# Mechanism-based inactivation of human aldehyde oxidase by erlotinib: Mechanistic insights from structural analogs and molecular docking

## TL;DR

Erlotinib and its metabolites irreversibly inactivate the enzyme AOX1, affecting drug metabolism.

## Contribution

The study reveals the mechanism of AOX1 inactivation by erlotinib using structural analogs and molecular docking.

## Key findings

- Erlotinib and its metabolites inactivate AOX1 in a time- and concentration-dependent manner.
- Molecular docking suggests covalent binding of erlotinib metabolites to the molybdenum cofactor of AOX1.
- Modifications at the C2-position of erlotinib affect inactivation potency and efficiency.

## Abstract

Aldehyde oxidase (AOX1) is a cytosolic molybdo-flavoenzyme that metabolizes azaheterocyclic drugs. Erlotinib and gefitinib are azaheterocyclic drugs. We deployed structural analogs to investigate the molecular interaction between these drugs and AOX1. Erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib, but not gefitinib, O-desmethylgefitinib, or O-desmorpholinopropylgefitinib, decreased carbazeran 4-oxidation by liver cytosol (human, rat, and mouse) and human recombinant AOX1. Erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib exhibited time- and concentration-dependent inactivation with unbound inactivation potency (KI,u) of 1.52, 4.41, and 1.67 μM, respectively. The inactivation was not reversed after dialysis, not protected by nucleophilic trapping agents or scavengers of reactive oxygen species, not affected by an oxidizing or reducing agent, but was attenuated by an alternative AOX1 substrate (O6-benzylguanine) and competitive AOX1 inhibitor (gefitinib). The terminal alkyne group of erlotinib was essential for AOX1 inactivation, as suggested by the findings for 3-vinylerlotinib (less potent inactivator) and tetrahydroerlotinib (no inactivation). Molecular docking results predicted covalent binding of erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib to the molybdenum cofactor. Adding a 4′-methyl group to erlotinib increased the inactivation potency but decreased inactivation efficiency, whereas blocking the C2-position of erlotinib with a hydroxy group or a methyl group decreased inactivation potency and efficiency, suggesting that the C2-position of erlotinib plays a role in AOX1 inactivation. In mice, erlotinib increased carbazeran (Aox substrate) and decreased 4-oxo-carbazeran (metabolite) levels in blood, liver, and kidneys. Overall, our study provides molecular insights into the mechanism-based inactivation of AOX1 by erlotinib, O-desmethylerlotinib, and O-didesmethylerlotinib and the irreversible AOX1 inactivation by erlotinib on the pharmacokinetics of AOX1-metabolized drugs.

This study shows that erlotinib and select metabolites are mechanism-based inactivators of AOX1, provides insights into the mechanism of the inactivation by deploying structural analogs and molecular docking, and demonstrates the in vivo impact on AOX1-mediated drug metabolism.