http://europepmc.org/articles/PMC3912268?pdf=render

Chromatin and beyond: the multitasking roles for SIRT6.

Less than a decade ago, it was shown that bromodomains, acetyl lysine 'reader' modules found in proteins with varied functions, were highly tractable small-molecule targets. This is an unusual property for protein-protein or protein-peptide interaction domains, and it prompted a wave of chemical probe discovery to understand the biological potential of new agents that targeted bromodomains. The original examples, inhibitors of the bromodomain and extra-terminal (BET) class of bromodomains, showed enticing anti-inflammatory and anticancer activities, and several compounds have since advanced to human clinical trials. Here, we review the current state of BET inhibitor biology in relation to clinical development, and we discuss the next wave of bromodomain inhibitors with clinical potential in oncology and non-oncology indications. The lessons learned from BET inhibitor programmes should affect efforts to develop drugs that target non-BET bromodomains and other epigenetic readers.

Bromodomains: a new target class for drug development

Aberrations in the epigenetic landscape are a hallmark of cancer. Alterations in enzymes that are "writers," "erasers," or "readers" of histone modification marks are common. Bromodomains are "readers" that bind acetylated lysines in histone tails. Their most important function is the regulation of gene transcription by the recruitment of different molecular partners. Moreover, proteins containing bromodomains are also epigenetic regulators, although little is known about the specific function of these domains. In recent years, there has been increasing interest in developing small molecules that can target specific bromodomains. First, this has helped clarify biological functions of bromodomain-containing proteins. Secondly, it opens a new front for combatting cancer. In this review we will describe the structures and mechanisms associated with Bromodomain and Extra-Terminal motif (BET) inhibitors and non-BET inhibitors, their current status of development, and their promising role as anti-cancer agents.

https://www.tandfonline.com/doi/pdf/10.1080/15592294.2016.1265710

Bromodomain inhibitors and cancer therapy: From structures to applications.

1.1. Discovery of Sirtuins
The founding member of sirtuin is the yeast-silencing information regulator 2 (SIR2) protein, one of four proteins (SIR1–4) required for silencing the mating-type information loci in yeast.1 SIR2–4, but not SIR1, are also required for gene silencing at telomeres.2 SIR2 also mediates gene silencing at the rDNA (rDNA) loci, which was shown to be independent of other SIR proteins.3,4 Immunofluorescence imaging showed that SIR2 is mainly in the nucleolus and telomeres in yeast.5 It was demonstrated that the silenced genetic loci have low histone acetylation levels compared to loci that are not silenced. Mutation in Sir2–4 increased histone acetylation levels and overexpression of SIR2 but not other SIR proteins led to decreases in histone H4, H2B, and H3 acetylation.6 Analyzing the sensitivity of yeast chromatin to micrococcal nuclease and dam methyltransferase indicated that Sir2 mutation affects the chromatin structure in the rDNA and mating-type loci.7

These interesting discoveries on SIR2 were further elevated by two discoveries made by Guarente and co-workers. The first one was that SIR2 is important for the replicative life span of yeast cells,8 a finding that was later extended to higher eukaryotic species,9,10 although the role of sirtuins in life span is highly controversial11 and may depend on genetic background and diet conditions.12,13 The second discovery was that SIR2 is an NAD-dependent histone deacetylase (Figure ​(Figure11),14 which established SIR2 as a mechanistically novel lysine deacetylase and revealed the connection between gene silencing and cellular metabolism. Indeed, it was later found that the life span extension effect of SIR2 is dependent on NAD level and NAD metabolism.15−17 Several other groups similarly reported the enzymatic activity of SIR2.18 These landmark discoveries established sirtuins as important players in epigenetics and triggered the explosion of research interest in sirtuins.



Figure 1

Enzymatic function of sirtuins. (A) NAD-dependent protein lysine deacylation activity of different sirtuins. (B) Enzymatic reaction mechanism of sirtuins. (C) Structure of a ternary sirtuin-NAD-acetyl peptide complex (PDB ID 2H4F). NAD, acetyl lysine, ...

Sirtuins in Epigenetic Regulation

Bromodomains, small protein modules that recognize acetylated lysine on histones, play a significant role in the epigenome, where they function as “readers” that ultimately determine the functional outcome of the post-translational modification. Because the initial discovery of selective BET inhibitors have helped define the role of that protein family in oncology and inflammation, BET bromodomains have continued to garner the most attention of any other bromodomain. More recently, non-BET bromodomain inhibitors that are potent and selective have been disclosed for ATAD2, CBP, BRD7/9, BRPF, BRPF/TRIM24, CECR2, SMARCA4, and BAZ2A/B. Such novel inhibitors can be used to probe the physiological function of these non-BET bromodomains and further understanding of their role in certain disease states. Here, we provide an update to the progress in identifying selective bromodomain inhibitors and their use as biological tools, as well as our perspective on the field.

Disrupting Acetyl-Lysine Recognition: Progress in the Development of Bromodomain Inhibitors

Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.

Fragment-Based Discovery of Low-Micromolar ATAD2 Bromodomain Inhibitors

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.

/pdf/over-expression-of-wild-type-or-a-catalytically-dead-mutant-48zlfqg56c.pdf

Over expression of wild type or a catalytically dead mutant of Sirtuin 6 does not influence NFκB responses.

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Bernadette Mouzon

Papers

Fragment-Based Discovery of Low-Micromolar ATAD2 Bromodomain Inhibitors

Over expression of wild type or a catalytically dead mutant of Sirtuin 6 does not influence NFκB responses.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.