Development of a high-throughput in vitro assay to identify selective inhibitors for human ALDH1A1
Introduction
The aldehyde dehydrogenase (ALDH) superfamily of enzymes primarily catalyze the NAD(P)+-dependent oxidation of an aldehyde to its corresponding carboxylic acid [1]. The human genome has at least 19 ALDHs. A primary function of the ALDH1A subfamily (ALDH1A1, ALDH1A2, and ALDH1A3), whose members share over 70% protein sequence identity, is the oxidation of retinaldehyde to retinoic acid, a critical regulator in a number of cell growth and differentiation pathways. Other aldehydes also serve as substrates for ALDH1A1, including acetaldehyde during ethanol metabolism, 3,4-dihydroxyphenylacetaldehyde (DOPAL) in dopamine metabolism, and (±)-4-hydroxy-2E-nonenal (4-HNE), a toxic by-product of oxidative stress pathways. ALDH1A1 has been associated with a number of diseases. Down-regulation of ALDH1A1 has been reported in Parkinson’s disease, possibly due to the build-up of the neurotoxic aldehyde DOPAL in dopamine metabolism [2], [3]. ALDH1A1 knockout mice are able to resist diet-induced obesity [4], while rodents given the nonselective ALDH1A1 inhibitor citral also exhibit reduced weight gain [5], indicating that ALDH1A1 is playing a role in obesity and/or adipogenesis. Up-regulation of ALDH1A1 is a biomarker for both normal and cancer stem cells, but the role of ALDH1A1 in establishing and/or maintaining stem cells is not known [6], [7], [8], [9]. ALDH1A1 and ALDH3A1 have long been linked to cancer drug resistance due to their roles in the metabolism of the anticancer agent cyclophosphamide [10]. It is evident that ALDH1A1 is involved in a number of biological processes, but its contributions to both normal and disease states, including retinoid-dependent processes, are not clearly understood. The development of selective activators and inhibitors of ALDH1A1 would provide chemical tools to help decipher the role of this enzyme. However, at this time there are no commercially available, ALDH1A1-selective modulators.
The development of compounds that selectively target ALDH1A1 has proven to be difficult as the ALDH superfamily of enzymes shares many common structural and mechanistic features. These members generally function as homodimers or homotetramers, with each subunit containing three structural domains, a catalytic domain, a cofactor binding domain, and an oligomerization domain. The NAD(P)+ binding domain is a Rossmann-fold, a nucleotide binding site that consists of two sets of parallel beta sheets and alpha helices. The Rossmann-fold structure motif is found in the NAD+ binding domains of multiple dehydrogenase families, including ALDHs, lactate dehydrogenases, alcohol dehydrogenases, and glyceraldehyde-3-phosphate dehydrogenase [11], [12], [13]. There are differences in the Rossmann fold between ALDH and other oxidoreductases that could possibly be exploited for the development of small molecule modulators of various ALDH isoenzymes compared to other NAD+-binding enzyme families [14]. However, there exists much structural similarity in the NAD+-binding site within the ALDH family and the development of selective modulators that target this site may present difficulties.
A number of ALDH’s, including ALDH1A1 also possess esterase activity. Based on the ALDH2 sequence, site-directed mutagenesis has shown that Cys-302 is the essential nucleophile for both the esterase and dehydrogenase reaction, with Glu-268 acting as the general base to activate Cys-302 [15], [16]. The proposed catalytic steps for both the dehydrogenase and esterase reactions have been recently reviewed [17], although minor details still need to be resolved including the roles of second sphere residues in assisting proton transfer to solvent [18], [19]. The use of common active site residues for the two reactions makes it likely that modulators of the esterase reaction would also modulate aldehyde oxidation activity. In support of this hypothesis, the ALDH2 activator Alda-1 activates both the esterase and dehydrogenase activity of the enzyme and daidzen inhibits both reactions [13], [20], [21]. An additional advantage of the esterase reaction is that it does not require the cofactor NAD+ to be present, and so allows the screen to be less influenced by compounds binding to this site.
The human ALDH1 family, which shares over 60% protein sequence identity, is a particularly difficult challenge for inhibitor development since it contains the highest number of orthologs in the genome at seven (ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, and ALDH2). Compounds such as diethylaminobenzaldehyde (DEAB) and disulfiram are potent inhibitors of ALDH1A1, with IC50’s in the nM range, but both also inhibit ALDH2 [17], [22]. DEAB is also a relatively potent inhibitor for a number of other ALDH1 family members, although not ALDH1L1 [22], [23]. An in vitro high throughput screen (HTS) is one method of discovering novel, small molecule modulators for a particular enzyme. Typically, the rate of aldehyde oxidation by ALDHs is studied by monitoring the formation of NADH at 340 nm on a spectrophotometer (molar extinction coefficient of 6220 M−1 cm−1) (Fig. 1A). However, this approach is not ideal for the screening assay as it is common for compounds in the libraries to absorb light in the same wavelength range as NADH and leads to interference in this analytical approach. Therefore, another assay design is needed for an ALDH1A1 HTS. One approach is to couple aldehyde oxidation to a second reaction that can be monitored by either fluorescence or UV/Vis spectrophotometry. For example, the dehydrogenase activity of ALDH2 was coupled to the NADH-dependent reduction of resazurin to resorufin to discover the ALDH2 activator Alda-1 [20]. However, a second approach would be to use the inherent esterase activity of ALDH1A1 to identify modulators. The ALDH1A1 ester substrate para-nitrophenylacetate (pNPA) is hydrolyzed to p-nitrophenol, which absorbs light at 405 nm and can be monitored spectrophotometrically, with minimal interference from library compounds (Fig. 1B).
In this paper, we used an in vitro esterase assay to identify compounds that modulate ALDH1A1 activity but have little to no effect on either ALDH2, an isoenzyme that has approximately 70% protein sequence identity with ALDH1A1, or ALDH3A1, a more distantly related isoenzyme with 30% protein sequence identity. Comparison of the cofactor binding sites of human ALDH1A1 and ALDH2 points to a high degree of similarity, suggesting that development of selective modulators that bind at this location would be challenging. Use of the esterase assay allowed us to minimize two potential problems: (1) identification of compounds that bind to the highly conserved cofactor site, and (2) monitor activity at a wavelength with minimal spectral overlap to that of the library compounds. Of the 64,000 compounds screened, 256 were identified as modulators of ALDH1A1 esterase activity. We examined the dehydrogenase activity and selectivity of 67 hits and nearly half selectively inhibited ALDH1A1 dehydrogenase activity. These results indicate that this simple esterase-based in vitro HTS was successful in identifying novel, selective inhibitors of ALDH1A1.
Section snippets
Materials
All chemicals and reagents including para-nitrophenylacetate, propionaldehyde, NAD+, and buffers were purchased from Sigma Aldrich unless where noted otherwise.
Expression and purification of ALDH proteins
ALDH1A1, ALDH2, and ALDH3A1 were prepared as described elsewhere [24], [25], [26]. Protein used for kinetics was flash frozen in liquid nitrogen and stored at −80 °C. ALDH1A1 protein used for X-ray crystallography was stored at −20 °C in a 50% (v/v) solution with glycerol and dialyzed against 10 mM Na+-ACES pH 6.6 and 1 mM dithiothreitol at 4
Structure of human ALDH1A1
X-ray crystallography was used to compare the structure of human ALDH1A1 with other members of the ALDH enzyme superfamily. The structure of human ALDH1A1 had not been previously reported (Fig. 2 and Table 1, PDB Code 4WJ9). As expected, it is highly similar to both the human ALDH2 enzyme (PDB Code 3N80), with which ALDH1A1 shares about 70% sequence identity, and the sheep ALDH1A1 (PDB Code 1BXS), with over 90% sequence identity. The structure of ALDH1A1 with NADH was determined to a resolution
Discussion
Comparison of the structures of human ALDH1A1, ALDH2, and ALDH3A1 indicate they exhibit a high degree of structural similarity, but demonstrate distinct differences within their substrate binding sites. In contrast, their respective coenzyme binding sites are less dissimilar, especially between ALDH1A1 and ALDH2 (Fig. 4). This supports our screening approach to avoid identifying compounds that interact at this location, as they are less likely to be selective for ALDH1/2 class members. However,
Conclusion
Aldehyde dehydrogenases are critical enzymes involved in the metabolism of a variety of aldehyde substrates. ALDH1A1 has been identified as a marker for both normal and cancer stem cells and has been linked to such diseases as obesity, Parkinson’s disease, and cancer. Small molecule probes are urgently needed to elicit the role of this enzyme in both normal and disease states. However at this time there are no commercially available small molecules that selectively modulate ALDH1A1 activity
Conflict of Interest
Thomas D. Hurley holds significant financial equity in SAJE Pharma, LLC. However, none of the work described in this study is related to, based on or supported by the company.
Transparency Document
Acknowledgments
The authors would like to thank Indiana University Chemical Genomics Core Facility (especially Lan Chen, Ph.D. and Andrea Gunawan) for assistance with the high-throughput screen, including access to the chemical library and use of their facility. Special thanks to Lanmin Zhai and Bibek Parajuli for assistance making ALDH proteins. The coordinates and structure factors for human ALDH1A1 and its complex with NADH have been deposited with the RCSB under codes 4WJ9 and 4WB9. This research was
References (39)
ALDH1 mRNA: presence in human dopamine neurons and decreases in substantia nigra in Parkinson’s disease and in the ventral tegmental area in schizophrenia
Neurobiol. Dis.
(2003)Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells
Blood
(1990)Assessment of aldehyde dehydrogenase in viable cells
Blood
(1995)The crystal structure of a ternary complex of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa. Provides new insight into the reaction mechanism and shows a novel binding mode of the 2′-phosphate of NADP+ and a novel cation binding site
J. Mol. Biol.
(2009)The enzymatic activity of human aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by Aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance
Chem. Biol. Interact.
(2012)Discovery of novel regulators of aldehyde dehydrogenase isoenzymes
Chem. Biol. Interact.
(2011)- et al.
Processing of X-ray diffraction data collected in oscillation mode
Macromol. Crystallogr. Pt. A
(1997) Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases
J. Biol. Chem.
(2011)Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases
Structure
(1998)- et al.
The aldehyde dehydrogenase gene superfamily resource center
Hum. Genomics
(2009)
Inflammatory pathways in Parkinson’s disease; a BNE Microarray Study
Parkinson’s Dis.
Retinaldehyde represses adipogenesis and diet-induced obesity
Nat. Med.
Toxicology and carcinogenesis studies of microencapsulated citral in rats and mice
Toxicol. Sci.
Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform
Cell Cycle
Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity
Proc. Natl. Acad. Sci. U.S.A.
Cyclophosphamide and cancer: golden anniversary
Nat. Rev. Clin. Oncol.
Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant
Nat. Struct. Mol. Biol.
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