Structural and biochemical evidence that ATP inhibits the cancer biomarker human aldehyde dehydrogenase 1A3

Human aldehyde dehydrogenase (ALDH) participates in the oxidative stress response and retinoid metabolism, being involved in several diseases, including cancer, diabetes and obesity. The ALDH1A3 isoform has recently elicited wide interest because of its potential use as a cancer stem cell biomarker and drug target. We report high-resolution three-dimensional ALDH1A3 structures for the apo-enzyme, the NAD+ complex and a binary complex with ATP. Each subunit of the ALDH1A3-ATP complex contains one ATP molecule bound to the adenosine-binding pocket of the cofactor-binding site. The ATP complex also shows a molecule, putatively identified as a polyethylene glycol aldehyde, covalently bound to the active-site cysteine. This mimics the thioacyl-enzyme catalytic intermediate, which is trapped in a dead enzyme lacking an active cofactor. At physiological concentrations, ATP inhibits the dehydrogenase activity of ALDH1A3 and other isoforms, with a Ki value of 0.48 mM for ALDH1A3, showing a mixed inhibition type against NAD+. ATP also inhibits esterase activity in a concentration-dependent manner. The current ALDH1A3 structures at higher resolution will facilitate the rational design of potent and selective inhibitors. ATP binding to ALDH1A3 enables activity modulation by the energy status of the cell and metabolic reprogramming, which may be relevant in several disease conditions

Inhibitors of aldehyde dehydrogenases of the 1A subfamily as putative anticancer agents : Kinetic characterization and effect on human cancer cells

Aldehyde dehydrogenases (ALDHs) are enzymes catalyzing the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. High ALDH activity has been related to some important features of cancer stem cells. ALDH1A enzymes, involved in the retinoic acid signaling pathway, are promising drug targets for cancer therapy, and the design of selective ALDH1A inhibitors has a growing pharmacological interest. In the present work, two already known compounds (DEAB and WIN 18,446) and novel thiazolidinedione and pyrimido quinoline acetic acid derivatives (compounds 5a and 64, formerly described as aldo-keto reductase inhibitors) were tested as inhibitors of the ALDH1A enzymes (namely, ALDH1A1, ALDH1A2 and ALDH1A3) as a first step to develop some potential drugs for cancer therapy. The inhibitory capacity of these compounds against the ALDH1A activity was characterized in vitro by using purified recombinant proteins. The IC50 values of each compound were determined indicating that the most potent inhibitors against ALDH1A1, ALDH1A2 and ALDH1A3 were DEAB, WIN 18,446 and compound 64, respectively. Type of inhibition and Ki values were determined for DEAB against ALDH1A1 (competitive, Ki = 0.13 μM) and compound 64 against ALDH1A3 (non-competitive, Ki = 1.77 μM). The effect of these inhibitors on A549 human lung cancer cell viability was assessed, being compound 64 the only inhibitor showing an important reduction of cell survival. We also tested the effect of the ALDH substrate, retinaldehyde, which was cytotoxic above 10 μM. This toxicity was enhanced in the presence of DEAB. Both DEAB and compound 64 were able to inhibit the ALDH1A activity in A549 cells. The current work suggests that, by blocking ALDH activity, drug inactivation may be avoided. Thus these results may be relevant to design novel combination therapies to fight cancer cell chemoresistance, using both enzyme inhibitors and chemotherapeutic agents

Structural and kinetic features of aldehyde dehydrogenase 1A (ALDH1A) subfamily members, cancer stem cell markers active in retinoic acid biosynthesis

Aldehyde dehydrogenases catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower Km values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of kcat/Km. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity

Design, synthesis, biological evaluation and in silico study of benzyloxybenzaldehyde derivatives as selective aldh1a3 inhibitors

Altres ajuts: This research was funded by Al-Zaytoonah University of Jordan, grant number 2019- 2018/18/03 and by UoB International Development Fund Scheme PhD, grant code studentship to E.B. R.J. is a recipient of a PIF predoctoral fellowship from Universitat Autònoma de Barcelona.Aldehyde dehydrogenase 1A3 (ALDH1A3) has recently gained attention from researchers in the cancer field. Several studies have reported ALDH1A3 overexpression in different cancer types, which has been found to correlate with poor treatment recovery. Therefore, finding selective inhibitors against ALDH1A3 could result in new treatment options for cancer treatment. In this study, ALDH1A3-selective candidates were designed based on the physiological substrate resemblance, synthesized and investigated for ALDH1A1, ALDH1A3 and ALDH3A1 selectivity and cytotoxicity using ALDH-positive A549 and ALDH-negative H1299 cells. Two compounds (ABMM- 15 and ABMM-16), with a benzyloxybenzaldehyde scaffold, were found to be the most potent and selective inhibitors for ALDH1A3, with IC50 values of 0.23 and 1.29 μM, respectively. The results also show no significant cytotoxicity for ABMM-15 and ABMM-16 on either cell line. However, a few other candidates (ABMM-6, ABMM-24, ABMM-32) showed considerable cytotoxicity on H1299 cells, when compared to A549 cells, with IC50 values of 14.0, 13.7 and 13.0μM, respectively. The computational study supported the experimental results and suggested a good binding for ABMM- 15 and ABMM-16 to the ALDH1A3 isoform. From the obtained results, it can be concluded that benzyloxybenzaldehyde might be considered a promising scaffold for further drug discovery aimed at exploiting ALDH1A3 for therapeutic intervention

Structural and kinetic features of three human aldehyde dehydrogenases, ALDH1A1, ALDH1A2 and ALDH1A3, active in retinoic acid biosynthesis

La superfamília de les aldehid deshidrogenases (ALDH) comprèn un elevat nombre de proteïnes dimèriques i tetramèriques, amb un pes molecular aproximat de 55 kDa per subunitat i amb diferents localitzacions cel·lulars (citoplasmàtica, mitocondrial i reticle endoplasmàtic). Les ALDH inclouen un grup evolutivament relacionat d’enzims dependents de NAD(P)+, que catalitzen l’oxidació d’un ampli espectre de substrats aldehídics, generats a partir de diversos precursors endògens i exògens, als seus corresponents àcids carboxílics. Aquesta Tesi Doctoral forma part dels estudis estructurals i funcionals realitzats pel nostre grup de recerca sobre el paper de les oxidoreductases en el metabolisme dels retinoides. En aquests estudis s’han determinat les seves constants catalítiques amb aquests substrats, després de la solubilització amb albúmina sèrica bovina i mitjançant l’anàlisi d’activitat utilitzant una metodologia basada en HPLC. La Tesi pretén realitzar una anàlisi estructural i cinètica, exhaustiva i robusta, sobre els enzims humans implicats en l’oxidació irreversible del retinaldehid a àcid retinoic. La primera part tracta sobre la comparació de la butxaca d’unió al substrat dels enzims humans ALDH1A, els quals van mostrar topologies similars i volums decreixents en les seves butxaques d’unió al substrat. Els tres enzims van ser subclonats, expressats i purificats en la seva forma soluble i activa. Pel que fa als valors de kcat/Km amb alcanals i alquenals, ALDH1A3 és l’enzim que presenta els valors més baixos per a tots els substrats, suggerint un paper moderat en l’oxidació fisiològica d’aquests aldehids. Els valors de kcat/Km d’ALDH1A1 i ALDH1A2 indiquen un paper potencialment important en la transformació d’aquests aldehids, encara que amb una especificitat de substrat lleugerament diferent. Per mesurar l’activitat de les ALDH1A amb un substrat fisiològic, el retinal, es va realitzar una optimització de la metodologia d’extracció en solvents orgànics. A partir dels mètodes avaluats, la barreja hexà/dioxà/isopropanol va permetre recuperar aproximadament el 100% de retinal i d’àcid retinoic. Els tres enzims van ser actius amb els dos isòmers del retinaldehid i van seguir una cinètica de Michaelis-Menten, amb valors de Km en el rang micromolar. En relació als valors de kcat, aquests van ser més elevats per al tot-trans-retinal (ALDH1A2 i ALDH1A3) o similar per a ambdós isòmers (ALDH1A1). ALDH1A3 va ser el millor enzim en termes d’eficiència catalítica, seguit per ALDH1A2. A més, s’ha descrit per primera vegada l’activitat enzimàtica dels enzims ALDH1A amb apo-β-carotenals, derivats de la digestió excèntrica del β-carotè. La segona part d’aquest treball es centra en el paper d’alguns residus específics en les propietats cinètiques dels enzims ALDH1A. Es va realitzar una mutagènesi dirigida, a partir de diferències estructurals en residus seleccionats de la butxaca d’unió al substrat. La substitució L114P en ALDH1A1 ha estat seleccionada per fer aquesta part de l’estructura més similar a ALDH1A2. Per altra banda, en ALDH1A2, es van realitzar quatre canvis de residus contigus, N475G, A476V, L477V i N478S, per imitar l'estructura d’ALDH1A1. Al mutant ALDH1A1 L114P, els valors de Km per a l’hexanal i el citral es van incrementar entre 50 i 100 vegades, en relació als valors d’ALDH1A1 salvatge. Per contra, l’ALDH1A2 mutant va mostrar una disminució de 50 vegades en el valor de la Km per al citral. A més, la disminució de 5 vegades en el valor de la kcat, va provocar que l’eficiència catalítica de l’ALDH1A2 mutant per al citral s’aproximés a l’observada a l’ALDH1A1 salvatge. En relació a les cinètiques amb els isòmers del retinal, els mutants no van mostrar diferències significatives amb els respectius enzims no mutats, de manera que els residus alterats no són crítics en l’especificitat per al retinal. Finalment, es van realitzar estudis d’inhibició dels enzims ALDH1A per trobar inhibidors nous, potents i selectius. Per primera vegada, s’han descrit alguns compostos que tenen efecte inhibidor sobre les ALDH1A. Aquests resultats preliminars semblen molt prometedors per desenvolupar nous farmacòfors en l’àmbit del disseny de fàrmacs basat en l’estructura.The aldehyde dehydrogenase (ALDH) superfamily comprises a large number of dimeric and tetrameric proteins with a subunit molecular weight of approximately 55 kDa and different subcellular localization (cytoplasm, mitochondria and endoplasmic reticulum). ALDHs include a cluster of evolutionarily related NAD(P)+-dependent enzymes catalyzing the oxidation of a wide spectrum of aldehydic substrates, generated from various endogenous and exogenous precursors, to their corresponding carboxylic acids. The Thesis dissertation is a part of the structural and functional studies performed by our group on the role of oxidoreductases in retinoid metabolism, where their catalytic constants with retinoids were determined after solubilization with bovine serum albumin and by activity analysis using an HPLC-based methodology. The Thesis aims to perform an exhaustive and robust structural and kinetic analysis on the human enzymes involved in the irreversible oxidation of retinaldehyde to retinoic acid. The first part deals with a comparison of the substrate-binding pocket of the human ALDH1A enzymes, which exhibited similar topologies and decreasing volumes in their substrate-binding pockets. The three enzymes were subcloned, overexpressed and affinity purified in their soluble and active form. Their enzymatic activity was characterized with alkanals and alkenals as substrates. In terms of kcat/Km values, ALDH1A3 exhibits the lowest values for all substrates, suggesting a moderate role in the physiological oxidation of these aldehydes. The kcat/Km values of ALDH1A1 and ALDH1A2 indicate a potentially major role in the transformation of these substrates with slightly different substrate specificity. In order to measure activity with a physiological substrate of ALDH1As, retinaldehyde, an optimization of the solvent extraction methodology was carried out. From the evaluated methods, extraction with hexane/dioxane/isopropanol was chosen, since it was the most efficient in recovering retinaldehyde and retinoic acid from the activity buffer, with a yield near 100%. The three enzymes were active with two retinaldehyde isomers and followed Michaelis-Menten kinetics, with Km values in the micromolar range. Related to the kcat values, they were higher for the all-trans isomer (ALDH1A2 and ALDH1A3) or similar for the two isomers (ALDH1A1). ALDH1A3 was the best enzyme in terms of catalytic efficiency, followed by ALDH1A2. Moreover, the activity of ALDH1A enzymes with apo-β-carotenals, derived from the eccentric cleavage of β-carotene, was described for the first time. The second part of this work is centered on the role of specific residues in the kinetic properties of ALDH1A enzymes. We performed site-directed mutagenesis, based on structural differences of selected residues from the substrate-binding pocket. The substitution L114P in ALDH1A1 was selected to make this part of the structure more similar to that of ALDH1A2. Likewise, in ALDH1A2, four contiguous residue changes, N475G, A476V, L477V and N478S were made to mimic the structure of ALDH1A1. In the ALDH1A1 L114P mutant, the Km values for hexanal and citral were increased by 50-100 fold related to those of the wild-type ALDH1A1. Conversely, the mutant ALDH1A2 exhibited a 50-fold decrease in the Km value for citral. In addition, the 5-fold decrease in the kcat value made the catalytic efficiency of mutant ALDH1A2 for citral to become similar to that of wild-type ALDH1A1. In regard to kinetics with retinaldehyde isomers, the mutants did not show significant differences with the respective wild-type forms, thus the mutated residues are not critical for retinaldehyde specificity. Finally, inhibition studies of ALDH1A enzymes were performed in order to find novel, potent and selective inhibitors against ALDH1A1, ALDH1A2 and ALDH1A3. For the first time, we described some compounds as ALDH1A inhibitors and these preliminary results appear to be very promising to develop new pharmacophores by using structure-based drug design

Structural and kinetic features of three human aldehyde dehydrogenases, ALDH1A1, ALDH1A2 and ALDH1A3, active in retinoic acid biosynthesis /

La superfamília de les aldehid deshidrogenases (ALDH) comprèn un elevat nombre de proteïnes dimèriques i tetramèriques, amb un pes molecular aproximat de 55 kDa per subunitat i amb diferents localitzacions cel·lulars (citoplasmàtica, mitocondrial i reticle endoplasmàtic). Les ALDH inclouen un grup evolutivament relacionat d'enzims dependents de NAD(P)+, que catalitzen l'oxidació d'un ampli espectre de substrats aldehídics, generats a partir de diversos precursors endògens i exògens, als seus corresponents àcids carboxílics. Aquesta Tesi Doctoral forma part dels estudis estructurals i funcionals realitzats pel nostre grup de recerca sobre el paper de les oxidoreductases en el metabolisme dels retinoides. En aquests estudis s'han determinat les seves constants catalítiques amb aquests substrats, després de la solubilització amb albúmina sèrica bovina i mitjançant l'anàlisi d'activitat utilitzant una metodologia basada en HPLC. La Tesi pretén realitzar una anàlisi estructural i cinètica, exhaustiva i robusta, sobre els enzims humans implicats en l'oxidació irreversible del retinaldehid a àcid retinoic. La primera part tracta sobre la comparació de la butxaca d'unió al substrat dels enzims humans ALDH1A, els quals van mostrar topologies similars i volums decreixents en les seves butxaques d'unió al substrat. Els tres enzims van ser subclonats, expressats i purificats en la seva forma soluble i activa. Pel que fa als valors de kcat/Km amb alcanals i alquenals, ALDH1A3 és l'enzim que presenta els valors més baixos per a tots els substrats, suggerint un paper moderat en l'oxidació fisiològica d'aquests aldehids. Els valors de kcat/Km d'ALDH1A1 i ALDH1A2 indiquen un paper potencialment important en la transformació d'aquests aldehids, encara que amb una especificitat de substrat lleugerament diferent. Per mesurar l'activitat de les ALDH1A amb un substrat fisiològic, el retinal, es va realitzar una optimització de la metodologia d'extracció en solvents orgànics. A partir dels mètodes avaluats, la barreja hexà/dioxà/isopropanol va permetre recuperar aproximadament el 100% de retinal i d'àcid retinoic. Els tres enzims van ser actius amb els dos isòmers del retinaldehid i van seguir una cinètica de Michaelis-Menten, amb valors de Km en el rang micromolar. En relació als valors de kcat, aquests van ser més elevats per al tot-trans-retinal (ALDH1A2 i ALDH1A3) o similar per a ambdós isòmers (ALDH1A1). ALDH1A3 va ser el millor enzim en termes d'eficiència catalítica, seguit per ALDH1A2. A més, s'ha descrit per primera vegada l'activitat enzimàtica dels enzims ALDH1A amb apo-β-carotenals, derivats de la digestió excèntrica del β-carotè. La segona part d'aquest treball es centra en el paper d'alguns residus específics en les propietats cinètiques dels enzims ALDH1A. Es va realitzar una mutagènesi dirigida, a partir de diferències estructurals en residus seleccionats de la butxaca d'unió al substrat. La substitució L114P en ALDH1A1 ha estat seleccionada per fer aquesta part de l'estructura més similar a ALDH1A2. Per altra banda, en ALDH1A2, es van realitzar quatre canvis de residus contigus, N475G, A476V, L477V i N478S, per imitar l'estructura d'ALDH1A1. Al mutant ALDH1A1 L114P, els valors de Km per a l'hexanal i el citral es van incrementar entre 50 i 100 vegades, en relació als valors d'ALDH1A1 salvatge. Per contra, l'ALDH1A2 mutant va mostrar una disminució de 50 vegades en el valor de la Km per al citral. A més, la disminució de 5 vegades en el valor de la kcat, va provocar que l'eficiència catalítica de l'ALDH1A2 mutant per al citral s'aproximés a l'observada a l'ALDH1A1 salvatge. En relació a les cinètiques amb els isòmers del retinal, els mutants no van mostrar diferències significatives amb els respectius enzims no mutats, de manera que els residus alterats no són crítics en l'especificitat per al retinal. Finalment, es van realitzar estudis d'inhibició dels enzims ALDH1A per trobar inhibidors nous, potents i selectius. Per primera vegada, s'han descrit alguns compostos que tenen efecte inhibidor sobre les ALDH1A. Aquests resultats preliminars semblen molt prometedors per desenvolupar nous farmacòfors en l'àmbit del disseny de fàrmacs basat en l'estructura.The aldehyde dehydrogenase (ALDH) superfamily comprises a large number of dimeric and tetrameric proteins with a subunit molecular weight of approximately 55 kDa and different subcellular localization (cytoplasm, mitochondria and endoplasmic reticulum). ALDHs include a cluster of evolutionarily related NAD(P)+-dependent enzymes catalyzing the oxidation of a wide spectrum of aldehydic substrates, generated from various endogenous and exogenous precursors, to their corresponding carboxylic acids. The Thesis dissertation is a part of the structural and functional studies performed by our group on the role of oxidoreductases in retinoid metabolism, where their catalytic constants with retinoids were determined after solubilization with bovine serum albumin and by activity analysis using an HPLC-based methodology. The Thesis aims to perform an exhaustive and robust structural and kinetic analysis on the human enzymes involved in the irreversible oxidation of retinaldehyde to retinoic acid. The first part deals with a comparison of the substrate-binding pocket of the human ALDH1A enzymes, which exhibited similar topologies and decreasing volumes in their substrate-binding pockets. The three enzymes were subcloned, overexpressed and affinity purified in their soluble and active form. Their enzymatic activity was characterized with alkanals and alkenals as substrates. In terms of kcat/Km values, ALDH1A3 exhibits the lowest values for all substrates, suggesting a moderate role in the physiological oxidation of these aldehydes. The kcat/Km values of ALDH1A1 and ALDH1A2 indicate a potentially major role in the transformation of these substrates with slightly different substrate specificity. In order to measure activity with a physiological substrate of ALDH1As, retinaldehyde, an optimization of the solvent extraction methodology was carried out. From the evaluated methods, extraction with hexane/dioxane/isopropanol was chosen, since it was the most efficient in recovering retinaldehyde and retinoic acid from the activity buffer, with a yield near 100%. The three enzymes were active with two retinaldehyde isomers and followed Michaelis-Menten kinetics, with Km values in the micromolar range. Related to the kcat values, they were higher for the all-trans isomer (ALDH1A2 and ALDH1A3) or similar for the two isomers (ALDH1A1). ALDH1A3 was the best enzyme in terms of catalytic efficiency, followed by ALDH1A2. Moreover, the activity of ALDH1A enzymes with apo-β-carotenals, derived from the eccentric cleavage of β-carotene, was described for the first time. The second part of this work is centered on the role of specific residues in the kinetic properties of ALDH1A enzymes. We performed site-directed mutagenesis, based on structural differences of selected residues from the substrate-binding pocket. The substitution L114P in ALDH1A1 was selected to make this part of the structure more similar to that of ALDH1A2. Likewise, in ALDH1A2, four contiguous residue changes, N475G, A476V, L477V and N478S were made to mimic the structure of ALDH1A1. In the ALDH1A1 L114P mutant, the Km values for hexanal and citral were increased by 50-100 fold related to those of the wild-type ALDH1A1. Conversely, the mutant ALDH1A2 exhibited a 50-fold decrease in the Km value for citral. In addition, the 5-fold decrease in the kcat value made the catalytic efficiency of mutant ALDH1A2 for citral to become similar to that of wild-type ALDH1A1. In regard to kinetics with retinaldehyde isomers, the mutants did not show significant differences with the respective wild-type forms, thus the mutated residues are not critical for retinaldehyde specificity. Finally, inhibition studies of ALDH1A enzymes were performed in order to find novel, potent and selective inhibitors against ALDH1A1, ALDH1A2 and ALDH1A3. For the first time, we described some compounds as ALDH1A inhibitors and these preliminary results appear to be very promising to develop new pharmacophores by using structure-based drug design