Biology subjects

Pequerul, R.

Publications and source records attributed to Pequerul, R..

4 recordsLinked to original sources

bioRxiv2026

A covalent irreversible inhibitor binds in two mutually exclusive conformations to the active-site cysteine residue of human aldehyde dehydrogenase 1A3

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family. Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 [A] resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/738401v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1d9ebd2org.highwire.dtl.DTLVardef@96470org.highwire.dtl.DTLVardef@a110c7org.highwire.dtl.DTLVardef@548a93_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Aldehyde dehydrogenase 1A3 detection in extracellular vesicles from breast cancer cell lines by nano-flow cytometry

1.Exosomes are nanosized extracellular vesicles that carry bioactive molecules reflective of their cells of origin. Developing methods to detect functional enzymatic activity within exosomes can provide a new generation of rapid and informative diagnostic tools. Aldehyde dehydrogenase (ALDH) enzymes, particularly ALDH1A3, are overexpressed in several cancers and contribute to tumor aggressiveness and drug resistance. However, their presence and functionality in cancer-derived exosomes remain poorly characterized. Here, we developed a nano-flow cytometry-based method to detect ALDH activity directly within individual exosomes derived from breast cancer cell lines (SKBR3, MDA-MB-231, and MCF7). Exosomes were isolated by differential ultracentrifugation and validated by nanoparticle tracking analysis, cryogenic transmission electron microscopy, and bead-based immunophenotyping of canonical markers. ALDH enzymatic activity was detected using a resorufin-based fluorescent substrate capable of crossing the exosomal membrane. To ensure specificity, assays were performed in the presence or absence of a selective ALDH inhibitor, confirming that the fluorescent signal originated from ALDH activity within the vesicles. This work provides the first functional evidence of ALDH1A3 enzymatic activity in cancer-derived exosomes and establishes a proof-of-concept platform for rapid, activity-based detection of exosomal enzymes, opening new perspectives for exosome-based diagnostics in breast cancer.

Cell Biology↗

bioRxiv2026

A sensitive fluorometric assay to detect aldo-keto reductase and carbonyl reductase activity based on a naphthaldehyde derivative

We have developed a fluorometric assay for detecting reductase activity in biological samples through 4-methoxy-1-naphthalenemethanol (MONOL-41) formation. The enzyme carbonyl reductase 1 (CBR1) and four members of the aldo-keto reductase (AKR) 1 family (AKR1A1, AKR1B1, AKR1B10, AKR1C3) were evaluated for their ability to reduce 4-methoxy-1-naphthaldehyde (MONAL-41). AKR1B1 and CBR1 followed Michaelis-Menten kinetics, whereas AKR1B10, AKR1A1, and AKR1C3 showed substrate inhibition above 10 {micro}M (70 {micro}M for AKR1C3). Among the tested enzymes, AKR1B10 displayed the highest catalytic efficiency in the absence of substrate inhibition. The MONOL-41 assay was compared with the standard NADPH-based method, showing improved sensitivity, robustness, and lower detection limits (0.77 {micro}g/mL vs. 1.49 {micro}g/mL). These results confirm its suitability for monitoring AKR1B10 activity. The assay was then applied to A549 cell extracts, which express multiple reductases. Activity decreased at substrate concentrations above 10 {micro}M, suggesting a predominant role of AKR1B10. Inhibition studies using tolrestat and high MONAL-41 concentrations indicated a limited contribution of CBR1 ([~]7-8%). Considering both catalytic efficiency and expression levels, AKR1B10 appears to be the main contributor to reductase activity in this model. In A549 living cells, MONAL-41 showed no cytotoxicity up to 50 {micro}M and enabled real-time monitoring due to its membrane permeability. However, oxidation by aldehyde dehydrogenases can generate MONOIC-41, which has similar spectral properties but a lower quantum yield, potentially affecting signal interpretation. Overall, this assay represents a sensitive and cost-effective tool for detecting reductase activity and screening inhibitors.

biochemistry↗

bioRxiv2024

ALDH1 subtype-specific inhibitor targets key cell populations in triple negative breast cancer

Aggressive breast cancer subtypes, particularly triple-negative breast cancer (TNBC), lack effective targeted therapies, requiring novel approaches. This study focuses on the aldehyde dehydrogenase 1A (ALDH1A) subfamily, comprising ALDH1A1, ALDH1A2, and ALDH1A3, and their roles in tumor biology and the tumor microenvironment. Comprehensive transcriptomic and single-cell analyses revealed subtype- and cell-specific expression patterns of ALDH1A isoforms, with ALDH1A3 predominantly expressed in epithelial cancer cells of basal-like tumors, while ALDH1A1 and ALDH1A2 were expressed in stromal and immune-associated subpopulations. Guided by these findings, we developed ABD0171, a selective ALDH1A3 inhibitor that demonstrated potent isoform-specific activity. ABD0171 effectively disrupted key pathways in TNBC cells in vitro, including IL6/JAK/STAT3, tPA and Src/FAK, and exhibited superior selectivity, robust antitumor and antimetastatic effects, and a favorable safety profile in vivo. These results establish ALDH1A3 as a promising therapeutic target and validate ABD0171 as a candidate to address current challenges in aggressive breast cancers.

cancer biology↗