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Histone/protein deacetylases (HDAC) 1 and 2 are typically viewed as structurally and functionally similar enzymes present within various co-regulatory complexes. We tested differential effects of these isoforms in renal ischemia reperfusion injury (IRI) using inducible knockout mice and found no significant change in ischemic tolerance with HDAC1 deletion, but mitigation of ischemic injury with HDAC2 deletion. Restriction of HDAC2 deletion to the kidney via transplantation or PAX8-controlled proximal renal tubule-specific Cre resulted in renal IRI protection. Pharmacologic inhibition of HDAC2 increased histone acetylation in the kidney but did not extend renal protection. Protein analysis demonstrated increased HDAC1-associated CoREST protein in HDAC2-/- versus WT cells, suggesting that in the absence of HDAC2, increased CoREST complex occupancy of HDAC1 can stabilize this complex. In vivo administration of a CoREST inhibitor exacerbated renal injury in WT mice and eliminated the benefit of HDAC2 deletion. Gene expression analysis of endothelin showed decreased endothelin levels in HDAC2 deletion. These data demonstrate that contrasting effects of HDAC1 and 2 on CoREST complex stability within renal tubules can affect outcomes of renal IRI and implicate endothelin as a potential downstream mediator.

Niemann-Pick disease type C1 (NPC1) is a rare genetic cholesterol storage disorder caused by mutations in the NPC1 gene. Mutations in this transmembrane late endosome protein lead to loss of normal cholesterol efflux from late endosomes and lysosomes. It has been shown that broad spectrum histone deacetylase inhibitors (HDACi's) such as Vorinostat correct the cholesterol accumulation phenotype in the majority of NPC1 mutants tested in cultured cells. In order to determine the optimal specificity for HDACi correction of the mutant NPC1s, we screened 76 HDACi's of varying specificity. We tested the ability of these HDACi's to correct the excess accumulation of cholesterol in patient fibroblast cells that homozygously express NPC1 I1061T , the most common mutation. We determined that inhibition of HDACs 1, 2, and 3 is important for correcting the defect, and combined inhibition of all three is needed to achieve the greatest effect, suggesting a need for multiple effects of the HDACi treatments. Identifying the specific HDACs involved in the process of regulating cholesterol trafficking in NPC1 will help to focus the search for more specific druggable targets.

Copyright © 2020 The Authors, some rights reserved. Fragile X syndrome is caused by FMR1 gene silencing and loss of the encoded fragile X mental retardation protein (FMRP), which binds to mRNA and regulates translation. Studies in the Fmr1-/y mouse model of fragile X syndrome indicate that aberrant cerebral protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5) signaling contributes to disease pathogenesis, but clinical trials using mGluR5 inhibitors were not successful. Animal studies suggested that treatment with lithium might be an alternative approach. Targets of lithium include paralogs of glycogen synthase kinase 3 (GSK3), and nonselective small-molecule inhibitors of these enzymes improved disease phenotypes in a fragile X syndrome mouse model. However, the potential therapeutic use of GSK3 inhibitors has been hampered by toxicity arising from inhibition of both α and β paralogs. Recently, we developed GSK3 inhibitors with sufficient paralog selectivity to avoid a known toxic consequence of dual inhibition, that is, increased β-catenin stabilization. We show here that inhibition of GSK3α, but not GSK3β, corrected aberrant protein synthesis, audiogenic seizures, and sensory cortex hyperexcitability in Fmr1-/y mice. Although inhibiting either paralog prevented induction of NMDA receptor–dependent long-term depression (LTD) in the hippocampus, only inhibition of GSK3α impaired mGluR5-dependent and protein synthesis–dependent LTD. Inhibition of GSK3α additionally corrected deficits in learning and memory in Fmr1-/y mice; unlike mGluR5 inhibitors, there was no evidence of tachyphylaxis or enhanced psychotomimetic-induced hyperlocomotion. GSK3α selective inhibitors may have potential as a therapeutic approach for treating fragile X syndrome.

Chronic stress promotes depression in some individuals, but has no effect in others. Susceptible individuals exhibit social avoidance and anxious behavior and ultimately develop depression, whereas resilient individuals live normally. Exercise counteracts the effects of stress. Our objective was to examine whether lactate, a metabolite produced during exercise and known to reproduce specific brain exercise-related changes, promotes resilience to stress and acts as an antidepressant. To determine whether lactate promotes resilience to stress, male C57BL/6 mice experienced daily defeat by a CD-1 aggressor, for 10 days. On the 11th day, mice were subjected to behavioral tests. Mice received lactate before each defeat session. When compared with control mice, mice exposed to stress displayed increased susceptibility, social avoidance and anxiety. Lactate promoted resilience to stress and rescued social avoidance and anxiety by restoring hippocampal class I histone deacetylase (HDAC) levels and activity, specifically HDAC2/3. To determine whether lactate is an antidepressant, mice only received lactate from days 12-25 and a second set of behavioral tests was conducted on day 26. In this paradigm, we examined whether lactate functions by regulating HDACs using co-treatment with CI-994, a brain-permeable class I HDAC inhibitor. When administered after the establishment of depression, lactate behaved as antidepressant. In this paradigm, lactate regulated HDAC5 and not HDAC2/3 levels. On the contrary, HDAC2/3 inhibition was antidepressant-like. This indicates that lactate mimics exercise's effects and rescues susceptibility to stress by modulating HDAC2/3 activity and suggests that HDAC2/3 play opposite roles before and after establishment of susceptibility to stress.

Selective inhibition of histone deacetylase 3 (HDAC3) prevents glucolipotoxicity-induced &beta, cell dysfunction and apoptosis by alleviation of proapoptotic endoplasmic reticulum (ER) stress-signaling, but the precise molecular mechanisms of alleviation are unexplored. By unbiased microarray analysis of the &beta, cell gene expression profile of insulin-producing cells exposed to glucolipotoxicity in the presence or absence of a selective HDAC3 inhibitor, we identified Enhancer of zeste homolog 2 (EZH2) as the sole target candidate. &beta, Cells were protected against glucolipotoxicity-induced ER stress and apoptosis by EZH2 attenuation. Small molecule inhibitors of EZH2 histone methyltransferase activity rescued human islets from glucolipotoxicity-induced apoptosis. Moreover, EZH2 knockdown cells were protected against glucolipotoxicity-induced downregulation of the protective non-canonical Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF&kappa, B) pathway. We conclude that EZH2 deficiency protects from glucolipotoxicity-induced ER stress, apoptosis and downregulation of the non-canonical NF&kappa, B pathway, but not from insulin secretory dysfunction. The mechanism likely involves transcriptional regulation via EZH2 functioning as a methyltransferase and/or as a methylation-dependent transcription factor.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPβCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.

The regulation of epigenetic factors is an emerging therapeutic target of immune function in a variety of osteolytic pathologies. Histone deacetylases (HDAC) modify core histone proteins and transcriptional processes, in addition to nonhistone protein activity. The activated immune response in rheumatoid arthritis, periodontitis, and prosthetic implant particle release stimulates the catabolic activity of osteoclasts. In this study, we investigated the effects of novel therapeutic agents targeting HDAC isozymes (HDAC 1, 2, and 5), previously shown to be upregulated in inflammatory bone disorders, in cytokine-stimulated human monocytes and osteoclasts in vitro. Inhibiting HDAC 1 and 2 significantly reduced gene expression of IL-1β, TNF, MCP-1, and MIP-1α in TNF-stimulated monocytes, while suppressing secretions of IL-1β, IL-10, INF-γ, and MCP-1 (P

Modulation of histone deacetylase (HDAC) activity has been implicated as a potential therapeutic strategy for multiple diseases. However, it has been difficult to dissect the role of individual HDACs due to a lack of selective small-molecule inhibitors. Here, we report the synthesis of a series of highly potent and isoform-selective class I HDAC inhibitors, rationally designed by exploiting minimal structural changes to the clinically experienced HDAC inhibitor CI-994. We used this toolkit of isochemogenic or chemically matched inhibitors to probe the role of class I HDACs in β-cell pathobiology and demonstrate for the first time that selective inhibition of an individual HDAC isoform retains beneficial biological activity and mitigates mechanism-based toxicities. The highly selective HDAC3 inhibitor BRD3308 suppressed pancreatic β-cell apoptosis induced by inflammatory cytokines, as expected, or now glucolipotoxic stress, and increased functional insulin release. In addition, BRD3308 had no effect on human megakaryocyte differentiation, while inhibitors of HDAC1 and 2 were toxic. Our findings demonstrate that the selective inhibition of HDAC3 represents a potential path forward as a therapy to protect pancreatic β-cells from inflammatory cytokines and nutrient overload in diabetes.

Aiming towards the development of novel nootropic therapeutics to address the cognitive impairment common to a range of brain disorders, we set out to develop highly selective small molecule inhibitors of HDAC2, a chromatin modifying histone deacetylase implicated in memory formation and synaptic plasticity. Novel ortho-aminoanilide inhibitors were designed and evaluated for their ability to selectively inhibit HDAC2 versus the other Class I HDACs. Kinetic and thermodynamic binding properties were essential elements of our design strategy and two novel classes of ortho-aminoanilides, that exhibit kinetic selectivity (biased residence time) for HDAC2 versus the highly homologous isoform HDAC1, were identified. These kinetically selective HDAC2 inhibitors (BRD6688 and BRD4884) increased H4K12 and H3K9 histone acetylation in primary mouse neuronal cell culture assays, in the hippocampus of CK-p25 mice, a model of neurodegenerative disease, and rescued the associated memory deficits of these mice in a cognition behavioural model. These studies demonstrate for the first time that selective pharmacological inhibition of HDAC2 is feasible and that inhibition of the catalytic activity of this enzyme may serve as a therapeutic approach towards enhancing the learning and memory processes that are affected in many neurological and psychiatric disorders.

Preservation of insulin-secreting β-cells is an important goal for therapies aimed at restoring normoglycemia in patients with diabetes. One approach, the inhibition of histone deacetylases (HDACs), has been reported to suppress pancreatic islet inflammation and β-cell apoptosis in vitro. In this report, we demonstrate the efficacy of HDAC inhibitors (HDACi) in vivo. We show that daily administration of BRD3308, an isoform-selective HDAC3 inhibitor, for 2 weeks to female nonobese diabetic (NOD) mice, beginning at 3 weeks of age, followed by twice-weekly injections until age 25 weeks, protects the animals from diabetes. The preservation of β-cells was because of a significant decrease in islet infiltration of mononuclear cells. Moreover, the BRD3308 treatment increased basal insulin secretion from islets cultured in vitro. All metabolic tissues tested in vehicle- or BRD3308-treated groups showed virtually no sign of immune cell infiltration, except minimal infiltration in white adipose tissue in animals treated with the highest BRD3308 dose (10 mg/kg), providing additional evidence of protection from immune attack in the treated groups. Furthermore, pancreata from animals treated with 10 mg/kg BRD3308 exhibited significantly decreased numbers of apoptotic β-cells compared with those treated with vehicle or low-dose BRD3308. Finally, animals treated with 1 or 10 mg/kg BRD3308 had enhanced β-cell proliferation. These in vivo results point to the potential use of selective HDAC3 inhibitors as a therapeutic approach to suppress pancreatic islet infiltration and prevent β-cell death with the long-term goal of limiting the progression of type 1 diabetes.

Histone deacetylase (HDAC) inhibition improves function and extends survival in rodent models of a host of neurological conditions, including stroke, and neurodegenerative diseases. Our understanding, however, of the contribution of individual HDAC isoforms to neuronal death is limited. In this study, we used selective chemical probes to assess the individual roles of the Class I HDAC isoforms in protectingMus musculusprimary cortical neurons from oxidative death. We demonstrated that the selective HDAC8 inhibitor PCI-34051 is a potent neuroprotective agent; and by taking advantage of both pharmacological and genetic tools, we established that HDAC8 is not critically involved in PCI-34051's mechanism of action. We used BRD3811, an inactive ortholog of PCI-34051, and showed that, despite its inability to inhibit HDAC8, it exhibits robust neuroprotective properties. Furthermore, molecular deletion of HDAC8 proved insufficient to protect neurons from oxidative death, whereas both PCI-34051 and BRD3811 were able to protect neurons derived from HDAC8 knock-out mice. Finally, we designed and synthesized two new, orthogonal negative control compounds, BRD9715 and BRD8461, which lack the hydroxamic acid motif and showed that they stably penetrate cell membranes but are not neuroprotective. These results indicate that the protective effects of these hydroxamic acid-containing small molecules are likely unrelated to direct epigenetic regulation via HDAC inhibition, but rather due to their ability to bind metals. Our results suggest that hydroxamic acid-based HDAC inhibitors may mediate neuroprotection via HDAC-independent mechanisms and affirm the need for careful structure–activity relationship studies when using pharmacological approaches.

Histone deacetylase (HDAC) enzymes have been demonstrated as critical components in maintaining chromatin homeostasis, CNS development, and normal brain function. Evidence in mouse models links HDAC expression to learning, memory, and mood-related behaviors; small molecule HDAC inhibitor tool compounds have been used to demonstrate the importance of specific HDAC subtypes in modulating CNS-disease-related behaviors in rodents. So far, no direct evidence exists to understand the quantitative changes in HDAC target engagement that are necessary to alter biochemistry and behavior in a living animal. Understanding the relationship between target engagement and in vivo effect is essential in refining new ways to alleviate disease. We describe here, using positron emission tomography (PET) imaging of rat brain, the in vivo target engagement of a subset of class I/IIb HDAC enzymes implicated in CNS-disease (HDAC subtypes 1, 2, 3, and 6). We found marked differences in the brain penetrance of tool compounds from the hydroxamate and benzamide HDAC inhibitor classes and resolved a novel, highly brain penetrant benzamide, CN147, chronic treatment with which resulted in an antidepressant-like effect in a rat behavioral test. Our work highlights a new translational path for understanding the molecular and behavioral consequences of HDAC target engagement.

Epigenetic enzymes are now targeted to treat the underlying gene expression dysregulation that contribute to disease pathogenesis. Histone deacetylases (HDACs) have shown broad potential in treatments against cancer and emerging data supports their targeting in the context of cardiovascular disease and central nervous system dysfunction. Development of a molecular agent for non-invasive imaging to elucidate the distribution and functional roles of HDACs in humans will accelerate medical research and drug discovery in this domain. Herein, we describe the synthesis and validation of an HDAC imaging agent, [(11)C]6. Our imaging results demonstrate that this probe has high specificity, good selectivity, and appropriate kinetics and distribution for imaging HDACs in the brain, heart, kidney, pancreas, and spleen. Our findings support the translational potential for [(11)C]6 for human epigenetic imaging.

Despite being extensively characterized structurally and biochemically, the functional role of histone deacetylase 8 (HDAC8) has remained largely obscure due in part to a lack of known cellular substrates. Herein, we describe an unbiased approach using chemical tools in conjunction with sophisticated proteomics methods to identify novel non-histone nuclear substrates of HDAC8, including the tumor suppressor ARID1A. These newly discovered substrates of HDAC8 are involved in diverse biological processes including mitosis, transcription, chromatin remodeling, and RNA splicing and may help guide therapeutic strategies that target the function of HDAC8.

Lysine acetylation is an ancient, evolutionarily conserved, reversible post-translational modification. A multitude of diverse cellular functions are regulated by this dynamic modification, including energy and metabolism, protein folding, transcription, and translation. Gene expression can be manipulated through changes in histone acetylation status, and this process is controlled by the function of 2 opposing enzymes: histone acetyl transferases and histone deacetylases (HDACs). The zinc-dependent HDACs are a family of hydrolases that remove acetyl groups from lysines, and their function can be modulated by the action of small molecule ligands. Inhibition through competitive binding of the catalytic domain of these enzymes has been achieved by a diverse array of small molecule chemotypes. Structural biology has aided the development of potent, and in some cases highly isoform-selective, inhibitors that have demonstrated utility in a number of neurological disease models. Continued development and characterization of highly optimized small molecule inhibitors of HDAC enzymes will help refine our understanding of their function and, optimistically, lead to novel therapeutic treatment alternatives for a host of neurological disorders.

Increasing evidence supports a role for epigenetic involvement in some of the neurobiological alterations observed in neurodegenerative and psychiatric disorders including schizophrenia. In particular, there is mounting evidence implicating dysfunction in acetylation status, a chromatin modification mediated in part by HDACs, as a possible contributing factor to certain facets of this debilitating disease. Additional data support the notion that small molecule inhibition of HDACs may provide therapeutic alternatives to treating many of the symptoms associated with schizophrenia, particularly cognitive deficits. However, the development of highly potent and selective inhibitors of the individual HDAC isoforms will be necessary to delineate the associated biological effects and test the feasibility of such an approach for this complex and chronically treated disease. Here, we summarize current evidence for the role of HDAC isoforms in schizophrenia and highlight the state of the art in developing selective inhibitors of these isoforms as a potential treatment for schizophrenia.

The purpose of this work--the first of its kind--was to evaluate the impact of chronic selective histone deacetylase (HDAC) inhibitor treatment on brain activity using uptake of the radioligand (18)F-fluorodeoxyglucose and positron emission tomography ((18)FDG-PET). HDAC dysfunction and other epigenetic mechanisms are implicated in diverse CNS disorders and animal research suggests HDAC inhibition may provide a lead toward developing improved treatment. To begin to better understand the role of the class I HDAC subtypes HDAC 1, 2 and 3 in modulating brain activity, we utilized two benzamide inhibitors from the literature, compound 60 (Cpd-60) and CI-994 which selectively inhibit HDAC 1 and 2 or HDACs 1, 2 and 3, respectively. One day after the seventh treatment with Cpd-60 (22.5 mg/kg) or CI-994 (5 mg/kg), (18)FDG-PET experiments (n=11-12 rats per treatment group) revealed significant, local changes in brain glucose utilization. These 2-17% changes were represented by increases and decreases in glucose uptake. The pattern of changes was similar but distinct between Cpd-60 and CI-994, supporting that (18)FDG-PET is a useful tool to examine the relationship between HDAC subtype activity and brain activity. Further work using additional selective HDAC inhibitors will be needed to clarify these effects as well as to understand how brain activity changes influence behavioral response.

[ (3)H]CI-994, a radioactive isotopologue of the benzamide CI-994, a class I histone deacetylase inhibitor (HDACi), was evaluated as an autoradiography probe for ex vivo labeling and localizing of class I HDAC (isoforms 1-3) in the rodent brain. After protocol optimization, up to 80% of total binding was attributed to specific binding. Notably, like other benzamide HDACi, [ (3)H]CI-994 exhibits slow binding kinetics when measured in vitro with isolated enzymes and ex vivo when used for autoradiographic mapping of HDAC1-3 density. The regional distribution and density of HDAC1-3 was determined through a series of saturation and kinetics experiments. The binding properties of [ (3)H]CI-994 to HDAC1-3 were characterized and the data were used to determine the regional Bmax of the target proteins. Kd values, determined from slice autoradiography, were between 9.17 and 15.6 nM. The HDAC1-3 density (Bmax), averaged over whole brain sections, was of 12.9 picomol · mg(-1) protein. The highest HDAC1-3 density was found in the cerebellum, followed by hippocampus and cortex. Moderate to low receptor density was found in striatum, hypothalamus and thalamus. These data were correlated with semi-quantitative measures of each HDAC isoform using western blot analysis and it was determined that autoradiographic images most likely represent the sum of HDAC1, HDAC2, and HDAC3 protein density. In competition experiments, [ (3)H]CI-994 binding can be dose-dependently blocked with other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA). In summary, we have developed the first known autoradiography tool for imaging class I HDAC enzymes. Although validated in the CNS, [ (3)H]CI-994 will be applicable and beneficial to other target tissues and can be used to evaluate HDAC inhibition in tissues for novel therapies being developed. [ (3)H]CI-994 is now an enabling imaging tool to study the relationship between diseases and epigenetic regulation.

Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates the development of germinal center (GC)–derived lymphomas in mice. In both human and murine lymphomas, CREBBP loss-of-function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses, including class II MHC. Mechanistically, CREBBP-regulated enhancers are counter-regulated by the BCL6 transcriptional repressor in a complex with SMRT and HDAC3, which we found to bind extensively to MHC class II loci. HDAC3 loss-of-function rescued repression of these enhancers and corresponding genes, including MHC class II, and more profoundly suppressed CREBBP-mutant lymphomas in vitro and in vivo. Hence, CREBBP loss-of-function contributes to lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, suggesting HDAC3-targeted therapy as a precision approach for CREBBP-mutant lymphomas. Significance: Our findings establish the tumor suppressor function of CREBBP in GC lymphomas in which CREBBP mutations disable acetylation and result in unopposed deacetylation by BCL6/SMRT/HDAC3 complexes at enhancers of B-cell signaling and immune response genes. Hence, inhibition of HDAC3 can restore the enhancer histone acetylation and may serve as a targeted therapy for CREBBP-mutant lymphomas. Cancer Discov; 7(1); 38–53. ©2016 AACR. See related commentary by Höpken, p. 14. This article is highlighted in the In This Issue feature, p. 1

Aim: Caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE), the major constituent of propolis, is able to increase the survival of the nematode Caenorhabditis elegans after infection with the fungal pathogen Candida albicans. Results: CAPE increases the expression of several antimicrobial proteins involved in the immune response to C. albicans. Structural derivatives of CAPE were synthesized to identify structure–activity relationships and decrease metabolic liability, ultimately leading to a compound that has similar efficacy, but increased in vivo stability. The CED-10(Rac-1)/PAK1 pathway was essential for immunomodulation by CAPE and was a critical component involved in the immune response to fungal pathogens. Conclusion: Caenorhabditis elegans is an efficient heterologous host to evaluate immunomodulatory compounds and identify components of the pathway(s) involved in the mode of action of compounds.

Inhibitors of zinc-dependent histone deacetylases (HDACs) profoundly affect cellular function by altering gene expression via changes in nucleosomal histone tail acetylation. Historically, investigators have employed pan-HDAC inhibitors, such as the hydroxamate trichostatin A (TSA), which simultaneously targets members of each of the three zinc-dependent HDAC classes (classes I, II, and IV). More recently, class- and isoform-selective HDAC inhibitors have been developed, providing invaluable chemical biology probes for dissecting the roles of distinct HDACs in the control of various physiologic and pathophysiological processes. For example, the benzamide class I HDAC-selective inhibitor, MGCD0103 [N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide], was shown to block cardiac fibrosis, a process involving excess extracellular matrix deposition, which often results in heart dysfunction. Here, we compare the mechanisms of action of structurally distinct HDAC inhibitors in isolated primary cardiac fibroblasts, which are the major extracellular matrix–producing cells of the heart. TSA, MGCD0103, and the cyclic peptide class I HDAC inhibitor, apicidin, exhibited a common ability to enhance histone acetylation, and all potently blocked cardiac fibroblast cell cycle progression. In contrast, MGCD0103, but not TSA or apicidin, paradoxically increased expression of a subset of fibrosis-associated genes. Using the cellular thermal shift assay, we provide evidence that the divergent effects of HDAC inhibitors on cardiac fibroblast gene expression relate to differential engagement of HDAC1- and HDAC2-containing complexes. These findings illustrate the importance of employing multiple compounds when pharmacologically assessing HDAC function in a cellular context and during HDAC inhibitor drug development.

Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite β-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of β-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of β-hydroxybutyrate. Electrophysiological measurements indicate that β-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF. DOI: http://dx.doi.org/10.7554/eLife.15092.001, eLife digest Exercise is not only good for our physical health but it benefits our mental health and abilities too. Physical exercise can affect how much of certain proteins are made in the brain. In particular, the levels of a protein called brain derived neurotrophic factor (or BDNF for short) increase after exercise. BDNF has already been shown to enhance mental abilities at the same time as acting against anxiety and depression in mice, and might act in similar way in humans. Nevertheless, it is currently not clear how exercise increases the production of BDNF by cells in the brain. Sleiman et al. have now investigated this question by comparing mice that were allowed to use a running wheel for 30 days with control mice that did not exercise. The comparison showed that the exercising mice had higher levels of BDNF in their brains than the control mice, which confirms the results of previous studies. Next, biochemical experiments showed that this change occurred when enzymes known as histone deacetylases stopped inhibiting the production of BDNF. Therefore Sleiman et al. hypothesised that exercise might produce a chemical that itself inhibits the histone deacetylases. Indeed, the exercising mice produced more of a molecule called β-hydroxybutyrate in their livers, which travels through the blood into the brain where it could inhibit histone deacetylases. Further experiments showed that injecting β-hydroxybutyrate directly into the brains of mice led to increase in BDNF. These new findings reveal with molecular detail one way in which exercise can affect the expression of proteins in the brain. This new understanding may provide ideas for new therapies to treat psychiatric diseases, such as depression, and neurodegenerative disorders, such as Alzheimer’s disease. DOI: http://dx.doi.org/10.7554/eLife.15092.002

The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders.

The synthesis and diversification of a densely functionalized azetidine ring system to gain access to a wide variety of fused, bridged, and spirocyclic ring systems is described. The in vitro physicochemical and pharmacokinetic properties of representative library members are measured in order to evaluate the use of these scaffolds for the generation of lead-like molecules to be used in targeting the central nervous system. The solid-phase synthesis of a 1976-membered library of spirocyclic azetidines is also described.

Cytokine-induced beta-cell apoptosis is important to the etiology of type-1 diabetes. Although previous reports have shown that general inhibitors of histone deacetylase (HDAC) activity, such as suberoylanilide hydroxamic acid and trichostatin A, can partially prevent beta-cell death, they do not fully restore beta-cell function. To understand HDAC isoform selectivity in beta cells, we measured the cellular effects of eleven structurally diverse HDAC inhibitors on cytokine-induced apoptosis in the rat INS-1E cell line. All eleven compounds restored ATP levels and reduced nitrite secretion. However, caspase-3 activity was reduced only by MS-275 and CI-994, both of which target HDAC1, 2, and 3. Importantly, both MS-275 and genetic knock-down of Hdac3 alone were sufficient to restore glucose-stimulated insulin secretion in the presence of cytokines. These results suggest that HDAC3-selective inhibitors may be effective in preventing cytokine-induced beta-cell apoptosis.

High grade epithelial ovarian cancers are relatively sensitive to DNA damaging platinum-based chemotherapy, suggesting that the dependencies of ovarian tumors on DNA damage response pathways can be harnessed for therapeutic purposes. Our goal was to determine if the DNA damage mark gamma-H2AX phosphorylation (pH2AX) could be used to identify suitable cytotoxic histone deacetylase inhibitors (HDACi) for ovarian cancer treatment. Nineteen chemically diverse HDACi compounds were tested in 7 ovarian cancer cell lines. Fluorescent, biochemical and cell-based assays were performed to assess DNA damage by induction of pH2AX and to measure cell viability and apoptosis. The relationships between pH2AX and the cellular effects of cell viability and apoptosis were calculated. Selected HDACi were tested in combination with cisplatin and other DNA damaging agents to determine if the HDACi improved upon the effects of the DNA damaging agents. The HDACi compounds induced differing levels of pH2AX expression. High levels of pH2AX in HDACi-treated ovarian cancer cells were tightly associated with decreased cell viability and increased apoptosis. Consequently, a ketone-based HDACi was chosen and found to enhance the effects of cisplatin, even in ovarian cancer cells with extreme resistance to DNA damaging drugs. In conclusion, a fluorescent-based assay for pH2AX can be used to determine cellular responses to HDACi in vitro and may be a useful tool to identify potentially more effective HDACi for the treatment of ovarian cancer. In addition, these results lend support to the inclusion of ketone-derived HDACi compounds for future development.

SummaryHdac3 is essential for efficient DNA replication and DNA damage control. Deletion of Hdac3 impaired DNA repair and greatly reduced chromatin compaction and heterochromatin content. These defects corresponded to increases in histone H3K9,K14ac; H4K5ac; and H4K12ac in late S phase of the cell cycle, and histone deposition marks were retained in quiescent Hdac3-null cells. Liver-specific deletion of Hdac3 culminated in hepatocellular carcinoma. Whereas HDAC3 expression was downregulated in only a small number of human liver cancers, the mRNA levels of the HDAC3 cofactor NCOR1 were reduced in one-third of these cases. siRNA targeting of NCOR1 and SMRT (NCOR2) increased H4K5ac and caused DNA damage, indicating that the HDAC3/NCOR/SMRT axis is critical for maintaining chromatin structure and genomic stability.

[reaction: see text] Syntheses of conduritols B-D and F and d-(+)-chiro- and neo-inositols from cyclohexenylsilane intermediates are described. The key cyclohexylsilane intermediates 5 and 14 were synthesized by stereoselective olefin dihydroxylation of the corresponding cyclohexenylsilanes. Selective Peterson elimination reactions and Fleming-Tamao oxidations of 5 and 14 then delivered the targeted cyclitol derivatives.

Histone deacetylase (HDAC) inhibitors have shown enormous promise for treating various disease states, presumably due to their ability to modulate acetylation of histone and non-histone proteins. Many of these inhibitors contain functional groups capable of strongly chelating metal ions. We demonstrate that several members of one such class of compounds, the hydroxamate-based HDAC inhibitors, can protect neurons from oxidative stress via an HDAC-independent mechanism. This previously unappreciated antioxidant mechanism involves the insitu formation of hydroxamate-iron complexes that catalyze the decomposition of hydrogen peroxide in a manner reminiscent of catalase. We demonstrate that while many hydroxamate-containing HDAC inhibitors display a propensity for binding iron, only a subset formactive catalase mimetics capable of protecting neurons from exogenous H2O2. In addition to their impact on stroke and neurodegenerative disease research, these results highlight the possibility that HDAC-independent factors might play a role in the therapeutic effects of hydroxamate-based HDAC inhibitors.

Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary – albeit often ineffective – treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity., Stanley Medical Research Institute, National Institutes of Health (U.S.) (R01DA028301), National Institutes of Health (U.S.) (R01DA030321)

Gene expression in eukaryotic organisms can be influenced by a host of internal and external stimuli. The magnitude and duration of the response can often determine the effect of a diverse array of biological functions, many of which can be driven by gene expression. The catalog of possibilities encoded within an organism's primary DNA sequence is heritable and, by design, is immutable in response to these temporally defined internal and external factors and events. It has become increasingly evident that the primary sequence alone is not the only determinant of phenotype and function. Rather, epigenetic mechanisms can influence gene expression without directly affecting the primary DNA sequence. These epigenetic mechanisms can affect gene expression directly via the modification of DNA, or indirectly via the modification of chromatin (storage state of DNA). The acetylation and deacetylation of the N-terminal tails of histones (the core protein component of chromatin) represents an important epigenetic mechanism which affects chromatin structure and, ultimately genomic response, via gene expression. The regulation of histone acetylation status is tightly balanced by the opposing function of two families of enzymes: the HATs (histone acetyltransferases) and the HDACs (histone deacetylases). The function and regulation of these enzymes, which is highly conserved in evolution, can have a profound effect on a multitude of biological processes including transcription, DNA repair and replication, and cellular differentiation. Keywords: Chromatin; Euchromatin; Heterochromatin; Nucleosome; Histones; Acetylation; Deacetylation; HDACs (histone deacetylases); HATs (histone acetyltranferases); Sirtuins

Bipolar disorder (BP) is a debilitating psychiatric disorder, affecting ∼2% of the worldwide population, for which the etiological basis, pathogenesis, and neurocircuitry remain poorly understood. Individuals with BP suffer from recurrent episodes of mania and depression, which are commonly treated with the mood stabilizer lithium. However, nearly half of BP patients do not respond adequately to lithium therapy and the clinically relevant mechanisms of lithium for mood stabilization remain elusive. Here, we modeled lithium responsiveness using cellular assays of glycogen synthase kinase 3 (GSK-3) signaling and mood-related behavioral assays in inbred strains of mice that differ in their response to lithium. We found that activating AKT through phosphosrylation of a key regulatory site (Thr308) was associated with lithium response—activation of signaling pathways downstream of GSK-3 in cells and attenuation of mood-related behaviors in mice—and this response was attenuated by selective and direct inhibition of AKT kinase activity. Conversely, the expression of constitutively active AKT1 in both the cellular and behavioral assays conferred lithium sensitivity. In contrast, selective and direct GSK-3 inhibition by the ATP-competitive inhibitor CHIR99021 bypassed the requirement for AKT activation and modulated behavior in both lithium-responsive and non-responsive mouse strains. These results distinguish the mechanism of action of lithium from direct GSK-3 inhibition both in vivo and in vitro, and highlight the therapeutic potential for selective GSK-3 inhibitors in BP treatment.

There is an urgent need for new antifungal agents that are both effective and non-toxic in the therapy of systemic mycoses. The model nematode Caenorhabditis elegans has been used both to elucidate evolutionarily conserved components of host-pathogen interactions and to screen large chemical libraries for novel antimicrobial compounds. Here we review the use of C. elegans models in drug discovery and discuss caffeic acid phenethyl ester, a novel antifungal agent identified using an in vivo screening system. C. elegans bioassays allow high-throughput screens of chemical libraries in vivo. This whole-animal system may enable the identification of compounds that modulate immune responses or affect fungal virulence factors that are only expressed during infection. In addition, compounds can be simultaneously screened for antifungal efficacy and toxicity, which may overcome one of the main obstacles in current antimicrobial discovery. A pilot screen for antifungal compounds using this novel C. elegans system identified 15 compounds that prolonged survival of nematodes infected with the medically important human pathogen Candida albicans. One of these compounds, caffeic acid phenethyl ester (CAPE), was an effective antifungal agent in a murine model of systemic candidiasis and had in vitro activity against several fungal species. Interestingly, CAPE is a potent immunomodulator in mammals with several distinct mechanisms of action. The identification of CAPE in a C. elegans screen supports the hypothesis that this model can identify compounds with both antifungal and host immunomodulatory activity.

Isoform-selective enzyme targeting poses a potential clinical challenge when there is significant homology in the protein activity domains. Glycogen synthase kinase 3-alpha (GSK3α) has been identified previously as a therapeutic target to promote differentiation in acute myeloid leukemia (AML) (Banerji et al, JCI, 2012). The GSK3 family consists of two highly homologous, but functionally non-redundant, isoforms (α and β). These proteins are serine-threonine kinases implicated in a diversity of cellular processes. Concurrent inhibition of both isoforms is known to induce β-catenin stabilization and can therefore increase self-renewal of leukemia initiating cells. Isoform-selective GSK3 suppression does not increase β-catenin levels (Doble et al, Dev Cell, 2007) and thus may offer a new therapeutic approach. GSK3α selective inhibitors, with cell-based activity, have not been reported. Our collaborative study aimed to identify first-in-class GSK3α selective inhibitors and evaluate their activity in AML, a disease in great need for new therapeutic approaches. A series of potent and selective inhibitors for GSK3α/β were discovered through a screening campaign supported by the NIH's Molecular Libraries Probe Centers Network (MLPCN) program. While these compounds are equipotent for GSK3α/β, they demonstrate remarkable selectivity versus the larger kinome (> 100x selective vs 311 kinases). Analysis of the high resolution x-ray crystal structure obtained with BRD9421, one of the lead compounds, and GSK3β revealed a tridentate hydrogen bond binding mode within the ATP catalytic domain. One of these interactions, is to aspartic 133 in GSK3β. This same residue in GSK3α is a glutamic acid and represents one of the two amino acid differences within the ATP binding domain between these two enzymes. GSK3α and β share 98% homology in their catalytic domains, but the exploitation of this hydrogen bond interaction through rational drug design offered an excellent opportunity to produce selective inhibitors for GSK3α and GSK3β. BRD0705 was thus identified as an 8-fold GSK3α isoform-selective inhibitor over GSK3β (IC50 GSK3α: 0.045 vs. GSK3β: 0.350 μM). We first validated BRD0705 isoform-selectivity in a panel of AML cell lines and primary patients (n=3 cell lines and n= 2 patient samples). Treatment with BRD0705 resulted in a dose- and time-dependent decrease in tyr279 GSK3α phosphorylation without any effect on tyr216 GSK3β phosphorylation. No increase in β-catenin protein levels by western blotting and no nuclear localization by immunofluorescence were observed. GSK3α inhibition with BRD0705 led to morphological and surface marker changes (CD11b, CD11c, CD14, CD117) consistent with AML differentiation in a panel of AML cell lines (n=4) and primary AML samples (n=5). BRD0705 also induced a G2/M arrest and impaired methylcellulose colony formation in AML cell lines and primary patient samples. BRD0705 possesses favorable pharmacokinetic properties and was well tolerated without any observed adverse events in C57BL/6 mice with repeated dosing. In vivo efficacy studies in AML models are ongoing. In conclusion, we identified a first-in-class GSK3α isoform-selective inhibitor and conducted preclinical studies validating BRD0705 as a promising new differentiation therapeutic candidate in AML. Small-molecule inhibition of GSK3α induced myeloid differentiation, cell cycle arrest and impaired colony formation without β-catenin stabilization in AML. Disclosures DeAngelo: Incyte: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; Agios: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy. Stone:Sunesis: Consultancy, Other: DSMB for clinical trial; Roche/Genetech: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Merck: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

[reaction: see text] A convergent and stereocontrolled synthesis of spiroketal 15 corresponding to the C-D fragment of spongistatin 1 has been accomplished by a sequence utilizing a kinetically controlled intramolecular iodo-spiroketalization of glycal 2, which in turn was synthesized via a ring-closing metathesis reaction.

The affinities of small molecules for proteins in megadalton complexes can be measured by mass spectrometry.

Background Targeting the epigenetic apparatus has become a successful treatment paradigm for the treatment of T-cell lymphomas (TCL), as demonstrated by the FDA approval of two histone deacetylase (HDAC) inhibitors, romidepsin and vorinostat. Romidepsin and vorinostat are pan-HDAC inhibitors, and their lack of enzyme specificity may be associated with toxicities that limit their clinical application. There is emerging evidence that the 11 classical HDAC isoforms, HDAC1-11, play different roles in oncogenesis, depending on the disease context. For example, HDAC3 has been strongly implicated in the survival of diffuse large B cell lymphoma, by interacting with STAT3. In T cells, HDAC1 and HDAC2 suppress lymphomagenesis in a dosage dependent manner, and are required to maintain the survival of TCL. Our hypothesis is that if different HDAC isoforms are involved in distinct cellular functions, then isoform selective HDAC inhibitors may be active in select subtypes of lymphoma, and that unique combinations of isofrom selective inhibitors could be used to more precisely target the relevant biology. Methods Cytotoxicity of isoform selective and nonselective HDAC inhibitors was determined in a panel of T cell lymphoma cell lines using a High Throughput Screening approach. Growth inhibition was determined using Cell TiterGlo that measures ATP produced by live and proliferating cells. The concentrations required to reach 50% of inhibition (IC50) were calculated from cells treated for 48 hours, using the CalcuSyn software. Cell death was determined by flow cytometry, using both Annexin V and propidium iodide as the probes. Finally, drug : drug synergy was determined by calculating relative risk ratio (RRR), as published previously. Values below 1 indicates synergy, and the smaller the RRR values the higher the level of synergy. Results Isoform selective HDAC inhibitors as a rule were much less potent than nonselective HDAC inhibitors in their ability to inhibit the growth of TCL cells (Table 1). However, two HDAC1/2 selective inhibitors, BRD2283 and BRD6597, demonstrated IC50 values comparable to vorinostat in the H9 cell line, but not in any other cell lines. This may be due to unique epigenetic features in H9 that are sufficiently sensitive to the inhibition of only HDAC1 and 2. In contrast, other cell lines may have complex epigenetic abnormalities that are not sensitive to the inhibition of only HDAC1 and 2, but were nevertheless sensitive to inhibition of a broader spectrum of HDAC isoforms. To further confirm that inhibiting a broader array of HDAC enzymes may lead to more efficient growth inhibition, we investigated the effect of combining the HDAC1/2 inhibitor BRD2283 and the selective HDAC3 inhibitor repligen-136 on TCL cell growth. Figure 1 demonstrates that repligen-136 at the concentration of 4uM produced only 30% growth inhibition at 48-hour exposure. BRD2283 as a single agent produced 20, 30, and 65% of growth inhibition at the concentration of 0.5, 1, and 2uM, respectively. BRD2283 at these concentrations, combined with the mildly active repligen-136 at 4uM, produced 80, 90, and 95% of growth inhibition, respectively. The calculated RRR values were 0.35, 0.20, and 0.13, respectively, highly significant for synergistic drug : drug interaction. These results serve as a proof of principle that inhibition of a narrow spectrum of isoform specific HDAC enzymes may be sufficient to inhibit select lymphoma characterized by 'uncomplicated' epigenetic apparatus. In contrast, lymphoma with 'complicated' epigenetic abnormalities may require simultaneous inhibition of multiple HDAC isoforms, which may be achieved by combining HDAC inhibitors that target different HDAC enzymes. The combination approach, as compared to a single, non-selective HDAC inhibitor, may allow for more opportunity of personalized cancer treatment based on improved understanding of the precise epigenetic abnormalities in the lymphoma. We have begun a broader combinatorial screening approach to explore what specific set of isoform selective inhibitors recapitulate, or perhaps even improve upon, the activity seen with the pan HDAC inhibitors. C-: Negative control Rep: Repligen-136 2283: BRD2283 RRR: Relative risk ratio Disclosures: No relevant conflicts of interest to declare.