Studies of the Binding of Modest Modulators of the Human Enzyme, Sirtuin 6, by STD NMR

Abstract

Pyrazinamide (PZA), an essential constituent of short-course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite the structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 by different mechanisms and through different binding sites, as suggested by saturation transfer difference (STD) NMR. Available experimental evidence, such as that derived from crystal structures and kinetic experiments, has been of only limited utility in elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For instance, crystallographic structural analysis of sirtuin binding sites does not help us understand important differences in binding affinities among sirtuins or capture details of such dynamic process. Hence, STD NMR was utilized throughout this study. Our results not only agreed with the binding kinetics experiments but also gave a qualitative insight into the binding process. The data presented herein suggested some details about the geometry of the binding epitopes of the ligands in solution with the apo- and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates might be possible. These derivatives might be accessible by employing the malleability of this scaffold to assist in the identification by STD NMR of the motifs that interact with the apo- and holoenzymes in solution.

Emerging roles of SIRT6 in human diseases and its modulators

“…Several amino acid residues (e.g., Ile 61, Pro62, Phe64/82/86, Val70, Asn114, Val115, and Asp116) form a hydrophobic pocket to anchor the molecules in the pocket with polar contact and potential H‐bonds. Most modulators binding in the active site have partial overlap with the pocket where NAM moiety of NAD + occupies (Figure 1A, C site), and thus no ADPr competition but statistically significant NAD + or NAM competition with them are detected in the activity assays 54,296–300 . Importantly, SIRT6 activators binding in this pocket primarily exerts concentration‐dependent deacetylation activity.…”

“…To the best of our knowledge, small molecule modulators including both inhibitors and activators which bind in the active site, are reported to act on SIRT6 by binding to the pocket locating at the distal end of SIRT6′s extended acyl binding channel near the N‐terminal loop 296–298 . Several amino acid residues (e.g., Ile 61, Pro62, Phe64/82/86, Val70, Asn114, Val115, and Asp116) form a hydrophobic pocket to anchor the molecules in the pocket with polar contact and potential H‐bonds.…”

Emerging Therapeutic Potential of SIRT6 Modulators

“…Remarkably, these compounds did not show NAD + competition, hence indicating a different mechanism of action from 11a . 150 11c was reported to be active toward SIRT1, but not SIRT2/3, while 11b was not evaluated against SIRT1–3. Nonetheless, selectivity against other SIRTs and HDACs need to be ascertained.…”

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

“…A variety of methods, including mass spectrometry (MS)‐based approaches, have been developed for the identification of antibody‐specific epitopes and for providing structural information about antigen–antibody complexes. These methods also include X‐ray crystallography and NMR spectroscopy, immunoanalytical characterization of antigen–antibody interactions, and the design and synthesis of peptide–epitope mimotopes . Proteolytic excision–extraction mass spectrometry (PROTEX‐MS), hydrogen–deuterium exchange mass spectrometry (HDX‐MS) of peptide backbone hydrogen atoms, and fast‐photochemical oxidation of proteins are the major techniques for MS‐based elucidation of antibody epitopes, but these tools alone do not provide quantitative affinity data .…”