# Lipid Dependence of CYP3A4 Activity in Nanodiscs

## TL;DR

This study shows that the activity of the CYP3A4 enzyme, important for drug metabolism, is influenced by the type of lipids in its membrane environment, depending on the substrate used.

## Contribution

The study reveals that lipid composition affects CYP3A4 activity differently based on the substrate, highlighting the enzyme's membrane interactions.

## Key findings

- Lipid composition significantly affects CYP3A4 activity with progesterone but not with midazolam.
- Nanodiscs with liver polar extract and phosphatidylserine showed the highest CYP3A4 activity.
- Midazolam hydroxylation ratios were unaffected by lipid composition, suggesting no allosteric perturbation.

## Abstract

About a dozen cytochrome P450 enzymes metabolize more than a half of all pharmaceuticals in the human liver. The most important of these enzymes is CYP3A4, which is involved in removal of hundreds of drugs and other chemical compounds from the human body. Cytochrome P450 enzymes are connected to the lipid membranes and various lipid head groups affect their functional properties. We used CYP3A4 incorporated in the soluble membrane Nanodiscs with controlled lipid compositions to probe the effect of various lipids on activity of this enzyme. Our results show that the effect depends on the structure of substrate used for the functional studies. Very small changes were detected with the specific substrate midazolam, which is used as a probe in clinical practice. However, a significant dependence of activity of CYP3A4 on the lipid composition was observed with progesterone as a substrate, because of modulation of the enzyme functional properties by the binding of this molecule at the protein–membrane interface. These novel results shed light on the important role of CYP3A4 interactions with the lipid membrane in drug metabolism studies.

All human cytochrome P450 enzymes are incorporated into a membrane with important functional implications. In some P450 enzymes, interactions of the F-G loops with lipid head groups create a peripheral allosteric site at this protein–lipid interface. In addition, the redox potential of CYP3A4 and cytochrome P450 reductase (CPR) is modulated by negatively charged lipids in the membrane. In order to investigate the effect of lipid headgroups on human CYP3A4 activity, we studied the rate of NADPH oxidation and product formation using progesterone and midazolam as substrates, assembled into Nanodiscs with different lipid compositions. Preparation of a polar lipid extract provided a modest acceleration of both NADPH oxidation and the rate of progesterone hydroxylation, compared to the zwitterionic palmitoyl-oleoyl phosphatidyl choline (POPC). The highest activity was found in Nanodiscs assembled with liver polar extract lipids together with negatively charged phosphatidylserine (PS). No effect of lipid composition was detected for the regiospecific 1OH/4OH hydroxylation ratio of midazolam (MDZ) metabolites, indicating the absence of membrane composition perturbation of the allosteric effects in MDZ hydroxylation. The magnitude of the observed effect of lipid composition on the catalytic rate of CYP3A4 strongly depends on the substrate used as a probe.