Validation of Slow Off-Kinetics of Sirtuin Rearranging Ligands (SirReals) by Means of the Label-Free Electrically Switchable Nanolever Technology

Recently, we have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase Sirt2. In previous studies, using a biotinylated SirReal analogue in combination with biolayer interferometry, we observed a slow dissociation rate of the inhibitor-enzyme complex, which had been postulated to be the key to the high affinity and selectivity of SirReals. However, for the attachment of biotin to the SirReal core, we introduced a triazole as a linking moiety, which was shown by X-ray co-crystallography to interact with Arg97 of the cofactor binding loop. This study now is directed to answer the question, whether the observed long residence time of the SirReals is induced mainly by triazole incorporation or is an inherent characteristic of the SirReal inhibitor core. Therefore, we used the novel label-free switchSENSE® technology, based on electrically switchable DNA nanolevers, to validate that the long residence time of the SirReals is caused by the core scaffold.