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Epigenetic factors are enzymes or proteins that confer, remove or recognize covalent modifications to chromatin DNA or proteins. They can be divided into three broad groups, commonly referred to as the 'writers', 'erasers' and 'readers'. The HDACs and sirtuins, which remove acetyl groups from the ɛ-amino of protein lysine residues, fall into the 'eraser' category. Due to their important effects on gene expression and involvement in various disease states, these enzymes have been the subjects of many assay development efforts in recent years. Commonly used techniques include mass spectrometry, antibody-based methods and protease-coupled assays with fluorogenic peptide substrates. Recent advances include the development of synthetic substrates for the assay of various newly discovered non-acetyl deacylation activities among the sirtuins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Methylation of histone H3 lysine-4 (H3K4) is an important, regulatory, epigenetic post-translational modification associated with actively transcribed genes. In humans, the principal mediators of this modification are part of the MLL/SET1 family of methyltransferases, which comprises six members, MLLs1-4 and SET1A/SET1B. Aberrations in the structure, expression, and regulation of these enzymes are implicated in various disease states, making them important potential targets for drug discovery, particularly for oncology indications. The MLL/SET1 family members are most enzymatically active when part of a "core complex," the catalytic SET-domain-containing subunits bound to a subcomplex consisting of the proteins WDR5, RbBP5, Ash2L and a homodimer of DPY-30 (WRAD2). The necessity of MLL/SET1 members to bind WRAD2 for full activity is the basis of a particular drug development strategy, which seeks to disrupt the interaction between the MLL/SET1 subunits and WDR5. This strategy is not without its theoretical and practical drawbacks, some of which relate to the ease with which complexes of Escherichia coli-expressed MLL/SET1 and WRAD2 fall apart. As an alternative strategy, we explore ways to stabilize the complex, focusing on the use of an excess of WRAD2 to drive the binding equilibria toward complex formation while maintaining low concentrations of the catalytic subunits. The purpose of this approach is to seek inhibitors that bind the SET domain, an approach proven successful with the related, but inherently more stable, enhancer of zeste homolog 2 (EZH2) complex.

Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-L-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpot(SM) platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC₅₀ values at a low μM range.

A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

Many plant molecules interact with and modulate key regulators of mammalian physiology in ways that are beneficial to health, but why? We propose that heterotrophs (animals and fungi) are able to sense chemical cues synthesized by plants and other autotrophs in response to stress. These cues provide advance warning about deteriorating environmental conditions, allowing the heterotrophs to prepare for adversity while conditions are still favorable.

This past decade has seen the identification of numerous conserved genes that extend lifespan in diverse species, yet the number of compounds that extend lifespan is relatively small. A class of compounds called STACs, which were identified as activators of Sir2/SIRT1 NAD+-dependent deacetylases, extend the lifespans of multiple species in a Sir2-dependent manner and can delay the onset of age-related diseases such as cancer, diabetes and neurodegeneration in model organisms. Plant-derived STACs such as fisetin and resveratrol have several liabilities, including poor stability and relatively low potency as SIRT1 activators. To develop improved STACs, stilbene derivatives with modifications at the 4' position of the B ring were synthesized using a Horner-Emmons-based synthetic route or by hydrolyzing deoxyrhapontin. Here, we describe synthetic STACs with lower toxicity toward human cells, and higher potency with respect to SIRT1 activation and lifespan extension in Saccharomyces cerevisiae. These studies show that it is possible to improve upon naturally occurring STACs based on a number of criteria including lifespan extension.

A major cause of aging is thought to result from the cumulative effects of cell loss over time. In yeast, caloric restriction (CR) delays aging by activating the Sir2 deacetylase. Here we show that expression of mammalian Sir2 (SIRT1) is induced in CR rats as well as in human cells that are treated with serum from these animals. Insulin and insulin-like growth factor 1 (IGF-1) attenuated this response. SIRT1 deacetylates the DNA repair factor Ku70, causing it to sequester the proapoptotic factor Bax away from mitochondria, thereby inhibiting stress-induced apoptotic cell death. Thus, CR could extend life-span by inducing SIRT1 expression and promoting the long-term survival of irreplaceable cells.

Calorie restriction (CR) slows aging in numerous species. In the yeast Saccharomyces cerevisiae, this effect requires Sir2, a conserved NAD+-dependent deacetylase. We report that CR reduces nuclear NAD+ levels in vivo. Moreover, the activity of Sir2 and its human homologue SIRT1 are not affected by physiological alterations in the NAD+:NADH ratio. These data implicate alternate mechanisms of Sir2 regulation by CR.

In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan. In the budding yeast Saccharomyces cerevisiae, calorie restriction extends lifespan by increasing the activity of Sir2 (ref. 1), a member of the conserved sirtuin family of NAD(+)-dependent protein deacetylases. Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan, and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor. Here we report the discovery of three classes of small molecules that activate sirtuins. We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD(+), and increases cell survival by stimulating SIRT1-dependent deacetylation of p53. In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.

The glycolate/glycerate transporter of spinach (Spinacia oleracea L.) chloroplast inner envelope membranes was solubilized by treatment of the membranes with sodium cholate. Mixtures of the cholate extracts and soy asolectin were subjected to gel filtration to remove the detergent. The reconstituted vesicles were frozen, thawed, and sonicated in a buffer that contained 10 millimolar d-glycerate and, usually, [(3)H]sucrose as an internal space indicator. The dilution of the vesicles into a medium that contained 0.4 millimolar [(14)C]d-glycerate resulted in a rapid accumulation of labeled glycerate, followed by a much slower loss of [(14)C]d-glycerate from the vesicles. This behavior is characteristic of counterflow. The accumulation of [(14)C]d-glycerate was strongly inhibited by HgCl(2), which blocks glycolate/glycerate transport in intact chloroplasts. In the absence of proton ionophores, the extent of [(14)C]glycolate accumulation under similar conditions was much greater than that of [(14)C]d-glycerate. External glycolate inhibited d-glycerate counterflow and external d-glycerate inhibited glycolate counterflow. The external pH dependence of the efflux of [(14)C]d-glycerate accumulated in vesicles by counterflow and its inhibition by external l-mandelate are characteristics displayed by glycolate transport in intact chloroplasts. Partial purification of the transporter was achieved by glycerol gradient centrifugation. The solubilized glycolate and glycerate counterflow activities, assayed by reconstitution into vesicles, were found to sediment similarly.

Experiments in which [1-(14)C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C(3) plants.

A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H(+) co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies.

The transport of glycolate and d-glycerate, the substrate and end product of the photorespiratory carbon pathway, respectively, by isolated intact pea (Pisum sativum) chloroplasts has been compared. d-Glycerate uptake was inhibited by the 2-hydroxymonocarboxylates, glycolate, glyoxylate, and d-lactate. Phosphate and phosphoglycerate and triose phosphates were without effect when the assays were carried out for two seconds. Glycolate was found to be a competitive inhibitor of d-glycerate uptake and the presence of glycolate in the chloroplast stroma strongly enhanced d-glycerate uptake from the medium. For optimal rates of d-glycerate-dependent O(2) evolution by pea chloroplasts, phosphate, ADP or ATP, and a mediator of cyclic electron flow had to be added. The inhibition of d-glycerate-dependent O(2) evolution by triose phosphates and 2-hydroxymonocarboxylates was tested. The inhibition of d-glycerate-dependent O(2) evolution by these metabolites did not correlate with their effects on glycerate transport. Thus, metabolism of d-glycerate, rather than its transport, limits the rate of glycerate-dependent oxygen evolution. The ramifications of d-glycerate metabolism on the interpretation of d-glycerate uptake data obtained with prolonged incubations are discussed. We conclude that d-glycerate and glycolate transport are mediated by the same transport system.