Development of a high-throughput in vitro assay to identify selective inhibitors for human ALDH1A1

Abstract

The human aldehyde dehydrogenase (ALDH) superfamily consists of at least 19 enzymes that metabolize endogenous and exogenous aldehydes. Currently, there are no commercially available inhibitors that target ALDH1A1 but have little to no effect on the structurally and functionally similar ALDH2. Here we present the first human ALDH1A1 structure, as the apoenzyme and in complex with its cofactor NADH to a resolution of 1.75 Å and 2.1 Å, respectfully. Structural comparisons of the cofactor binding sites in ALDH1A1 with other closely related ALDH enzymes illustrate a high degree of similarity. In order to minimize discovery of compounds that inhibit both isoenzymes by interfering with their conserved cofactor binding sites, this study reports the use of an in vitro, NAD+-independent, esterase-based high-throughput screen (HTS) of 64,000 compounds to discover novel, selective inhibitors of ALDH1A1. We describe 256 hits that alter the esterase activity of ALDH1A1. The effects on aldehyde oxidation of 67 compounds were further analyzed, with 30 selectively inhibiting ALDH1A1 compared to ALDH2 and ALDH3A1. One compound inhibited ALDH1A1 and ALDH2, while another inhibited ALDH1A1, ALDH2, and the more distantly related ALDH3A1. The results presented here indicate that this in vitro enzyme activity screening protocol successfully identified ALDH1A1 inhibitors with a high degree of isoenzyme selectivity. The compounds identified via this screen plus the screening methodology itself represent a starting point for the development of highly potent and selective inhibitors of ALDH1A1 that may be utilized to better understand the role of this enzyme in both normal and disease states.

Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1

“…The MD simulation results also indicate that the monitored hydrophobic residues exposure is due not only to the locally flexible active site property but also by interruption of subunit dimerization by urea. The recent crystal structure [12] and a previous study [28] directly supported our findings that the hydrophobic interaction of subunit surface acts critical role in the formation of tetramers and the C-terminal modification directly induces the overall stability of ALDH1.…”

“…Since the three-dimensional structure of human ALDH1 has recently been determined, the structure of ALDH1 (PDB entry ID: 4wj9) [12] was used as a template for ALDH1-urea docking simulation and MD simulations. The urea structure was prepared using the Marvin program, ChemAxon (http://www.chemaxon.com).…”

“…As ALDH1 has been found to be overexpressed in diseases, its downregulation via regulation of gene expression or through enzymatic inhibition via inhibitors is a possible strategic target for treatment. The crystal structure of human ALDH1 (PDB ID: 4WJ9) was depicted recently [12] as a homotetramer, with each subunit containing a catalytic domain, a cofactor binding domain, and an oligomerization domain. The X-ray crystal structure of ALDH1 showed high similarity to the human ALDH2 isozyme (PDB ID: 3N80) including a high degree of similarity for NADH cofactor binding sites.…”

Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors

“…eliminate compounds that compete with structurally conserved elements of the cofactor binding site. CM026 and CM037 emerged from the HTS as esterase modulators of ALDH1A1 40 . CM026 (Figure 1A) has a molecular weight of 442.5 Daltons and CM037 (Figure 2A) has a molecular weight of 431.6 Daltons.…”

“…For instance, the control inhibitor used in the ALDEFLUOR assay, diethylaminobenzaldehyde (DEAB), is an effective inhibitor of at least three of these isoenzymes 38,39 . From a high-throughput screen (HTS) 40 , we have identified two classes of ALDH1A1 inhibitors and have determined the structural and kinetic basis for their selectivity toward ALDH1A1.…”