Discovery of a series of aromatic lactones as ALDH1/2-directed inhibitors
Introduction
Aldehydes are found throughout the body as a product of dietary metabolism and the biotransformation of neurotransmitters, carbohydrates, lipids, and endogenous amino acids [1], [2], [3]. In addition numerous aldehydes are present in the environment in smog, motor vehicle exhaust, and formed during the production of plastics [4], [5]. The accumulation of aldehydes within the body can lead to cytotoxicity and carcinogenesis [3], [4], [6], [7]. The human body has multiple systems of enzymes to alleviate aldehyde stress, one of these being the aldehyde dehydrogenases (ALDHs). The human genome has 19 functional genetic loci for members of the ALDH superfamily, most of which catalyze the NAD(P)+-dependent oxidation of aldehydes to their respective carboxylic acids, except for ALDH6A1, which catalyzes the formation of their respective CoA esters [4], [8]. ALDHs are separated into families and subfamilies based on their sequence similarity [9]. The 19 ALDHs share similar but distinct functions within the body due to their varying substrate specificities and gene expression differences. Some gene products, such as ALDH1A1 and ALDH2, are ubiquitously expressed, whereas others are expressed preferentially in certain tissues or during certain periods of development. Naturally occurring mutations within various ALDHs cause human diseases or aversive conditions such as the alcohol flush reaction (ALDH2), Sjogren–Larsson syndrome (ALDH3A2), type II hyperprolinemia (ALDH4A1), and 4-hydroxybutyricaciduria (ALDH5A1) [10], [11], [12]. In contrast, members of the ALDH1 and ALDH2 families possess broad and somewhat overlapping substrate specificities making specific assignment of function difficult. Due to the apparent overlap in their function, ALDH-selective chemical probes could aid in gaining a better understanding of the function of these ALDHs, especially those which are within the same family or subfamily.
For several years our lab has been interested in finding novel selective compounds for ALDH1A1, ALDH2, and ALDH3A1 [13], [14], [15]. ALDH1A1 and ALDH3A1 are cytosolic proteins expressed in many cell types, including ocular tissues where they appear to function as corneal crystallins. Both ALDH1A1 and ALDH3A1 are implicated in providing resistance to certain anti-cancer agents, such as cyclophosphamide [16], [17], [18]. ALDH1B1, a mitochondrial enzyme most similar to ALDH2, has recently been shown to be a potential biomarker for colon cancer [19]. ALDH1A1, along with the related cytosolic isoenzymes ALDH1A2 and ALDH1A3, contribute to retinoid metabolism [20]. ALDH1A2 and ALDH1A3 perform crucial functions during embryogenesis, as individual genetic knockout of these two genes in mice are not viable [21], [22]. ALDH2 is a mitochondrial enzyme which is most well-known for its role in acetaldehyde metabolism [23]. However, other members of the ALDH family can contribute to acetaldehyde metabolism, especially when ALDH2 activity is reduced by the presence of the ALDH2∗2 allele [24]. These other isoenzymes include ALDH1B1 and ALDH1A1 [25], [26]. ALDH2, along with ALDH1A1, is implicated in the metabolism of the neurotransmitter dopamine [27]. In addition to these oxidative functions, ALDH2 can contribute to cardiovascular function through its ability to bioactivate nitroglycerin by acting as a nitrate reductase, and has been associated with cardioprotection from ischemic damage by limiting the damage from lipid peroxidation products [28]. Many of the other isoenzymes in the ALDH1, ALDH2, and ALDH3 families are also known to have a cytoprotective role against lipid peroxidation products [1]. The discovery and development of isoenzyme-selective inhibitors or activators could prove useful in evaluating the relative contributions that these related ALDH isoenzymes make toward these common substrates.
Here we report the results of a high-throughput screen designed to discover selective modulators of ALDH2 activity. The screen identified 53 compounds from a library of 63,000 compounds that modulated the esterase activity of ALDH2. Commercially available compounds were then tested for their effects on the oxidation of aldehyde substrate by ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH1B1, ALDH3A1, ALDH4A1, and ALDH5A1. This screen discovered a set of four aromatic lactones which exhibit potent inhibition of the ALDH1/2 family members but do not inhibit ALDH3A1, ALDH4A1, or ALDH5A1 activity. Two compounds in particular show selectivity for ALDH2 versus ALDH1A1, and one of those two shows selectivity for ALDH2 versus all other tested isoenzymes. Future characterization will include determining the mechanism by which these aromatic lactones inhibit the ALDH1/2 family of isoenzymes.
Section snippets
Materials
All materials were purchased from Sigma–Aldrich, unless otherwise noted.
Expression of ALDH
ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH1B1, ALDH3A1, ALDH4A1, and ALDH5A1 were prepared and purified as previously described [[13], [15], [29], Morgan et al., Chem-Biol. Inter. (this issue)].
High-throughput screening
63,000 compounds from ChemDiv were present in 20 μL aliquots in 2% DMSO at 25 μM concentration in 384 well plates. Primary screening was completed in 384 well clear bottom plates by measuring the increase in absorbance of para-nitrophenol
High-throughput ALDH2 esterase screen
Primary screening identified 1495 compounds from a 63,000 compound library which either activated the esterase activity of ALDH2 at least 1.3-fold or inhibited it by at least 35%. Of these 1495 compounds, only 57 of them were identified as activators of the esterase activity. In the secondary screen, 53 compounds, 11 activators and 42 inhibitors, modulated the esterase activity of ALDH2 greater than 25%. Fig. 1A depicts the primary screen results for these 53 hit compounds.
ALDH2 dehydrogenase screen
Out of the 53 lead
Conclusion
The aromatic lactones identified by this screen are potent (2P4 Ki ≈ 35 nM for ALDH2) inhibitors of the ALDH1/2 isoenzymes. These compounds, upon further development, could be used to better determine the functions of each isoenzyme. These compounds also have therapeutic potential as inhibitors of the ALDH1/2 family of enzymes have medicinal applications as modulators of dopamine metabolism, antidipsotropic drugs, and as chemotherapy sensitizers. However, a strictly ALDH2-selective compound
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
Acknowledgements
The authors would like to thank Cindy Morgan, Bibek Parajuli, and especially Lan Min Zhai for help with the production and purification of the various ALDH isozymes. The authors would also like to thank Lan Chen, PhD, and the Indiana University Chemical Genomics Core for providing access to their facilities to complete the high-throughput screening and the chemical libraries and John Turchi, PhD, for use of the Cary Eclipse fluorimeter. This research was supported by NIH R01-AA018123. Cameron
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