Abstract by Kathrin Sten Troelsen

The sirtuin (SIRT) enzymes are important regulatory deacylases with different acyl substrate preferences and subcellular localization. The enzymes are NAD⁺-dependent and catalyze the hydrolysis of ε-N-acyllysine posttranslational modifications (PTMs). SIRT3–5 are mainly located in the mitochondria and regulate acetylation (SIRT3) and succinylation (SIRT5) levels on a wide range of metabolic enzymes. The sirtuins have been considered as potential drug targets in certain cancers and metabolic diseases. However, the biological role of the mitochondrial sirtuins is still not fully understood. Thus, it was envisioned to develop chemical tools such as inhibitors and substrates to aid in the investigation of SIRT3–5. 

For investigation of SIRT5, mechanism-based inhibitors were developed and afforded potent and selective SIRT5 inhibitors with nanomolar affinity. Importantly, kinetic evaluation of the inhibitors revealed for the first time a slow, tight-binding mechanism of inhibition for SIRT5.

Selective inhibition of SIRT3 has proven challenging due to the high structural similarity between SIRT1–3. A novel strategy, which involved mitochondrial targeting of inhibitors, was developed to achieve selectivity for SIRT3 over SIRT1 and SIRT2 that are located in the nucleus and cytosol, respectively. Excellent mitochondrial localization was observed in HeLa cells as indicated by fluorophore-labeled inhibitor versions. Downstream effects on SIRT1–3 targets showed increased acetylation levels of the documented SIRT3 target manganese superoxide dismutase (MnSOD), while known targets of SIRT1 and SIRT2 remained unaffected. Finally, direct engagement between the lead inhibitor and SIRT3 was demonstrated by cellular thermal shift assays. This thorough biological evaluation showed that the lead inhibitor exhibited selectivity for SIRT3 in cells.

The substrate preference of SIRT4 was only recently uncovered and like SIRT5, this enzyme also cleaves negatively charged carboxyacyl modifications. This emphasized the possibility of additional undiscovered SIRT4 and SIRT5 substrates. Itaconyl and mesaconyl substrates were synthesized and evaluated against SIRT5. The itaconyl C4 substrates were cleaved by SIRT5 and kinetic analyses showed that the substrates performed similarly to an already known glutaryl SIRT5 substrate. The substrates exhibited excellent thermal stability and satisfactory chemical stability in the presence of glutathione.

Finally, acylating reagents for the introduction of more complex lysine modifications were designed using photolabile protecting groups for the carboxylic acids. The reagents were successfully used to modify small molecules and shorter peptides, however when more complex systems were evaluated the reagents failed to produce well-defined effects. 

It is expected that the developed tools will enable a more detailed investigation of the mitochondrial sirtuins and further unveil the biological functions regulated by these enzymes.