Antitumor activity of a small-molecule inhibitor of human silent information regulator 2 enzymes.

SIRT1 and other NAD-dependent deacetylases have been implicated in control of cellular responses to stress and in tumorigenesis through deacetylation of important regulatory proteins, including p53 and the BCL6 oncoprotein. Hereby, we describe the identification of a compound we named cambinol that inhibits NAD-dependent deacetylase activity of human SIRT1 and SIRT2. Consistent with the role of SIRT1 in promoting cell survival during stress, inhibition of SIRT1 activity with cambinol during genotoxic stress leads to hyperacetylation of key stress response proteins and promotes cell cycle arrest. Treatment of BCL6-expressing Burkitt lymphoma cells with cambinol as a single agent induced apoptosis, which was accompanied by hyperacetylation of BCL6 and p53. Because acetylation inactivates BCL6 and has the opposite effect on the function of p53 and other checkpoint pathways, the antitumor activity of cambinol in Burkitt lymphoma cells may be accomplished through a combined effect of BCL6 inactivation and checkpoint activation. Cambinol was well tolerated in mice and inhibited growth of Burkitt lymphoma xenografts. Inhibitors of NAD-dependent deacetylases may constitute novel anticancer agents.

In vitro assays for the determination of histone deacetylase activity.

Histone deacetylases are important regulators of transcription and an emerging target for anticancer drugs. We present an overview over various assay formats that include radiolabelled histones, oligopeptides, and small molecules as substrates. The advantages and disadvantages of the various formats in terms of, e.g., substrate availability, throughput or subtype selectivity are discussed. Detailed procedures for various assay types that can be used for different problems, such as library screening or fluorescent inhibitor testing, are given. We present a new protocol for a simple high-throughput assay for NAD+-dependent (class III) histone deacetylases, also termed sirtuins.

Effect of inhibitors of histone deacetylase on the induction of cell differentiation in murine and human erythroleukemia cell lines.

Histone deacetylase (HDAC) inhibitors are a novel class of promising anti-cancer agents. Little information is available on the capacity of structurally different HDAC inhibitors to induce terminal cell differentiation in different cell types in relation to enzyme inhibition and subtype selectivity. Consequently, the aim of this study was to provide a comprehensive comparison of these effects. New biarylalanine inhibitors of HDAC were synthesized and compared to a series of standard inhibitors from different laboratories. Chromatographically purified rat liver and immunoprecipitated FLAG-tagged recombinant human HDACs were used as sources of HDAC activity. Enzyme inhibition was studied using a fluorescent substrate and its conversion was monitored by high-performance liquid chromatography. The ability to induce cell differentiation was compared in murine (Friend DS-19) and human (K562) erythroleukemic cell lines, and was quantified by benzidine staining. Inhibition of cell proliferation was evaluated by cell counting. All HDAC inhibitors were identified as potent inhibitors of erythroleukemic cell proliferation. However, we observed a complex pattern of differentiation induction: structurally similar inhibitors resulted in disparate activity profiles, whereas similar profiles were detected within distinct structural classes. Among the newly synthesized biarylalanine compounds, a 3'-methoxy derivative was identified as a very effective inducer of terminal cell differentiation. We conclude that investigation of subtype selectivity of selected HDAC inhibitors does not provide a clear link between selectivity and the observed cellular activity profile. The predictive value of in vitro HDAC inhibition assays for identifying anti-proliferative compounds has been emphasized.

Substrate-specific activation of sirtuins by resveratrol.

Resveratrol, a small molecule found in red wine, is reported to slow aging in simple eukaryotes and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported to act as a sirtuin activator, and this property has been proposed to account for its anti-aging effects. We show here that resveratrol is a substrate-specific activator of yeast Sir2 and human SirT1. In particular, we observed that, in vitro, resveratrol enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a non-physiological fluorescent moiety, but has no effect on binding and deacetylation of acetylated peptides lacking the fluorophore. Consistent with these biochemical data we found that in three different yeast strain backgrounds, resveratrol has no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and life span. In light of these findings, the mechanism accounting for putative longevity effects of resveratrol should be reexamined.

Subtype selective substrates for histone deacetylases.

To probe the steric requirements for deacylation, we synthesized lysine-derived small molecule substrates and examined structure-reactivity relationships with various histone deacetylases. Rat liver, human HeLa, and human recombinant class I and II histone deacetylases (HDACs) as well as human recombinant NAD(+)-dependent SIRT1 (class III enzyme) were used in these studies. A benzyloxycarbonyl substituent on the alpha-amino group yielded the highest conversion rates. Replacing the epsilon-acetyl group with larger lipophilic acyl substituents led to a pronounced decrease in conversion by class I and II enzymes; the class III enzyme displayed a greater tolerance. Incubations with recombinant FLAG-tagged human HDACs 1, 3, and 6 showed a distinct subtype selectivity among small molecule substrates. The subtype selectivity of HDAC inhibitors could be predicted with these substrates and an easily obtainable mixture of HDAC subtypes.

Nonisotopic substrate for assaying both human zinc and NAD+-dependent histone deacetylases.

Histone deacetylases (HDACs) are involved in the regulation of transcription and their inhibitors are a promising class of new anticancer drugs. We have previously reported Boc(Ac)Lys-AMC, also termed MAL, as a fluorescent substrate for HDACs. Now we present a modification of MAL called Z-MAL that is characterized by an increased rate of conversion by histone deacetylases of classes I and II and the recently discovered sirtuins (histone deacetylases class III). MAL and Z-MAL are the first nonradioactive substrates for class III enzymes. The new substrate Z-MAL allows for shorter assay times in inhibitor screening and is applicable to diverse sources of deacetylase activity even with completely different catalytic mechanisms. Interestingly, MAL shows some relative preference toward class II, indicating that subtype selectivity in small-molecule HDAC substrates might be obtained.

A homogeneous nonisotopic histone deacetylase activity assay.

Histone deacetylases (HDACs) are important regulators of transcription, and their inhibitors are a promising class of anticancer agents. The methods for the determination of HDAC activity and its inhibition that are currently available suffer from various drawbacks, such as animal testing, radioactive substrates, or limited throughput. Therefore, a fast nonisotopic method for the measurement of HDAC activity is highly desirable. The authors present such an assay that relies on the fluorescent HDAC substrate developed previously in their group. After incubation of the substrate with the enzyme, a derivatization leads to efficient fluorescence quenching in the deacetylated metabolite. Thus, only the fluorescence emitted by the remaining substrate is detected, which allows for a convenient detection of HDAC activity in a homogeneous format that can be performed on multiwell plate readers. This procedure, called HDASH (histone deacetylase assay-homogeneous), should be a valuable tool in transcriptional research and especially drug discovery.

Hydroxamide derivatives of short-chain fatty acids are potent inducers of human fetal globin gene expression.

OBJECTIVE: To examine whether hydroxamic acids are inducers of fetal hemoglobin expression, we tested the effects on gamma gene expression of butyric and propionic hydroxamic acids and of two other hydroxamic acids (SBHA and SAHA), which are potent inhibitors of histone deacetylase (HDAC). We also investigated whether there is a correlation between HDAC inhibitory activity of the compounds and their ability to induce gamma-globin gene expression. MATERIALS AND METHODS: Effects on gamma-globin expression were assessed by two methods: 1) a screening assay in which specific gamma-globin gene inducers are recognized by their ability to increase gamma firefly luciferase activity significantly more than beta-renilla luciferase activity; and 2) measurements of gamma-globin mRNA and the frequency of fetal hemoglobin-positive erythroblasts in cultures of burst-forming unit erythroid (BFU-E) from normal individuals. HDAC in vitro activity was measured with a partially purified rat liver HDAC and a fluorogenic substrate. RESULTS: All compounds tested increased gamma firefly luciferase activity, gamma/gamma+beta mRNA ratios, and percentage of fetal hemoglobin-containing erythroblasts in BFU-E cultures, in a dose-dependent fashion. Butyryl-hydroxamic acid 100 microM increased the gamma/gamma+beta mRNA ratios by 5.8-fold and the frequency of fetal hemoglobin-containing erythroblasts by 4.1-fold. Propionyl-hydroxamic acid 150 microM increased the gamma/gamma+beta ratios by 6.3-fold and the fetal hemoglobin-containing erythroblasts by 3.9-fold. SBHA induced gamma-globin gene expression at very low concentrations, 5 to 20 microM in the luciferase system and 2 to 8 microM in BFU-E cultures; SAHA at 1 to 7.5 microM in the luciferase system and 1 to 2.5 microM in the BFU-E cultures. HDAC in vitro inhibition was observed in the millimolar range for propionate and butyrate. IC(50) determinations led to values of 384 microM for propionyl-hydroxamate, 47 microM for butyryl-hydroxamate, 0.93 microM for SBHA, and 0.26 microM for SAHA. CONCLUCION: Our data indicate that hydroxamic acid-based HDAC inhibitors are potent gamma-globin gene inducers and that the concentration range of their effects on gamma gene expression can be correlated roughly with their HDAC inhibitory potencies.

Eosin Y as an internal standard for a plate reader-based quantitation of a histone deacetylase substrate.

Ongoing interest in histone deacetylase (HDAC) inhibitors as potential anticancer drugs and mechanistic tools for the study of gene regulation is driving the improvement of assay techniques for the determination of HDAC activity. We previously reported the first non-isotopic substrate for HDAC. A plate reader-based determination of the substrate conversion utilized a boraindacene as an internal standard which is no longer commercially available. We report here that Eosin Y is a suitable replacement for that purpose, leading to a validated HDAC assay with increased throughput.

A microplate reader-based nonisotopic histone deacetylase activity assay.

Recent years have brought an enormous increase in knowledge concerning the involvement of histone deacetylase (HDAC) in gene regulation and the potential use of its inhibitors in transcription therapy. This also stimulates research toward new methods for the determination of HDAC activity and thus the potency of potential inhibitors. We have previously succeeded in developing a nonisotopic assay for HDAC using a fluorescent coumarin derivative of epsilon-acetyllysine. Here we present plate reader-based quantitation as an alternative means for the determination of substrate conversion. A new validated assay procedure with a boradiazaindacene (BODIPY 530/550) rather than a coumarin internal standard was established to allow for fluorescence measurement without chromatographic separation. The method is equal in its sensitivity, accuracy, and precision to the previously published HPLC method. A comparison with a new commercially available homogeneous plate reader assay leads to similar inhibition constants for the HDAC inhibitor trichostatin A. The commercial assay has a higher throughput but its procedure for the detection of HDAC activity could not be applied to our enzyme preparation, while our substrate is also converted by HeLa HDAC. This indicates a broader range of potential applications for our system.