Thiosuccinyl Peptides as Sirt5-Specific Inhibitors

Sirtuins, a class of enzymes known as nicotinamide adenine dinucleotide-dependent deacetylases, have been shown to regulate a variety of biological processes, including aging, transcription, and metabolism. Sirtuins are considered promising targets for treating several human diseases. There are seven sirtuins in humans (Sirt1–7). Small molecules that can target a particular human sirtuin are important for drug development and fundamental studies of sirtuin biology. Here we demonstrate that thiosuccinyl peptides are potent and selective Sirt5 inhibitors. The design of these inhibitors is based on our recent discovery that Sirt5 prefers to catalyze the hydrolysis of malonyl and succinyl groups, rather than an acetyl group, from lysine residues. Furthermore, among the seven human sirtuins, Sirt5 is the only one that has this unique acyl group preference. This study demonstrates that the different acyl group preferences of different sirtuins can be conveniently utilized to develop small molecules that selectively target different sirtuins

Dph3 Is an Electron Donor for Dph1-Dph2 in the First Step of Eukaryotic Diphthamide Biosynthesis

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from <i>S</i>-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C–C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1–Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis <i>in vitro</i>. Here we demonstrate by <i>in vitro</i> reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step <i>in vitro</i> in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells

An Immunosuppressive Antibody–Drug Conjugate

We have developed a novel antibody–drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib–antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC₅₀ and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology