Discovery of novel regulators of aldehyde dehydrogenase isoenzymes

Abstract

Over the past three years we have been involved in high-throughput screening in an effort to discover novel small molecular modulators of aldehyde dehydrogenase (ALDH) activity. In particular, we have been interested in both the activation and inhibitionof the three commonly studied isoenzymes, ALDH1A1, ALDH2 and ALDH3A1, as their distinct, yet overlapping substrate specificities, present a particularly difficult challenge for inhibitor discovery and design. Activation of ALDH2 has been shown to benefit cardiovascular outcome following periods of ischemia and renewed interest in specific inhibition of ALDH2 has application for alcohol aversion therapy, and more recently, in cocaine addiction. In contrast, inhibition of either ALDH1A1 or ALDH3A1 has application in cancer treatments where the isoenzymes are commonly over-expressed and serve as markers for cancer stem cells. We are taking two distinct approaches for these screens: in vitro enzyme activity screens using chemical libraries and virtual computational screens using the structures of the target enzymes as filters for identifying potential inhibitors, followed by in vitro testing of their ability to inhibit their intended targets. We have identified selective inhibitors of each of these three isoenzymes with inhibition constants in the high nanomolar to low micromolar range from these screening procedures. Together, these inhibitors provide proof for concept that selective inhibition of these broad specificity general detoxication enzymes through small molecule discovery and design is possible.

Aldehyde Dehydrogenase Inhibitors: a Comprehensive Review of the Pharmacology, Mechanism of Action, Substrate Specificity, and Clinical Application

“…Based on these observations, ALDH isozymes have been proposed as new pharmacological targets for the more effective treatment of cancer. In this regard, new agents are being developed and tested as ALDH inhibitors, particularly targeting the ALDH1A1 and ALDH3A1 isozymes (Parajuli et al, 2011).…”

Discovery of a Novel Class of Covalent Inhibitor for Aldehyde Dehydrogenases

“…Protein Purification-Human ALDH3A1 was expressed and purified as described elsewhere (19). The activity of the enzyme is also tested using a standard assay that measures production of NADPH (see description below).…”

“…Inhibitor Screens-The ALDH2 enzyme assay design has been reported previously (19). All tested compounds were dissolved in DMSO and screened at 10 M concentration prior to addition of the substrate.…”

“…IC 50 Determination Methods for ALDH2, ALDH1A1, and ALDH3A1-IC 50 inhibition curves for the inhibitors were measured using the activity of hALDH2, hALDH1A1, and hALDH3A1 as described elsewhere (19). IC 50 values were further determined for inhibitors Aldi-1, Aldi-2, and Aldi-4 against propionaldehyde oxidation (ALDH1A1, ALDH2) or benzaldehyde oxidation (ALDH3A1) by measuring NADH production over time at various concentrations ranging from 1 to 100 M of inhibitors following a 2-min pre-incubation.…”

α,β-Acetylenic Amino Thiolester Inhibitors of Aldehyde Dehydrogenases 1&3: Suppressors of Apoptogenic Aldehyde Oxidation and Activators of Apoptosis

“…In view of the very recent comprehensive review on the ALDHs: substrate specificities, crystallographic structures, and mechanisms of action of ALDH inhibitors [75], as well as earlier reports underlining the need for developing ALDH inhibitors as therapeutic compounds for cancer stem cells [76][77][78], some of the inhibitors cited could serve as lead compounds for the development of derivatives to try to meet this challenge.…”