The inhibition mechanism of aldehyde dehydrogenase 1A3 by the antitumor drug DIMATE
13 affiliations
- 1Faculty of Biosciences
- 2Universitat Autònoma de Barcelona
- 3Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona
- 4Department of Research and Development
- 5Advanced BioDesign
- 6Institut de Biologia Molecular de Barcelona (IBMB-CSIC
- 7Parc Científic de Barcelona
- 8Centre for Genomic Regulation (CRG
- 9Barcelona Institute of Science and Technology
- 10Institució Catalana de Recerca i Estudis Avançats (ICREA
- 11Departament de Química Inorgànica i Orgànica and IQTCUB
- 12Universitat de Barcelona
- 13Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica and IQTCUB
Abstract
Aldehyde dehydrogenase 1A (ALDH1A) enzymes play central roles in retinoic acid signaling and cancer stem cell biology, yet the molecular determinants governing isoform-selective covalent inhibition remain poorly understood. Here we investigate the binding and reactivity of the activated alkyne inhibitor S-methyl-4-(dimethylamino)-4-methylpent-2-ynethioate (DIMATE), a potent, irreversible inhibitor of ALDH1A currently in phase I clinical trials. Using QM/MM metadynamics simulations based on high-resolution crystal structures, enzyme kinetics and site-directed mutagenesis, we found that active-site cysteine reactivity and cofactor-binding channel occupancy control whether the inhibitor binds reversibly or irreversibly to the ALDH1A3 isoform. We identified two reaction pathways initiated by nucleophilic attack from the catalytic Cys314: transthioesterification at the carbonyl carbon and α-addition to the alkyne. Only the α-addition leads to irreversible inhibition, a process that critically depends on a vicinal cysteine (Cys313) acting as a proton donor. Consistently, experiments show that ALDH1A isoforms lacking this neighboring cysteine, as well as Cys313Ala mutants, display exclusively reversible inhibition. NAD+ binding further modulates reactivity by disfavoring α-addition and promoting hydrolysis of DIMATE to a non-inhibitory carboxylic acid product, establishing NAD+ as a switch between irreversible covalent modification and reversible turnover. Together, these results reveal how vicinal cysteine chemistry and cofactor-dependent activesite dynamics govern covalent reactivity in ALDH1A enzymes, providing a basis for the rational design of isoform-selective covalent inhibitors targeting cancer-associated ALDH enzymes.
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BibTeX
@misc{pequerul2026inhibition,
title = {{The inhibition mechanism of aldehyde dehydrogenase 1A3 by the antitumor drug DIMATE}},
author = {Raquel Pequerul and Daniela Covaleda and Marc Serveto and Sebastián Tanco and Julia Lorenzo and Mileidys Pérez-Alea and David Vizarraga and Ignacio Fita and Xavier Parés and Carme Rovira and Jaume Farrés and Marina Corbella},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15010967/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010967/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - The inhibition mechanism of aldehyde dehydrogenase 1A3 by the antitumor drug DIMATE
AU - Raquel Pequerul
AU - Daniela Covaleda
AU - Marc Serveto
AU - Sebastián Tanco
AU - Julia Lorenzo
AU - Mileidys Pérez-Alea
AU - David Vizarraga
AU - Ignacio Fita
AU - Xavier Parés
AU - Carme Rovira
AU - Jaume Farrés
AU - Marina Corbella
PY - 2026
DA - 2026/10/23
PB - ChemRxiv
DO - 10.26434/chemrxiv.15010967/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010967/v1
AB - Aldehyde dehydrogenase 1A (ALDH1A) enzymes play central roles in retinoic acid signaling and cancer stem cell biology, yet the molecular determinants governing isoform-selective covalent inhibition remain poorly understood. Here we investigate the binding and reactivity of the activated alkyne inhibitor S-methyl-4-(dimethylamino)-4-methylpent-2-ynethioate (DIMATE), a potent, irreversible inhibitor of ALDH1A currently in phase I clinical trials. Using QM/MM metadynamics simulations based on high-resolution crystal structures, enzyme kinetics and site-directed mutagenesis, we found that active-site cysteine reactivity and cofactor-binding channel occupancy control whether the inhibitor binds reversibly or irreversibly to the ALDH1A3 isoform. We identified two reaction pathways initiated by nucleophilic attack from the catalytic Cys314: transthioesterification at the carbonyl carbon and α-addition to the alkyne. Only the α-addition leads to irreversible inhibition, a process that critically depends on a vicinal cysteine (Cys313) acting as a proton donor. Consistently, experiments show that ALDH1A isoforms lacking this neighboring cysteine, as well as Cys313Ala mutants, display exclusively reversible inhibition. NAD+ binding further modulates reactivity by disfavoring α-addition and promoting hydrolysis of DIMATE to a non-inhibitory carboxylic acid product, establishing NAD+ as a switch between irreversible covalent modification and reversible turnover. Together, these results reveal how vicinal cysteine chemistry and cofactor-dependent activesite dynamics govern covalent reactivity in ALDH1A enzymes, providing a basis for the rational design of isoform-selective covalent inhibitors targeting cancer-associated ALDH enzymes.
ER -