better fit yielded a higher permanent activation of the enzyme.

Steroid bioconjugation to a CYP3A4 allosteric site and its effect on substrate binding and coupling efficiency.

Human cytochrome P450 3A4 (CYP3A4) is an important drug metabolizing enzyme involved in a number of drug-drug and food-drug interactions. As such, much effort has been devoted into investigating its mechanism of interaction with ligands. CYP3A4 has one of the highest levels of substrate promiscuity for an enzyme, and can even bind multiple ligands simultaneously. The location and orientation of these ligands depend on the chemical structure and stoichiometry, and are generally poorly understood. In the case of the steroid testosterone, up to three copies of the molecule can associate with the enzyme at once, likely two in the active site and one at a postulated allosteric site. Recently, we demonstrated that steroid bioconjugation at the allosteric site results in an increase in activity of CYP3A4 toward testosterone and 7-benzyloxy-4-trifluoromethylcoumarin oxidation. Here, using the established bioconjugation methodology, we show how steroid bioconjugation at the allosteric site affects the heme spin state, the binding affinity (KS) of CYP3A4 for testosterone, as well as the enzyme coupling efficiency.

Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation.

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of the majority of drugs. As such, it is implicated in many adverse drug-drug and food-drug interactions, and is of significant interest to the pharmaceutical industry. This enzyme is known to simultaneously bind multiple ligands and display atypical enzyme kinetics, suggestive of allostery and cooperativity. As well, evidence of a postulated peripheral allosteric binding site has provoked debate around its significance and location. We report the use of bioconjugation to study the significance of substrate binding at the proposed allosteric site and its effect on CYP3A4 activity. CYP3A4 mutants were created and covalently modified with various small molecules including progesterone. The labeled mutants displayed enhanced kinetic stability and improved activity in testosterone and 7-benzyloxy-(4-trifluoromethyl)coumarin oxidation assays. Our work applies a new strategy to study cytochrome P450 allostery and supports the hypothesis that substrate binding at the postulated allosteric site of CYP3A4 may induce functional cooperativity.

Use of chemical auxiliaries to control p450 enzymes for predictable oxidations at unactivated C-h bonds of substrates.

Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio- and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drug-metabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme.

Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis.

P450 enzymes (P450s) are well known for their ability to oxidize unactivated CH bonds with high regio- and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications.