Physiologically relevant fluorescent assay for identification of 17ß-hydroxysteroid dehydrogenase type 10 inhibitors.

Mitochondrial enzyme 17ß-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 (Aß)-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 17ß-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.

Correction to "C-3 Steroidal Hemiesters as Inhibitors of 17ß-Hydroxysteroid Dehydrogenase Type 10".

[This corrects the article DOI: 10.1021/acsomega.3c10148.].

C-3 Steroidal Hemiesters as Inhibitors of 17ß-Hydroxysteroid Dehydrogenase Type 10.

17ß-HSD10 is a mitochondrial enzyme that catalyzes the steroidal oxidation of a hydroxy group to a keto group and, thus, is involved in maintaining steroid homeostasis. The druggability of 17ß-HSD10 is related to potential treatment for neurodegenerative diseases, for example, Alzheimer's disease or cancer. Herein, steroidal derivatives with an acidic hemiester substituent at position C-3 on the skeleton were designed, synthesized, and evaluated by using pure recombinant 17ß-HSD10 converting 17ß-estradiol to estrone. Compounds 22 (IC50 = 6.95 ± 0.35 µM) and 23 (IC50 = 5.59 ± 0.25 µM) were identified as the most potent inhibitors from the series. Compound 23 inhibited 17ß-HSD10 activity regardless of the substrate. It was found not cytotoxic toward the HEK-293 cell line and able to inhibit 17ß-HSD10 activity also in the cellular environment. Together, these findings support steroidal compounds as promising candidates for further development as 17ß-HSD10 inhibitors.

Nanomolar Benzothiazole-Based Inhibitors of 17ß-HSD10 with Cellular Bioactivity.

Multifunctional mitochondrial enzyme 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is a potential drug target for the treatment of various pathologies. The most discussed is the pathology associated with Alzheimer's disease (AD), where 17ß-HSD10 overexpression and its interaction with amyloid-ß peptide contribute to mitochondrial dysfunction and neuronal stress. In this work, a series of new benzothiazole-derived 17ß-HSD10 inhibitors were designed based on the structure-activity relationship analysis of formerly published inhibitors. A set of enzyme-based and cell-based methods were used to evaluate the inhibitory potency of new compounds, their interaction with the enzyme, and their cytotoxicity. Most compounds exhibited significantly a higher inhibitory potential compared to published benzothiazolyl ureas and good target engagement in a cellular environment accompanied by low cytotoxicity. The best hits displayed mixed-type inhibition with half maximal inhibitory concentration (IC50) values in the nanomolar range for the purified enzyme (3-7, 15) and/or low micromolar IC50 values in the cell-based assay (6, 13-16).

Development of submicromolar 17ß-HSD10 inhibitors and their in vitro and in vivo evaluation.

17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is a multifunctional mitochondrial enzyme and putative drug target for the treatment of various pathologies including Alzheimer's disease or some types of hormone-dependent cancer. In this study, a series of new benzothiazolylurea-based inhibitors were developed based on the structure-activity relationship (SAR) study of previously published compounds and predictions of their physico-chemical properties. This led to the identification of several submicromolar inhibitors (IC50 ∼0.3 µM), the most potent compounds within the benzothiazolylurea class known to date. The positive interaction with 17ß-HSD10 was further confirmed by differential scanning fluorimetry and the best molecules were found to be cell penetrable. In addition, the best compounds weren't found to have additional effects for mitochondrial off-targets and cytotoxic or neurotoxic effects. The two most potent inhibitors 9 and 11 were selected for in vivo pharmacokinetic study after intravenous and peroral administration. Although the pharmacokinetic results were not fully conclusive, it seemed that compound 9 was bioavailable after peroral administration and could penetrate into the brain (brain-plasma ratio 0.56).