Ondrej Benek

17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a multifunctional mitochondrial enzyme and a potential drug target for the treatment of various pathologies, including Alzheimer’s disease (AD). In this study, five new benzothiazole-derived 17β-HSD10 inhibitors were developed based on structure–activity relationship (SAR) analyses of previously published compounds. To evaluate the inhibitory effects, cytotoxicity, and therapeutic potential of these new compounds, several enzyme- and cell-based methods were employed. All prepared compounds exhibited high inhibitory potential and confirmed good biomembrane permeation. Three inhibitors (9b, 9c, and 15a) showed lower IC₅₀ values in both enzyme- and cell-based assays than the formerly published hit compounds. The compounds were also found to reduce the pathological effects associated with 17β-HSD10 overexpression, although not the combined pathological effects of 17β-HSD10 overexpression within an amyloid-β rich environment.

Cyclophilins are a family of enzymes with peptidyl‐prolyl isomerase activity found in all cells of all organisms. To date, 17 cyclophilin isoforms have been identified in the human body, participating in diverse biological processes. Consequently, cyclophilins have emerged as promising targets for drug development to address a wide array of human diseases. This review describes the structural characteristics of individual cyclophilin isoforms and explores the roles that they play in human health and diseases, such as in viral infections, Alzheimer's disease, Parkinson's disease, cardiovascular diseases, or cancer. Additionally, the review addresses inhibition of cyclophilins, particularly focusing on the development of selective small‐molecule inhibitors of individual cyclophilins, which possess a significant potential as novel therapeutics.

The multifunctional mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10) plays an important role in the pathology of several diseases, of which Alzheimer’s disease (AD) is the most debated. HSD10 overexpression and its interplay with amyloid-β peptide (Aβ) are considered a factor contributing to mitochondrial damage and neuronal stress observed in AD patients. This study confirms that individual overexpression of HSD10 or APP (amyloid precursor protein that gives rise to Aβ) leads to cytotoxicity, and both pathological conditions are linked to mitochondrial damage. However, the metabolic changes caused by these two overexpressions significantly differ, particularly in their effect on the tricarboxylic acid cycle and β-oxidation. Furthermore, the enzymatic activity of HSD10 is identified as the primary factor of HSD10 cytotoxicity, which is significantly exacerbated in an Aβ-rich environment and can be partially reversed by HSD10 inhibitors. Notably, a previously published and competitive benzothiazole inhibitor was effective in restoring the viability of HSD10 overexpressing cells alone and in an Aβ-rich environment, implying the potential benefit of HSD10 inhibitors in mitochondrial diseases and/or AD treatment. GRAPHICAL

Cyclophilin D (CypD) is a mitochondrial enzyme widely accepted as a regulator of the mitochondrial permeability transition pore (mPTP). Excessive opening of mPTP is associated with mitochondrial dysfunction and the development of various diseases; thus, suppression of mPTP opening through CypD inhibition presents a promising therapeutic approach. However, only a limited number of selective CypD inhibitors are currently available. In this study, 10 derivatives of 2-(benzyloxy)arylurea similar or identical to previously published CypD/mPTP inhibitors were synthesized. Unlike the original reports that assessed the opening of mPTP at the cellular level, the compounds were tested directly on the purified CypD enzyme to validate their putative mechanism of action. Additionally, the effect of the selected compounds was tested on isolated mitochondria. The obtained results show that the compounds are only weak inhibitors of CypD and mPTP opening, which is in contrast to previous conclusions drawn from the unspecific cellular JC-1 assay.

Multifunctional mitochondrial enzyme 17 beta-hydroxysteroid dehydrogenase type 10 (17 beta-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 17 beta-HSD10 overexpression and its interaction with amyloid-beta peptide contribute to mitochondrial dysfunction and neuronal stress. In this work, a series of new benzothiazole-derived 17 beta-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).

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