Physiologically relevant fluorescent assay for identification of 17β‐hydroxysteroid dehydrogenase type 10 inhibitors

Abstract

Mitochondrial enzyme 17β‐hydroxysteroid dehydrogenase type 10 (HSD10) is a potential molecular target for treatment of mitochondrial‐related disorders such as Alzheimer's disease (AD). Its over‐expression in AD brains is one of the critical factors disturbing the homeostasis of neuroprotective steroids and exacerbating amyloid beta (Aβ)‐mediated mitochondrial toxicity and neuronal stress. This study was focused on revalidation of the most potent HSD10 inhibitors derived from benzothiazolyl urea scaffold using fluorescent‐based enzymatic assay with physiologically relevant substrates of 17β‐oestradiol and allopregnanolone. The oestradiol‐based assay led to the identification of two nanomolar inhibitors (IC50 70 and 346 nM) differing from HSD10 hits revealed from the formerly used assay. Both identified inhibitors were found to be effective also in allopregnanolone‐based assay with non‐competitive or uncompetitive mode of action. In addition, both inhibitors were confirmed to penetrate the HEK293 cells and they were able to inhibit the HSD10 enzyme in the cellular environment. Both molecules seem to be potential lead structures for further research and development of HDS10 inhibitors.image

Nanomolar Benzothiazole-Based Inhibitors of 17β-HSD10 with Cellular Bioactivity

“…To detect the ability of compounds to penetrate into cells and inhibit 17β-HSD10 activity inside the cellular environment, the fluorogenic probe (−)–CHANA was used. This probe is oxidized to the corresponding fluorescent ketone (CHANK), and it was previously shown as a specific substrate for 17β-HSD10. ,,, The inhibition inside the HEK293 cells overexpressing 17β-HSD10 (HEK293 17β-HSD10) was determined as the decrease in fluorescence intensity of CHANK production after the inhibitor treatment. All 14 compounds were screened at 10 and 25 μM concentrations, and all of them displayed the ability to penetrate the cells and inhibit 17β-HSD10 activity except for compound 11 , which interfered with the assay (Figure A,B; numerical data in Table S4).…”

“…The goal of this study was to generate new benzothiazolebased inhibitors with an enhanced inhibitory ability against the 17β-HSD10 enzyme and/or with improved physicochemical properties, that would be active also at the cellular level. For this purpose, we employed the newly established 17β-HSD10 enzymatic assays utilizing physiological substrates E2 and ALLOP, 28 as well as the cellular assay utilizing the (−)-cyclohexenyl amino naphthalene alcohol ((−)−CHANA) fluorogenic probe 29 in the HEK293 cell line overexpressing 17β-HSD10.…”

“…All four hits exhibited mixed-type inhibition with the E2 substrate, which is in line with previous findings on benzothiazolyl urea inhibitors obtained either in an AAC or E2 assay. 25,26,28 Mixed-type inhibition is a result of a partial competition between the inhibitor and the substrate and thus indicates noncovalent binding of inhibitor within the active site of 17β-HSD10. Compounds 5, 6, and 7 showed similar IC 50 values with the ALLOP substrate compared to E2 results, while compound 3 displayed a significantly lower IC 50 value of 95 nM.…”

“…Notably, an even more potent benzothiazolyl urea inhibitor was recently identified using the E2/ALLOP by Schmidt et al (IC 50 values of 70 nM in E2 and 19 nM in ALLOP assays). 28 However, this inhibitor contains an atypical thiocyanate substitution that is potentially troublesome from a selectivity point of view, as it was reported to react with cysteine residues of other enzymes. 32 Structure−activity relationship analysis of new inhibitors revealed several important trends.…”

“…However, a more physiological assay with E2 or ALLOP steroid substrates and NAD + cofactor (i.e., substrate oxidation) has been recently established. 28 Therefore, the new compounds were initially screened in both assays at 10 and 1 μM concentrations to depict potential differences in inhibition results among the two assays (Figure 5; numerical data in Table S1).…”