Design and application of synthetic 17B-HSD13 substrates reveals preserved catalytic activity of protective human variants

Several hydroxysteroid dehydrogenase 17-beta 13 variants have previously been identified as protective against metabolic dysfunction-associated steatohepatitis (MASH) fibrosis, ballooning and inflammation, and as such this target holds significant therapeutic potential. However, over 5 years later, the function of 17B-HSD13 remains unknown. Structure-aided design enables the development of potent and selective sulfonamide-based 17B-HSD13 inhibitors. In order to probe their inhibitory potency in endogenous expression systems like primary human hepatocytes, inhibitors are transformed into synthetic surrogate substrates with distinct selectivity advantages over substrates previously published. Their application to cells endogenously expressing 17B-HSD13 enables quantitative measures of enzymatic inhibition in primary human hepatocytes which has never been reported to date. Application to multiple cellular systems expressing the protective human variants reveals that the most prevalent IsoD variant maintains NAD-dependent catalytic activity towards some but not all substrates, contradicting reports that the truncation results in loss-of-function.