Improved antiproliferative activity of 1,3,4-thiadiazole-containing histone deacetylase (HDAC) inhibitors by introduction of the heteroaromatic surface recognition motif

A series of 1,3,4-thiadiazole-containing hydroxamic acids, in accord with the common pharmacophore of histone deacetylase (HDAC) inhibitors (a Zn²⁺ binding moiety–a linker–a surface recognition motif), was identified as submicromolar HDAC inhibitors by our group. In this study, we continued our efforts to develop 1,3,4-thiadiazole bearing hydroxamate analogues by modifying the surface recognition motif. We found that 1,3,4-thiadiazoles having a heteroaromatic substituent showed better HDAC inhibitory activity in enzymatic assay and higher antiproliferative potency in cellular assay compared to SAHA.     

Histone deacetylases: A novel class of therapeutic targets for pancreatic cancer

Pancreatic cancer is the seventh leading cause of cancer death worldwide, with a low 5-year survival rate. Novel agents are urgently necessary to treat the main pathological type, known as pancreatic ductal carcinoma (PDAC). The dysregulation of histone deacetylases (HDACs) has been identified in association with PDAC, which can be more easily targeted by small molecular inhibitors than gene mutations and may represent a therapeutic breakthrough for PDAC. However, the contributions of HDACs to PDAC remain controversial, and pharmacokinetic challenges have limited the application of HDAC inhibitors (HDACis) in PDAC. This review summarizes the mechanisms associated with success and failure of HDACis in PDAC and discusses the recent progress made in HDACi development and application, such as combination therapies designed to enhance efficacy. More precise strategies involving HDACis might eventually improve the outcomes of PDAC treatment.     

Design, synthesis, and activity of HDAC inhibitors with a N-formyl hydroxylamine head group

Histone deacetylases (HDAC) are promising targets for cancer chemotherapy. HDAC inhibitors are thought to act in part by disrupting normal cell cycle regulation, resulting in apoptosis and/or differentiation of transformed cells. Several HDAC inhibitors, which contain hydrophobic tails and the Zn(2+) chelator hydroxyamic acid as a head group, are potent inhibitors of HDACs both in vitro and in vivo. In this study, a related class of compounds with a N-formyl hydroxylamino head group has been synthesized and their ability to inhibit HDACs have been assayed in biochemical and cellular assays. These compounds were found to have comparable activities to suberoylanilide hydroxyamic acid (SAHA) in HDAC enzymatic assays and histone hyperacetylation cellular assays.     

Thiol-based SAHA analogues as potent histone deacetylase inhibitors

In order to find novel nonhydroxamate histone deacetylase (HDAC) inhibitors, a series of thiol-based compounds modeled after suberoylanilide hydroxamic acid (SAHA) was synthesized, and their inhibitory effect on HDACs was evaluated. Compound 6, in which the hydroxamic acid of SAHA was replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA.     

The structural requirements of histone deacetylase inhibitors: SAHA analogs modified at the C5 position display dual HDAC6/8 selectivity

Histone deacetylase (HDAC) proteins have emerged as important targets for anti-cancer drugs, with four small molecules approved for use in the clinic. Suberoylanilide hydroxamic acid (Vorinostat, SAHA) was the first FDA-approved HDAC inhibitor for cancer treatment. However, SAHA inhibits most of the eleven HDAC isoforms. To understand the structural requirements of HDAC inhibitor selectivity and develop isoform selective HDAC inhibitors, SAHA analogs modified in the linker at the C5 position were synthesized and tested for potency and selectivity. C5-modified SAHA analogs displayed dual selectivity to HDAC6 and HDAC8 over HDAC 1, 2, and 3, with only a modest reduction in potency. These findings are consistent with prior work showing that modification of the linker region of SAHA can alter isoform selectivity. The observed HDAC6/8 selectivity of C5-modified SAHA analogs provide guidance toward development of isoform selective HDAC inhibitors and more effective anti-cancer drugs.     

SelSA,selenium analogs of SAHA as potent histone deacetylase inhibitors

Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase (HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular, HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy. Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In particular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the literature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors; SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC₅₀ values of 196 nM. These results provided novel selenium-containing potent HDAC inhibitors.     

Design and synthesis of novel histone deacetylase inhibitor derived from nuclear localization signal peptide

We describe herein the synthesis and characterization of a new class of histone deacetylase (HDAC) inhibitors derived from conjugation of a suberoylanilide hydroxamic acid-like aliphatic-hydroxamate pharmacophore to a nuclear localization signal peptide. We found that these conjugates inhibited the histone deacetylase activities of HDACs 1, 2, 6, and 8 in a manner similar to suberoylanilide hydroxamic acid (SAHA). Notably, compound 7b showed a threefold improvement in HDAC 1/2 inhibition, a threefold increase in HDAC 6 selectivity and a twofold increase in HDAC 8 selectivity when compared to SAHA.     

Synthesis and biochemical analysis of 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-N-hydroxy-octanediamides as inhibitors of human histone deacetylases

Inhibition of human histone deacetylases (HDACs) has emerged as a novel concept in the chemotherapeutic treatment of cancer. Two chemical entities, SAHA (ZOLINZA, Merck) and romidepsin (Istodax, Celgene) have been recently approved by the FDA as first-in-class drugs against cutaneous T-cell lymphoma. Clinical use of these drugs revealed several side effects including gastro-intestinal symptoms, fatigue, thrombocytopenia, thrombosis. Romidepsin is associated with an yet unresolved cardiotoxicity issue. A general hypothesis for the diminishment of unwanted adverse effects and an improved therapeutical window suggests the development of more isotype selective inhibitors. In this study the first time HDAC inhibitors with perfluorinated spacers between the zinc chelating moiety and the aromatic capping group were synthesized and tested against representatives of HDAC classes I, IIa and IIb. Competitive binding assays and a combined approach by using blind docking and molecular dynamics support binding of the perfluorinated analogs of SAHA to the active site of the HDAC-like amidohydrolase from Bordetella/Alcaligenes and presumably also to human HDACs. In contrast to the alkyl spacer of SAHA and derivatives, the perfluorinated alkyl spacer seems to contribute to or facilitate the induction of selectivity for class II, particularly class IIa, HDACs even though the overall potency of the perfluorinated SAHA analogs in this study against human HDACs remained still rather moderate in the micromolar range.     

Identification of selective class II histone deacetylase inhibitors using a novel dual-parameter binding assay based on fluorescence anisotropy and lifetime

Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.     

Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is the most common soft tissue cancer in children. The prognosis of patients with relapsed or metastatic disease remains poor. ERMS genomes show few recurrent mutations, suggesting that other molecular mechanisms such as epigenetic regulation might play a major role in driving ERMS tumor biology. In this study, we have demonstrated the diverse roles of histone deacetylases (HDACs) in the pathogenesis of ERMS by characterizing effects of HDAC inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; also known as vorinostat) in vitro and in vivo. TSA and SAHA suppress ERMS tumor growth and progression by inducing myogenic differentiation as well as reducing the self-renewal and migratory capacity of ERMS cells. Differential expression profiling and pathway analysis revealed downregulation of key oncogenic pathways upon HDAC inhibitor treatment. By gain-of-function, loss-of-function, and chromatin immunoprecipitation (ChIP) studies, we show that Notch1- and EphrinB1-mediated pathways are regulated by HDACs to inhibit differentiation and enhance migratory capacity of ERMS cells, respectively. Our study demonstrates that aberrant HDAC activity plays a major role in ERMS pathogenesis. Druggable targets in the molecular pathways affected by HDAC inhibitors represent novel therapeutic options for ERMS patients.     

Identification of a potent non-hydroxamate histone deacetylase inhibitor by mechanism-based drug design

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, we synthesized several suberoylanilide hydroxamic acid (SAHA)-based compounds designed on the basis of the catalytic mechanism of HDACs. Among these compounds, 5b was found to be as potent as SAHA. Kinetic enzyme assays and molecular modeling suggested that the mercaptoacetamide moiety of 5b interacts with the zinc in the active site of HDACs and removes a water molecule from the reactive site of the deacetylation.     

An active site tyrosine residue is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme

HDACs (histone deacetylases) are considered to be among the most important enzymes that regulate gene expression in eukaryotic cells acting through deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones. In addition, both eukaryotic HDACs as well as their bacterial counterparts were reported to also act on non-histone targets. However, we are still far from a comprehensive understanding of the biological activities of this ancient class of enzymes. In the present paper, we studied in more detail the esterase activity of HDACs, focussing on the HDAH (histone deacetylase-like amidohydrolase) from Bordetella/Alcaligenes strain FB188. This enzyme was classified as a class 2 HDAC based on sequence comparison as well as functional data. Using chromogenic and fluorogenic ester substrates we show that HDACs such as FB188 HDAH indeed have esterase activity that is comparable with those of known esterases. Similar results were obtained for human HDAC1, 3 and 8. Standard HDAC inhibitors were able to block both activities with similar IC(50) values. Interestingly, HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) also showed inhibitory activity against porcine liver esterase and Pseudomonas fluorescens lipase. The esterase and the amidohydrolase activity of FB188 HDAH both appear to have the same substrate specificity concerning the acyl moiety. Interestingly, a Y312F mutation in the active site of HDAH obstructed amidohydrolase activity but significantly improved esterase activity, indicating subtle differences in the mechanism of both catalytic activities. Our results suggest that, in principle, HDACs may have other biological roles besides acting as protein deacetylases. Furthermore, data on HDAC inhibitors affecting known esterases indicate that these molecules, which are currently among the most promising drug candidates in cancer therapy, may have a broader target profile requiring further exploration.     

Fluoroalkene modification of mercaptoacetamide-based histone deacetylase inhibitors

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was replaced by (mercaptomethyl)fluoroalkene, and their HDAC inhibitory activity was evaluated. Subnanomolar inhibition was observed for all synthetic compounds.     

Histone deacetylase inhibitors: Potential in cancer therapy

The role of histone deacetylases (HDAC) and the potential of these enzymes as therapeutic targets for cancer, neurodegenerative diseases and a number of other disorders is an area of rapidly expanding investigation. There are 18 HDACs in humans. These enzymes are not redundant in function. Eleven of the HDACs are zinc dependent, classified on the basis of homology to yeast HDACs: Class I includes HDACs 1, 2, 3, and 8; Class IIA includes HDACs 4, 5, 7, and 9; Class IIB, HDACs 6 and 10; and Class IV, HDAC 11. Class III HDACs, sirtuins 1–7, have an absolute requirement for NAD⁺, are not zinc dependent and generally not inhibited by compounds that inhibit zinc dependent deacetylases. In addition to histones, HDACs have many nonhistone protein substrates which have a role in regulation of gene expression, cell proliferation, cell migration, cell death, and angiogenesis. HDAC inhibitors (HDACi) have been discovered of different chemical structure. HDACi cause accumulation of acetylated forms of proteins which can alter their structure and function. HDACi can induce different phenotypes in various transformed cells, including growth arrest, apoptosis, reactive oxygen species facilitated cell death and mitotic cell death. Normal cells are relatively resistant to HDACi induced cell death. Several HDACi are in various stages of development, including clinical trials as monotherapy and in combination with other anti‐cancer drugs and radiation. The first HDACi approved by the FDA for cancer therapy is suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza), approved for treatment of cutaneous T‐cell lymphoma. J. Cell. Biochem. 107: 600–608, 2009. © 2009 Wiley‐Liss, Inc.     

HDAC modulation and cell death in the clinic

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two opposing classes of enzymes, which finely regulate the balance of histone acetylation affecting chromatin packaging and gene expression. Imbalanced acetylation has been associated with carcinogenesis and cancer progression. In contrast to genetic mutations, epigenetic changes are potentially reversible. This implies that epigenetic alterations are amenable to pharmacological interventions. Accordingly, some epigenetic-based drugs (epidrugs) have been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for cancer treatment. Here, we focus on the biological features of HDAC inhibitors (HDACis), analyzing the mechanism(s) of action and their current use in clinical practice.