Fluorescent substrates enable specific detection and structure-function insights into human aldehyde dehydrogenase isoforms
Pequerul Pavón, Raquel (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Covaleda, Daniela (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Sánchez-Vaca, Andrés S. (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Torres, Laura (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Constantinescu, Andrada (Advanced BioDesign-ABD. Unit of Research in Cellular and Molecular Biology)
Pérez-Alea, Mileidys (Advanced BioDesign-ABD. Unit of Research in Cellular and Molecular Biology)
Parés i Casasampera, Xavier (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Farrés, Jaume (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Date:	2025
Abstract:	Aldehyde dehydrogenase (ALDH) isoforms are widely used as biomarkers and potential drug targets in cancer research. Quantitation of ALDH enzymatic activity in biological samples often relies on the use of commercially available assays that are quite unspecific and do not discriminate between the various ALDH isoforms. The availability of highly purified recombinant ALDH isoforms allowed us to perform a full kinetic characterization of ALDH isoforms with fluorogenic substrates, BODIPY™-aminoacetaldehyde (BAAA), the ALDEFLUOR™ assay reagent, and two naphthaldedyde derivatives, 6-methoxy-2-naphthaldehyde (MONAL-62) and 7-methoxy-1-naphthaldehyde (MONAL-71). All ALDH1A isoforms were active to different extend with BAAA, while ALDH3A1 did not show any activity. Remarkable kinetic differences between ALDH1A1, ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1 were observed with naphthaldehyde derivatives. Exquisite sensitivity was attained with MONAL-62 with a lower detection limit of 2 amol or 10 molecules of enzyme per microliter for ALDH1A1. The high substrate specificity of ALDH1A1 for MONAL-71 provides an alternative assay for the unambiguous identification of this isoform. Enzymatic properties of isoforms were accounted for by in silico simulations of substrate docking to the active site of ALDH structures. In addition to substrate specificity, inhibitor selectivity of each isoform, as assessed by incubation with DIMATE and ABD0171 inhibitors, provided additional information about isoform composition in low-activity samples isolated from cell extracts. The method was successfully applied to the detection of ALDH isoform activity in triple-negative breast cancer cells.
Grants:	Agencia Estatal de Investigación PID2020-119424RB-I00 Agencia Estatal de Investigación PID2023-150696NB-I00 Agència de Gestió d'Ajuts Universitaris i de Recerca 2021/SGR-00135
Note:	Altres ajuts: acords transformatius de la UAB
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Breast cancer ; Enzyme inhibition ; Fluorescence ; Kinetic properties ; Oxidoreductases ; Structure-function relationships
Published in:	Chemico-biological interactions, Vol. 418 (September 2025) , art. 111609, ISSN 1872-7786

DOI: 10.1016/j.cbi.2025.111609

9 p, 3.3 MB

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