{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Faculty of Biosciences",
    "Universitat Autònoma de Barcelona",
    "Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona",
    "Department of Research and Development",
    "Advanced BioDesign",
    "Institut de Biologia Molecular de Barcelona (IBMB-CSIC",
    "Parc Científic de Barcelona",
    "Centre for Genomic Regulation (CRG",
    "Barcelona Institute of Science and Technology",
    "Institució Catalana de Recerca i Estudis Avançats (ICREA",
    "Departament de Química Inorgànica i Orgànica and IQTCUB",
    "Universitat de Barcelona",
    "Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica and IQTCUB"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 12,
  "authors": [
    {
      "affs": [
        0,
        1
      ],
      "name": "Raquel Pequerul",
      "orcid": "0000-0002-0008-975X"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Daniela Covaleda",
      "orcid": "0009-0007-4919-0377"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Marc Serveto"
    },
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Sebastián Tanco",
      "orcid": "0000-0003-2130-4401"
    },
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Julia Lorenzo"
    },
    {
      "affs": [
        3,
        4
      ],
      "name": "Mileidys Pérez-Alea",
      "orcid": "0000-0002-8739-8087"
    },
    {
      "affs": [
        5,
        6,
        7,
        8
      ],
      "name": "David Vizarraga",
      "orcid": "0000-0002-4478-0155"
    },
    {
      "affs": [
        5,
        6
      ],
      "name": "Ignacio Fita",
      "orcid": "0000-0002-7521-2679"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Xavier Parés",
      "orcid": "0000-0002-5071-9465"
    },
    {
      "affs": [
        9,
        10,
        11
      ],
      "name": "Carme Rovira",
      "orcid": "0000-0003-1477-5010"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Jaume Farrés",
      "orcid": "0000-0001-9069-3987"
    },
    {
      "affs": [
        12,
        11
      ],
      "name": "Marina Corbella",
      "orcid": "0000-0001-9209-868X"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/97jdegx31bcdw.bib",
  "cited_by": 0,
  "doi": "10.26434/chemrxiv.15010967/v1",
  "fulltext_url": null,
  "id": "97jdegx31bcdw",
  "issns": [],
  "landing_url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010967/v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1312|Enzyme function and inhibition"
    ]
  ],
  "openalex": "W7223266301",
  "pdf_url": "https://chemrxiv.org/doi/pdf/10.26434/chemrxiv.15010967/v1",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-23",
  "publisher": "American Chemical Society (ACS)",
  "ris": "https://research.draconitic.com/paper/97jdegx31bcdw.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:26Z",
      "native_id": "10.26434/chemrxiv.15010967",
      "source": "chemrxiv",
      "url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010967/v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Enzyme function and inhibition",
      "subfield": "molecular-biology"
    }
  ],
  "title": "The inhibition mechanism of aldehyde dehydrogenase 1A3 by the antitumor drug DIMATE",
  "title_text": "The inhibition mechanism of aldehyde dehydrogenase 1A3 by the antitumor drug DIMATE",
  "type": "preprint",
  "updated_at": "2026-10-30T12:17:26Z",
  "url": "https://research.draconitic.com/paper/97jdegx31bcdw",
  "venue": "ChemRxiv",
  "versions": []
}