Small Molecule Histone Deacetylase Inhibitors Research Articles

The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair.

Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization.

Histone deacetylase inhibition is cytotoxic to oligodendrocyte precursor cells in vitro and in vivo

Histone deacetylase (HDAC) inhibition mediated by small molecule HDAC inhibitors (HDACi) has demonstrated divergent effects including toxicity towards transformed cell lines, neuroprotection in neurological disease models, and inhibition of oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes (OL). However, it remains unknown if transient HDAC inhibition may promote OPC survival. Using mouse cortical OPC primary cultures, we investigated the effects of the FDA approved pan-HDACi suberoylanilide hydroxamic acid (SAHA) on OPC survival. Initial studies showed differences in the HDAC expression pattern of multiple HDAC isoforms in OPCs relative to their terminally differentiated progeny cells, OLs and astrocytes. Treatment of OPCs with SAHA for up to 72h using a maximum concentration either at or lower than those necessary for cytotoxicity in most transformed cell lines resulted in over 67% reduction in viability relative to vehicle-treated OPCs. This was at least partly due to increased apoptosis as SAHA-treated cells displayed activated caspase 3 and were protected by the general caspase inhibitor Q-VD-OPH. Additionally, SAHA treatment of whole mice at postnatal day 5 induced apoptosis of cortical OPCs. These results suggest that SAHA negatively impacts OPC survival and may be detrimental to the myelinating brain and spinal cord. Such toxicity may be relevant in a clinical context as SAHA is currently involved in numerous clinical trials and is in consideration for use in the treatment of psychiatric and neurodegenerative conditions.

Modulating epigenetic HAT activity for reinstating acetylation homeostasis: A promising therapeutic strategy for neurological disorders.

Epigenetic mechanisms are emerging as a fundamental regulatory switch in neuronal function. Acetylation homeostasis governed by the antagonistic activities of HATs and HDACs plays a critical role in neuronal gene activity. It is now becoming increasingly clear that several neurodevelopmental, neurodegenerative, and neuropsychiatric disorders are caused by aberrant changes in chromatin acetylation. Several HATs have been shown to be vital for neuronal processes such as synaptic plasticity and memory formation. Thus not surprisingly, dysregulation of such HATs has been implicated in the pathogenesis of several neurodegenerative diseases including Huntington's disease (HD) and Alzheimer's disease (AD). The current therapeutic strategy involves the use of small-molecule histone deacetylase inhibitors to compensate the acetylation deficits arising due to loss of HAT activity. Despite the promising therapeutic effects, the lack of isoform (target) specificity of HDACi raises concerns regarding their applicability. Mounting evidences about the role of HATs in neuronal survival, learning and memory has triggered a new wave of modulating specific HATs as a novel therapeutic option to tackle neurodegenerative diseases. In this review we focus on different HAT families and the critical roles they play in neural development and how the altered acetylation homeostasis culminates in neurodegeneration. Further, we describe the HDACi-based therapeutic approach and its flip side in overcoming neurodegenerative diseases. Furthermore, we discuss the therapeutic potential of HAT modulators in reinstating acetylation homeostasis to ameliorate neurodegenerative disorders.

How do tumor cells respond to HDAC inhibition?

It is now well recognized that mutations, deregulated expression, and aberrant recruitment of epigenetic readers, writers, and erasers are fundamentally important processes in the onset and maintenance of many human tumors. The molecular, biological, and biochemical characteristics of a particular class of epigenetic erasers, the histone deacetylases (HDACs), have been extensively studied and small-molecule HDAC inhibitors (HDACis) have now been clinically approved for the treatment of human hemopoietic malignancies. This review explores our current understanding of the biological and molecular effects on tumor cells following HDACi treatment. The predominant responses include induction of tumor cell death and inhibition of proliferation that in experimental models have been linked to therapeutic efficacy. However, tumor cell-intrinsic responses to HDACi, including modulating tumor immunogenicity have also been described and may have substantial roles in mediating the antitumor effects of HDACi. We posit that the field has failed to fully reconcile the biological consequences of exposure to HDACis with the molecular events that underpin these responses, however progress is being made. Understanding the pleiotrophic activities of HDACis on tumor cells will hopefully fast track the development of more potent and selective HDACi that may be used alone or in combination to improve patient outcomes.

Microbiota and Neurological Disorders: A Gut Feeling.

In the past century, noncommunicable diseases have surpassed infectious diseases as the principal cause of sickness and death, worldwide. Trillions of commensal microbes live in and on our body, and constitute the human microbiome. The vast majority of these microorganisms are maternally derived and live in the gut, where they perform functions essential to our health and survival, including: digesting food, activating certain drugs, producing short-chain fatty acids (which help to modulate gene expression by inhibiting the deacetylation of histone proteins), generating anti-inflammatory substances, and playing a fundamental role in the induction, training, and function of our immune system. Among the many roles the microbiome ultimately plays, it mitigates against untoward effects from our exposure to the environment by forming a biotic shield between us and the outside world. The importance of physical activity coupled with a balanced and healthy diet in the maintenance of our well-being has been recognized since antiquity. However, it is only recently that characterization of the host–microbiome intermetabolic and crosstalk pathways has come to the forefront in studying therapeutic design. As reviewed in this report, synthetic biology shows potential in developing microorganisms for correcting pathogenic dysbiosis (gut microbiota–host maladaptation), although this has yet to be proven. However, the development and use of small molecule drugs have a long and successful history in the clinic, with small molecule histone deacetylase inhibitors representing one relevant example already approved to treat cancer and other disorders. Moreover, preclinical research suggests that epigenetic treatment of neurological conditions holds significant promise. With the mouth being an extension of the digestive tract, it presents a readily accessible diagnostic site for the early detection of potential unhealthy pathogens resident in the gut. Taken together, the data outlined herein provide an encouraging roadmap toward important new medicines and companion diagnostic platforms in a wide range of therapeutic indications.

Abstract A24: HDAC inhibition unmasks a microRNA-mediated mechanism of tumor suppression

Abstract Genetic lesions resulting in epigenetic dysregulation have a newly appreciated role in tumor initiation and progression. Alterations in epigenetic enzymes, such as histone deacetylases (HDACs), lead to dynamic changes in the epigenetic landscape that are postulated to alter the expression of critical mediators of tumorigenesis. HDACs not only alter the expression of protein-coding genes, but also affect noncoding RNAs, including microRNA (miRNA). Recent evidence suggests that miRNA are one of the largest classes of regulatory molecules and, given their broad impact on gene expression, it is not surprising they have a significant role in tumorigenesis. Although, small molecule inhibitors of HDACs have demonstrated therapeutic utility, the molecular events underlying the observed selective killing of cancer cells are incompletely resolved. We determined that HDAC inhibition in numerous hematopoietic malignancies relieved the Myc-mediated transcriptional repression of miRNA with known tumor suppressive properties, namely the miR-15 and let-7 families. Specifically, following HDAC inhibition, Myc, which normally represses the miR-15 and let-7 families in cancer cells, transcriptionally activated their expression. In addition, we demonstrated that Myc was required for this HDAC inhibition-induced miRNA upregulation. As a result, transcript levels of the miR-15 and let-7 families increased, which subsequently targeted and decreased the expression of anti-apoptotic Bcl-2 and Bcl-xL, respectively, inducing apoptosis in multiple hematopoietic malignancies. Unexpectedly, we demonstrated that, in normal cells, dysregulated Myc transcriptionally upregulated these miRNA as a means to prevent cellular transformation, revealing a novel mechanism whereby Myc induces apoptosis independent of the p53 tumor suppressor. Our results expose a previously unknown, and surprisingly general, mechanism of Myc-induced apoptosis that capitalizes on miRNA. Importantly, our data provide insight into the molecular underpinnings following HDAC inhibition and contribute new knowledge that should aid in the development of improved cancer therapeutics. Citation Format: Clare M. Adams, Scott W. Hiebert, Christine M. Eischen. HDAC inhibition unmasks a microRNA-mediated mechanism of tumor suppression. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A24.

Multicomponent Analysis of the Differential Induction of Secondary Metabolite Profiles in Fungal Endophytes.

Small molecule histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors are commonly used to perturb the production of fungal metabolites leading to the induction of the expression of silent biosynthetic pathways. Several reports have described the variable effects observed in natural product profiles in fungi treated with HDAC and DNMT inhibitors, such as enhanced chemical diversity and/or the induction of new molecules previously unknown to be produced by the strain. Fungal endophytes are known to produce a wide variety of secondary metabolites (SMs) involved in their adaptation and survival within higher plants. The plant-microbe interaction may influence the expression of some biosynthetic pathways, otherwise cryptic in these fungi when grown in vitro. The aim of this study was to setup a systematic approach to evaluate and identify the possible effects of HDAC and DNMT inhibitors on the metabolic profiles of wild type fungal endophytes, including the chemical identification and characterization of the most significant SMs induced by these epigenetic modifiers.

Histone deacetylases in monocyte/macrophage development, activation and metabolism: refining HDAC targets for inflammatory and infectious diseases.

Macrophages have central roles in danger detection, inflammation and host defense, and consequently, these cells are intimately linked to most disease processes. Major advances in our understanding of the development and function of macrophages have recently come to light. For example, it is now clear that tissue-resident macrophages can be derived from either blood monocytes or through local proliferation of phagocytes that are originally seeded during embryonic development. Metabolic state has also emerged as a major control point for macrophage activation phenotypes. Herein, we review recent literature linking the histone deacetylase (HDAC) family of enzymes to macrophage development and activation, particularly in relation to these recent developments. There has been considerable interest in potential therapeutic applications for small molecule inhibitors of HDACs (HDACi), not only for cancer, but also for inflammatory and infectious diseases. However, the enormous range of molecular and cellular processes that are controlled by different HDAC enzymes presents a potential stumbling block to clinical development. We therefore present examples of how classical HDACs control macrophage functions, roles of specific HDACs in these processes and approaches for selective targeting of drugs, such as HDACi, to macrophages. Development of selective inhibitors of macrophage-expressed HDACs and/or selective delivery of pan HDACi to macrophages may provide avenues for enhancing efficacy of HDACi in therapeutic applications, while limiting unwanted side effects.

A structure–activity relationship of non-peptide macrocyclic histone deacetylase inhibitors and their anti-proliferative and anti-inflammatory activities

Inhibition of the enzymatic activity of histone deacetylase (HDAC) is a promising therapeutic strategy for cancer treatment and several distinct small molecule histone deacetylase inhibitors (HDACi) have been reported. We have previously identified a new class of non-peptide macrocyclic HDACi derived from 14- and 15-membered macrolide skeletons. In these HDACi, the macrocyclic ring is linked to the zinc chelating hydroxamate moiety through a para-substituted aryl-triazole cap group. To further delineate the depth of the SAR of this class of HDACi, we have synthesized series of analogous compounds and investigated the influence of various substitution patterns on their HDAC inhibitory, anti-proliferative and anti-inflammatory activities. We identified compounds 25b and 38f with robust anti-proliferative activities and compound 26f (IC50 47.2 nM) with superior anti-inflammatory (IC50 88 nM) activity relative to SAHA.

Histone Deacetylases as Potential Targets for Cocaine Addiction.

Drug-induced changes in gene expression likely contribute to long-lasting structural and functional alterations in the brain's reward circuitry and the persistence of addiction. Modulation of chromatin structure through covalent histone modifications has emerged as an important regulator of gene transcription in brain and increasing evidence suggests that misregulation of histone acetylation contributes to the establishment and maintenance of aberrant neuronal gene programs and behaviors associated with cocaine or amphetamine exposure. In this review, we summarize evidence supporting a role for histone acetylation in psychostimulant-induced plasticity and discuss findings from preclinical studies investigating histone deacetylase (HDAC) action and the use of small-molecule HDAC inhibitors (HDACis) to correct drug-mediated transcriptional dysregulation.

The Potential of Histone Deacetylase Inhibitors in Breast Cancer Therapy

SUMMARY Breast cancer is the second leading cause of cancer-related mortality in women. Despite improvements in prevention, detection and treatment, breast cancer will be responsible for nearly 40,000 deaths in 2014. The function of histone deacetylases (HDACs) and their potential as therapeutic targets has become an area of intense investigation and small molecule inhibitors of HDACs (HDACi) are now being investigated as potential chemotherapeutics for breast cancer. In addition to altering chromatin structure through stabilization of histone acetylation, HDACi treatment induces the accumulation of acetylated isoforms of many nonhistone proteins, altering their structure and function. These structural changes influence protein–protein interactions and cellular processes including cell cycle arrest, apoptosis, autophagy, induction of reactive oxygen species and mitotic catastrophe. While the usefulness of these compounds as single agents for treatment of breast cancer is still under investigation, cotreatment with other therapies is being evaluated in a number of clinical trials.

Emerging roles for histone deacetylases in pulmonary hypertension and right ventricular remodeling (2013 Grover Conference series).

Reversible lysine acetylation has emerged as a critical mechanism for controlling the function of nucleosomal histones as well as diverse nonhistone proteins. Acetyl groups are conjugated to lysine residues in proteins by histone acetyltransferases and removed by histone deacetylases (HDACs), which are also commonly referred to as lysine deacetylases. Over the past decade, many studies have shown that HDACs play crucial roles in the control of left ventricular (LV) cardiac remodeling in response to stress. Small molecule HDAC inhibitors block pathological hypertrophy and fibrosis and improve cardiac function in various preclinical models of LV failure. Only recently have HDACs been studied in the context of right ventricular (RV) failure, which commonly occurs in patients who experience pulmonary hypertension (PH). Here, we review recent findings with HDAC inhibitors in models of PH and RV remodeling, propose next steps for this newly uncovered area of research, and highlight potential for isoform-selective HDAC inhibitors for the treatment of PH and RV failure.

Nanoparticle formulations of histone deacetylase inhibitors for effective chemoradiotherapy in solid tumors

Histone deacetylase inhibitors (HDACIs) represent a class of promising agents that can improve radiotherapy in cancer treatment. However, the full therapeutic potential of HDACIs as radiosensitizers has been restricted by limited efficacy in solid malignancies. In this study, we report the development of nanoparticle (NP) formulations of HDACIs that overcome these limitations, illustrating their utility to improve the therapeutic ratio of the clinically established first generation HDACI vorinostat and a novel second generation HDACI quisinostat. We demonstrate that NP HDACIs are potent radiosensitizers in vitro and are more effective as radiosensitizers than small molecule HDACIs in vivo using mouse xenograft models of colorectal and prostate carcinomas. We found that NP HDACIs enhance the response of tumor cells to radiation through the prolongation of γ-H2AX foci. Our work illustrates an effective method for improving cancer radiotherapy treatment.

Suppression of proinflammatory cytokine production by suberoylanilide hydroxamic acid an inhibitor of histone deacetylases

ABSRACT Chromatin remodeling by acetylation and deacetylation of histones play an essential role in regulation of the gene expression. The opposing activity of two type enzymes controls the acetylation degree of the chromatin structure. Histone acetylation by acetyltransferases leads to relaxed chromatin structure and this process supports the transcription. Deacetylation by histone deacetylases (HDACs) compact the chromatin structure and favors gene silence. Small molecule inhibitors of HDACs are a new class drugs used in clinical trials for the treatment of various malignancies. Emerging evidence suggest that HDAC inhibitors may have also anti-inflammatory properties, although the molecular mechanisms remain poorly defined. Our study investigates the effect of the HDACs inhibitor Suberoylanilide Hydroxamic Acid (SAHA) on Th1 and Th17 polarizing cytokines IL-12p40 and IL-23. For this purpose, human peripheral blood mononuclear cells (PBMC) were stimulated with LPS and C3bgp with or without SAHA. Cytokine production was determined in culture supernatants at 6 and 24 h, by ELISA. IL-12p40 and IL-23 synthesis was measured in stimulated PBMC at 6 and increased at 24 h. Early at 6 h, we detected significantly decreased IL-12p40 and IL-23 production in stimulated PBMC treated with SAHA and this tendency was retained at 24 h. In conclusion, our results demonstrated that the inhibition of HDACs by SAHA leads to suppression of IL-12p40 and IL-23 regardless of stimuli used. The downregulating effect of SAHA on the proinflammatory cytokine production may be beneficial during treatment of chronic inflammatory diseases which progression is mediated by Th17 immune response.

Acetyl-lysine erasers and readers in the control of pulmonary hypertension and right ventricular hypertrophy.

Acetylation of lysine residues within nucleosomal histone tails provides a crucial mechanism for epigenetic control of gene expression. Acetyl groups are coupled to lysine residues by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), which are also commonly referred to as "writers" and "erasers", respectively. In addition to altering the electrostatic properties of histones, lysine acetylation often creates docking sites for bromodomain-containing "reader" proteins. This review focuses on epigenetic control of pulmonary hypertension (PH) and associated right ventricular (RV) cardiac hypertrophy and failure. Effects of small molecule HDAC inhibitors in pre-clinical models of PH are highlighted. Furthermore, we describe the recently discovered role of bromodomain and extraterminal (BET) reader proteins in the control of cardiac hypertrophy, and provide evidence suggesting that one member of this family, BRD4, contributes to the pathogenesis of RV failure. Together, the data suggest intriguing potential for pharmacological epigenetic therapies for the treatment of PH and right-sided heart failure.

PET imaging demonstrates histone deacetylase target engagement and clarifies brain penetrance of known and novel small molecule inhibitors in rat.

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.

Selective HDAC Inhibition for the Disruption of Latent HIV-1 Infection

Selective histone deacetylase (HDAC) inhibitors have emerged as a potential anti-latency therapy for persistent human immunodeficiency virus type 1 (HIV-1) infection. We utilized a combination of small molecule inhibitors and short hairpin (sh)RNA-mediated gene knockdown strategies to delineate the key HDAC(s) to be targeted for selective induction of latent HIV-1 expression. Individual depletion of HDAC3 significantly induced expression from the HIV-1 promoter in the 2D10 latency cell line model. However, depletion of HDAC1 or −2 alone or in combination did not significantly induce HIV-1 expression. Co-depletion of HDAC2 and −3 resulted in a significant increase in expression from the HIV-1 promoter. Furthermore, concurrent knockdown of HDAC1, −2, and −3 resulted in a significant increase in expression from the HIV-1 promoter. Using small molecule HDAC inhibitors of differing selectivity to ablate the residual HDAC activity that remained after (sh)RNA depletion, the effect of depletion of HDAC3 was further enhanced. Enzymatic inhibition of HDAC3 with the selective small-molecule inhibitor BRD3308 activated HIV-1 transcription in the 2D10 cell line. Furthermore, ex vivo exposure to BRD3308 induced outgrowth of HIV-1 from resting CD4+ T cells isolated from antiretroviral-treated, aviremic HIV+ patients. Taken together these findings suggest that HDAC3 is an essential target to disrupt HIV-1 latency, and inhibition of HDAC2 may also contribute to the effort to purge and eradicate latent HIV-1 infection.

Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders.

Epigenetic aberrations, which are recognized as key drivers of several human diseases, are often caused by genetic defects that result in functional deregulation of epigenetic proteins, their altered expression and/or their atypical recruitment to certain gene promoters. Importantly, epigenetic changes are reversible, and epigenetic enzymes and regulatory proteins can be targeted using small molecules. This Review discusses the role of altered expression and/or function of one class of epigenetic regulators--histone deacetylases (HDACs)--and their role in cancer, neurological diseases and immune disorders. We highlight the development of small-molecule HDAC inhibitors and their use in the laboratory, in preclinical models and in the clinic.

Class-IIa Histone Deacetylase Inhibition Promotes the Growth of Neural Processes and Protects Them Against Neurotoxic Insult.

Small molecule histone deacetylase inhibitors (HDIs) hold much promise as pharmacological modifiers of the epigenetic status of the central nervous system (CNS), given their ability to cross the blood-brain barrier. This is particularly relevant given the lack of disease-modifying therapies for many neurodegenerative diseases and that epigenetic perturbations are increasingly recognised as playing a key role in their pathophysiology. In particular, emerging evidence in recent years has shown that epigenetic dysregulation may contribute to dopaminergic neuronal death in Parkinson's disease. As a result, a number of pan-HDIs have been explored as potential neuroprotective agents for dopaminergic neurons. However, it is not known if the neuroprotective effects of pan-histone deacetylase (HDAC) inhibition are a general phenomenon or if these effects require inhibition of specific classes of HDACs. Here, we examine the ability of class-specific HDIs to promote neurite growth in a variety of cellular contexts. We find that MC1568, a class IIa-specific HDI, promotes neurite growth and arbourisation and protects neurite arbours against neurotoxic insult. Furthermore, we show that class IIa-specific HDAC inhibition results in activation of the canonical Smad signalling pathway, which is known to promote the survival and growth of midbrain dopaminergic neurons. These results demonstrate the potential of class IIa-specific HDIs as regulators of neuronal structure and suggest they should be examined in animal models of Parkinson's disease as the next stage in rationalising their use as a potential therapy for this disorder.

HDAC inhibitor-mediated beta-cell protection against cytokine-induced toxicity is STAT1 Tyr701 phosphorylation independent.

Histone deacetylase (HDAC) inhibition protects pancreatic beta-cells against apoptosis induced by the combination of the proinflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ. Decreased expression of cell damage-related genes is observed on the transcriptional level upon HDAC inhibition using either IL-1β or IFN-γ alone. Whereas HDAC inhibition has been shown to regulate NFκB-activity, related primarily to IL-1β signaling, it is unknown whether the inhibition of HDACs affect IFN-γ signaling in beta-cells. Further, in non-beta-cells, there is a dispute whether HDAC inhibition regulates IFN-γ signaling at the level of STAT1 Tyr701 phosphorylation. Using different small molecule HDAC inhibitors with varying class selectivity, INS-1E wild type and stable HDAC1-3 knockdown pancreatic INS-1 cell lines, we show that IFN-γ-induced Cxcl9 and iNos expression as well as Cxcl9 and GAS reporter activity were decreased by HDAC inhibition in a STAT1 Tyr701 phosphorylation-independent fashion. In fact, knockdown of HDAC1 increased IFN-γ-induced STAT1 phosphorylation.