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Fluorescent substrates enable specific detection and structure-function insights into human aldehyde dehydrogenase

Raquel Pequerul1, Daniela Covaleda1, Andrés S Sánchez-Vaca1

  • 1Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Barcelona, 08193, Spain.

Chemico-Biological Interactions
|June 20, 2025
PubMed
Summary

New assays using specific substrates can now differentiate aldehyde dehydrogenase (ALDH) enzyme activity, improving cancer research and drug targeting by identifying specific ALDH isoforms.

Keywords:
Breast cancerEnzyme inhibitionFluorescenceKinetic propertiesOxidoreductasesStructure-function relationships

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Area of Science:

  • Biochemistry
  • Enzymology
  • Cancer Biology

Background:

  • Aldehyde dehydrogenase (ALDH) isoforms are crucial biomarkers and drug targets in cancer research.
  • Current assays lack specificity, failing to distinguish between different ALDH isoforms.
  • This limits accurate quantitation of ALDH activity in biological samples.

Purpose of the Study:

  • To kinetically characterize purified ALDH isoforms using novel fluorogenic substrates.
  • To develop specific assays for identifying and quantifying individual ALDH isoforms.
  • To apply these assays for detecting ALDH activity in cancer cells.

Main Methods:

  • Full kinetic characterization of recombinant ALDH isoforms (ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH3A1).
  • Utilized fluorogenic substrates: BODIPY™-aminoacetaldehyde (BAAA), ALDEFLUOR™ reagent, 6-methoxy-2-naphthaldehyde (MONAL-62), and 7-methoxy-1-naphthaldehyde (MONAL-71).
  • In silico simulations of substrate docking and inhibitor selectivity assays (DIMATE, ABD0171) were performed.

Main Results:

  • ALDH1A isoforms showed activity with BAAA; ALDH3A1 did not.
  • Significant kinetic differences observed with naphthaldehyde derivatives across isoforms.
  • MONAL-62 demonstrated high sensitivity (2 amol/µL for ALDH1A1); MONAL-71 showed high specificity for ALDH1A1.
  • Assays successfully detected ALDH isoform activity in triple-negative breast cancer cells.

Conclusions:

  • Novel naphthaldehyde substrates enable specific detection and kinetic characterization of ALDH isoforms.
  • High sensitivity and specificity achieved, particularly with MONAL-62 and MONAL-71.
  • Method applicable for isoform identification in complex biological samples, including cancer cell extracts.