Studying sirtuin inhibitors with in silico and in vitro approaches

Caloric restriction (CR) is the kind of diet where the intake of calories is suppressed but the amount of nutrients is maintained; it has long been touted as the recipe for a longer lifespan. After the yeast enzyme silent information regulator 2 (Sir2) was found to be responsible for the longevity effects of CR, the search commenced to identify its analogues in mammals. Even though the roles of mammalian Sir2 relatives, sirtuins 1-7 (SIRT1-7), in CR and lifespan extension have not been definitely confirmed, sirtuins have become a major research target in health sciences. The function of sirtuins is linked to a wide variety of cellular functions, such as gene expression, energy production and adaptation to environmental stimuli. Sirtuins form the class III of histone deacetylase enzymes (HDACs). They affect the function of their target proteins by mediating the deacetylation of lysine residues. During the last few years, some sirtuin subtypes have also been shown to possess other catalytic activities, such as demyristoylation and desuccinylation. All catalytic mechanisms of sirtuins involve the cofactor nicotinamide adenine dinucleotide (NAD+). NAD+ is a biomarker for cellular metabolism, and it is the utilization of this cofactor which places sirtuins in the crossroads of metabolism, gene expression and protein function. The involvement of sirtuins in a wide variety of biological processes has triggered the search for sirtuin modulators, which could elucidate the physiological roles of sirtuins and ultimately function as sirtuin targeting drugs. Changes in sirtuin function have been proposed to affect several major pathological conditions including cancer, neurodegenerative diseases and diabetes. For example in these conditions it could be beneficial to inhibit sirtuin function, and thus effective sirtuin inhibitors have been sought for more than a decade. In this thesis, modern computer aided drug design methods have been combined with in vitro methods in the investigation of the properties of sirtuin inhibitors. Often, sirtuin inhibitors are have been tested on SIRT1-3, whereas their effects on the lesser studied sirtuins i.e. SIRT4-7 have been ignored. Thus, as a starting point for the design of SIRT6 inhibitors, the effect of known sirtuin inhibitors on SIRT6 deacetylation was examined. In the subsequent screening for SIRT6 inhibitors, a new in vitro method was developed, based upon the natural deacetylation substrates of SIRT6. Then, data derived from interactions between substrate based inhibitors and SIRT1 was used in a quantitative structure-activity relationship study (QSAR) to design new inhibitors. Lastly, new small molecule sirtuin inhibitors were screened from a commercial database in silico to a putative allosteric binding site. This thesis has resulted in the identification of new sirtuin inhibitors and novel insights into sirtuin inhibitor specificity. Additionally, the developed in silico and in vitro methods represent useful approaches for sirtuin inhibitor screening and drug design. National Library of Medicine Classification: QU 26.5, QU 135, QU 143, QV 745 Medical Subject Headings: Caloric Restriction; Catalysis; Drug Design; Computational Biology; Histone Deacetylases; Inhibitors; NAD; Quantitative Structure-Activity Relationship; Sirtuins; In vitro Techniques