Sirtuin 1 Inhibiting Thiocyanates (S1th)-A New Class of Isotype Selective Inhibitors of NAD+ Dependent Lysine Deacetylases.

Sirtuin 1 (Sirt1) is a NAD+ dependent lysine deacetylase associated with the pathogenesis of various diseases including cancer. In many cancer types Sirt1 expression is increased and higher levels have been associated with metastasis and poor prognosis. However, it was also shown, that Sirt1 can have tumor suppressing properties and in some instances even a dual role for the same cancer type has been reported. Increased Sirt1 activity has been linked to extension of the life span of cells, respectively, organisms by promoting DNA repair processes and downregulation of tumor suppressor proteins. This may have the downside of enhancing tumor growth and metastasis. In mice embryonic fibroblasts depletion of Sirt1 was shown to decrease levels of the DNA damage sensor histone H2AX. Impairment of DNA repair mechanisms by Sirt1 can promote tumorigenesis but also lower chemoresistance toward DNA targeting therapies. Despite many biological studies, there is currently just one small molecule Sirt1 inhibitor in clinical trials. Selisistat (EX-527) reached phase III clinical trials for treatment of Huntington's Disease. New small molecule Sirt1 modulators are crucial for further investigation of the contradicting roles of Sirt1 in cancer. We tested a small library of commercially available compounds that were proposed by virtual screening and docking studies against Sirt1, 2 and 3. A thienopyrimidone featuring a phenyl thiocyanate moiety was found to selectively inhibit Sirt1 with an IC50 of 13 µM. Structural analogs lacking the thiocyanate function did not show inhibition of Sirt1 revealing this group as key for the selectivity and affinity toward Sirt1. Further analogs with higher solubility were identified through iterative docking studies and in vitro testing. The most active compounds (down to 5 µM IC50) were further studied in cells. The ratio of phosphorylated γH2AX to unmodified H2AX is lower when Sirt1 is depleted or inhibited. Our new Sirtuin 1 inhibiting thiocyanates (S1th) lead to similarly lowered γH2AX/H2AX ratios in mouse embryonic fibroblasts as Sirt1 knockout and treatment with the reference inhibitor EX-527. In addition to that we were able to show antiproliferative activity, inhibition of migration and colony forming as well as hyperacetylation of Sirt1 targets p53 and H3 by the S1th in cervical cancer cells (HeLa). These results reveal thiocyanates as a promising new class of selective Sirt1 inhibitors.

Azologization and repurposing of a hetero-stilbene-based kinase inhibitor: towards the design of photoswitchable sirtuin inhibitors.

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno analogue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out (E)-form. Irradiation of this isomer triggers isomerisation to the long-lived (Z)-configuration with a bent geometry causing a considerably shorter end-to-end distance. The resulting affinity shifts are intended to enable real-time photomodulation of sirtuins in vitro.

New chemical tools for probing activity and inhibition of the NAD+-dependent lysine deacylase sirtuin 2.

Sirtuins are NAD+-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Identification of Bichalcones as Sirtuin Inhibitors by Virtual Screening and In Vitro Testing.

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent class III histone deacetylases, which have been linked to the pathogenesis of numerous diseases, including HIV, metabolic disorders, neurodegeneration and cancer. Docking of the virtual pan-African natural products library (p-ANAPL), followed by in vitro testing, resulted in the identification of two inhibitors of sirtuin 1, 2 and 3 (sirt1-3). Two bichalcones, known as rhuschalcone IV (8) and an analogue of rhuschalcone I (9), previously isolated from the medicinal plant Rhus pyroides, were shown to be active in the in vitro assay. The rhuschalcone I analogue (9) showed the best activity against sirt1, with an IC50 value of 40.8 µM. Based on the docking experiments, suggestions for improving the biological activities of the newly identified hit compounds have been provided.

Chemically Induced Degradation of Sirtuin 2 (Sirt2) by a Proteolysis Targeting Chimera (PROTAC) Based on Sirtuin Rearranging Ligands (SirReals).

Here we report the development of a proteolysis targeting chimera (PROTAC) based on the combination of the unique features of the sirtuin rearranging ligands (SirReals) as highly potent and isotype-selective Sirt2 inhibitors with thalidomide, a bona fide cereblon ligand. For the first time, we report the formation of a PROTAC by Cu(I)-catalyzed cycloaddition of a thalidomide-derived azide to an alkynylated inhibitor. This thalidomide-derived azide as well as the highly versatile linking strategy can be readily adapted to alkynylated ligands of other targets. In HeLa cells, our SirReal-based PROTAC induced isotype-selective Sirt2 degradation that results in the hyperacetylation of the microtubule network coupled with enhanced process elongation. Thus, our SirReal-based PROTAC is the first example of a probe that is able to chemically induce the degradation of an epigenetic eraser protein.

Three-Component Aminoalkylations Yielding Dihydronaphthoxazine-Based Sirtuin Inhibitors: Scaffold Modification and Exploration of Space for Polar Side-Chains.

Nonpolar derivatives of heterocyclic aromatic screening hits like the non-selective sirtuin inhibitor splitomicin tend to be poorly soluble in biological fluids. Unlike sp3 -rich natural products, flat aromatic compounds are prone to stacking and often difficult to optimize into leads with activity in cellular systems. The aim of this work was to identify anchor points for the introduction of sp3 -rich fragments with polar functional groups into the newly discovered active (IC50 = 5 µM) but nonpolar scaffold 1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione by a molecular modeling approach. Docking studies were conducted with structural data from crystallized human SIRT2 enzyme. Subsequent evaluation of the in silico hypotheses through synthesis and biological evaluation of the designed structures was accomplished with the aim to discover new SIRT2 inhibitors with improved aqueous solubility. Derivatives of 8-bromo-1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione N-alkylated with a hydrophilic morpholino-alkyl chain at the thiocarbamate group intended for binding in the acetyl-lysine pocket of the enzyme appeared to be promising. Both the sulfur of the thiocarbamate and the bromo substituent were assumed to result in favorable hydrophobic interactions and the basic morpholino-nitrogen was predicted to build a hydrogen bond with the backbone Ile196. While the brominated scaffold showed moderately improved activity (IC50 = 1.8 µM), none of the new compounds displayed submicromolar activity. Synthesis and characterization of the new compounds are reported and the possible reasons for the outcome are discussed.