Novel Human Sirtuin Inhibitors And Inhibitory Strategies

The sirtuin family enzymes are able to catalyze the N ε-acyl-lysine side chain deacylation reaction on histone and non-histone protein substrates. Bacteria and archaea usually contains one or two sirtuins, however, the eukarya contains multiple sirtuin members. Since the discovery of founding member in 2000, the sirtuin-catalyzed deacylation reaction has been confirmed to play an important role in multiple crucial cellular processes such as gene transcription, DNA damage repair,and metabolism. This reaction has also been used in some human diseases such as cancer, metabolic and neurodegenerative diseases. Through a mechanistic view of the enzyme chemistry supported by several lines of experimental evidence, the unique NAD+-dependent nature of the sirtuin-catalyzed deacylation reaction has also been widely studied. In recent years, for this type of enzymatic deacylation reaction,different types of its inhibitors and inhibitory strategies have been reported, and these can help us to understand the catalytic mechanism of this enzymatic deacylation reaction which is of biological and pharmacological importance, and are important for the development of potential therapeutics for neurodegeneration, age-related diseases, and especially cancer.In my thesis work, I specifically explored the following on the basis of a solid past research foundation of my advisor’s research group:(1) By exploiting an open space observed in sirtuin 3-D structures that extended from the side chain of the bound substrate’s N?-acetyl-lysine residue, a series of bidentate compounds in which the Nε-acetyl-lysine part was covalenty linked to a distal functionality via a linker were designed and examined for their inhibitory potencies against SIRT1, the model sirtuin for this study. A few of such bidentate compounds were found to be stronger SIRT1 inhibitors than the N?-acetyl-lysine-containing monodentate counterpart,attesting to the feasibility of this design concenpt in furnishing potent sirtuin inhibitors. Moreover, the SIRT1 inhibition kinetics determination for a typical bidentate inhibitor indicated that it was a competitive inhibitor versus the N?-acetyl-lysine substrate and a non-competitive inhibitor versus NAD+. This findingwould be consistent with an ordered sequential kinetic mechanism for the SIRT1-catalyzed deacetylation reaction, in which the N?-acetyl-lysine substrate binding precedes the binding of NAD+. This finding also suggested that the bidentate SIRT1 inhibitors seem indeed to be able to occupy the open space observed in sirtuin structural analysis and thus prevent the binding of the N?-acetyl-lysine substrate and NAD+, which would consolidate the design rationale for the bidentate sirtuin inhibitors.(2)In this part of my thesis work, based on two SIRT1/2/3 pan-inhibitors that my advisor’s laboratory discovered previously, a series of their derivatives were design. These compounds harbored modified structural elements immediately surrounding the catalytic mechanism-based SIRT1/2/3 inhibitory warhead Nε-thioacetyl-lysine.Together with the derivatives prepared and assayed by one of my former labmates, we found a potent(low ?M) and selective(~10-fold versus SIRT1/2, >1,000-fold versus SIRT5, ~161-fold versus SIRT6) inhibitor against the SIRT3-catalyzed deacetylation reaction.(3) The third part of my thesis work is on our attempts to develop irreversible sirtuin inhibitors. With a few designed and prepared N?-haloacetyl-lysine compounds and SIRT1 as the model sirtuin for the work, we performed extensive SIRT1 inhibition assays, however, none of these compounds were found to be able to inhibit SIRT1 irreversibly. Nevertheless, this piece of work has laid a good foundation for future efforts in identifying irreversible sirtuin inhibitors.