The sirtuin enzymes are important regulatory deacylases in a variety of biochemical contexts and may therefore be potential therapeutic targets through either activation or inhibition by small molecules. Here, we describe the discovery of the most potent inhibitor of sirtuin 5 (SIRT5) reported to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more “drug-like” properties. Importantly, enzyme kinetic evaluation revealed a slow, tight-binding mechanism of inhibition, which is unprecedented for SIRT5. This is important information when applying inhibitors to probe mechanisms in biology.

SIRT3 is a mitochondrial lysine deacetylase enzyme, regulating the activity of numerous mitochondrial proteins. Here, we have designed inhibitors of this enzyme, which exhibit selective inhibition of SIRT3 in cells by specific organelle localizing.

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two b-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the b-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the b-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.Editor: David Eliezer. SIGNIFICANCE The complex between von Willebrand factor (VWF) and factor VIII (FVIII) is imperative for hemostasis, and failure to form this complex leads to bleeding diatheses such as hemophilia A and von Willebrand disease (VWD). The FVIII a3 acidic region is shown to interact with VWF TIL'E', and the a3 binding region is localized to two b-sheet regions on the periphery of TIL'. Characterizations of the interaction between FVIII and mutants of VWF TIL'E' further highlight the importance of the b-sheet region for binding. The insight into VWF:FVIII complex formation facilitate the design of improved hemophilia A treatments, whereas the analysis of VWD mutations provides a link between genetic pathology and clinical phenotype to facilitate targeted management of patients with VWD.

We report the synthesis and biological evaluation of a lightactivated (caged) prodrug of the KDAC inhibitor panobinostat (Zap-Pano). We demonstrate that addition of the 4,5-dimethoxy-2-nitrobenzyl group to the hydroxamic acid oxygen results in an inactive prodrug. In two cancer cell lines we show that photolysis of this compound releases panobinostat and an unexpected carboxamide analogue of panobinostat. Photolysis of Zap-Pano causes an increase in H3K9Ac and H3K18Ac, consistent with KDAC inhibition, in an oesophageal cancer cell line (OE21). Irradiation of OE21 cells in the presence of Zap-Pano results in apoptotic cell death. This compound is a useful research tool, allowing spatial and temporal control over release of panobinostat.