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Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin coupled assay determined that the tested inhibitors showed no competition towards NAD+ or the synthetic substrate analogue ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our initial SAR and docking