# Functional Dynamics of Cytochrome P4503A4

> **NIH NIH R01** · UNIVERSITY OF WASHINGTON · 2021 · $414,701

## Abstract

Project Summary
The proposed work aims to clarify longstanding mechanistic uncertainties about drug metabolizing cytochrome
P450s (CYPs). These CYPs are extraordinarily substrate promiscuous and they are major determinants of
xenobiotic detoxication, drug metabolism and drug interactions. Because of their role in drug metabolism, they
are also drug targets, wherein their pharmacological manipulation can afford control of therapy with other drugs.
One focus here is the knowledge gap concerning the relationship between ligand-dependent heme spin state
and catalytic properties. In the CYP canonical catalytic reaction cycle developed for substrate specific
isoforms, the substrate displaces heme bound water and causes a shift from low spin to high spin heme with
concomitant shift in the heme properties that facilitate reduction and progress through the catalytic cycle. In
contrast, with drug metabolizing CYPs, many drugs cause no shift to high spin heme, but they are efficiently
metabolized or cause hydrogen peroxide formation, both of which require progression through the catalytic
cycle. Recent results demonstrate that many of these substrates hydrogen bond to the axial water, rather than
displace it. The catalytic properties of these water-bridged complexes have not been determined. Therefore,
the proposed work will determine for CYP3A4 the heme redox properties and the catalytic competence of
water-bridged complexes, using computational approaches and advanced biochemical methods including
spectropotentiometry and stopped-flow reaction kinetics. Computational approaches will also be employed to
understand the effect of water-bridged complexes on heme reduction processes.
A second focus of the proposed work aims to clarify the role of conformational dynamics in the complex
allosteric behavior of CYPs and their remarkable substrate promiscuity. Both traits are linked to the protein
dynamics, which remain poorly characterized, and unclarified by the available crystal structures. The allosteric
properties confound prediction of drug clearance and drug interactions, so there is great interest in translational
models that better predict drug interactions based on refined allosteric models. Here, the experimental
methods of hydrogen-deuterium exchange mass spectrometry (H/DX) and pre-steady state ligand binding
methods with CYP3A4 in lipid bilayer nanodiscs are combined with computational approaches such as
accelerated Molecular Dynamics simulations (aMD) and steered molecular dynamics. The proposed studies fill
a significant gap in understanding the, previously experimentally inaccessible, CYP dynamics in a lipid
membrane and the role of conformational dynamics in achieving substrate promiscuity and allostery.

## Key facts

- **NIH application ID:** 10205098
- **Project number:** 5R01GM130810-04
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** WILLIAM M ATKINS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $414,701
- **Award type:** 5
- **Project period:** 2018-09-18 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10205098

## Citation

> US National Institutes of Health, RePORTER application 10205098, Functional Dynamics of Cytochrome P4503A4 (5R01GM130810-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10205098. Licensed CC0.

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