Antimalarial and antileishmanial activities of histone deacetylase inhibitors with triazole-linked cap group

Histone deacetylase inhibitors (HDACi) are endowed with plethora of biological functions including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. Parsing the structure–activity relationship (SAR) for each disease condition is vital for long-term therapeutic applications of HDACi. We report in the present study specific cap group substitution patterns and spacer-group chain lengths that enhance the antimalarial and antileishmanial activity of aryltriazolylhydroxamates-based HDACi. We identified many compounds that are several folds selectively cytotoxic to the plasmodium parasites compared to standard HDACi. Also, a few of these compounds have antileishmanial activity that rivals that of miltefosine, the only currently available oral agent against visceral leishmaniasis. The anti-parasite properties of several of these compounds tracked well with their anti-HDAC activities. The results presented here provide further evidence on the suitability of HDAC inhibition as a viable therapeutic option to curb infections caused by apicomplexan protozoans and trypanosomatids.     

Aza-PBHA,a potent histone deacetylase inhibitor,inhibits human gastric-cancer cell migration via PKCα-mediated AHR-HDAC interactions

Recently, histone deacetylase inhibitors (HDACi) have become widely used in anti-cancer treatment; however, due to acquired drug resistance and their relatively low specificity, they are largely ineffective against late-stage cancer. Thus, it is critical to elucidate the molecular mechanisms underlying these issues, so as to identify novel therapeutic targets to prevent late-stage cancer progression and resistance acquisition. The present study investigated the Aryl hydrocarbon receptor (AHR), that has been shown to mediate histone acetylation by regulating histone deacetylase (HDAC) activity during HDACi treatment in human gastric-cancer cell lines (i.e. AGS and NCI-N87 cells). The potent HDACi, Aza-PBHA, was thus shown to upregulate AHR expression in both AGS and NCI-N87 cell lines, and to increase histone acetylation levels by facilitating AHR/HDAC interactions. Conversely, AHR knockdown increased HDAC activity. Aza-PBHA also increased PKCα phosphorylation and membrane translocation; however, interestingly, PKCα inhibition reduced the Aza-PBHA-increased AHR and histone acetylation levels, and inhibited the formation of the AHR/HDAC complex, likely upregulating Aza-PBHA-inhibited cell migration. Thus, our results suggest that Aza-PBHA treatment increased AHR levels to suppress HDAC activity, and inhibited cell migration by activating PKCα activation. These findings support the use of drugs to control AHR-related epigenetic regulation as a promising potential method to prevent acquired resistance to cancer treatments.     

Inhibition of histone deacetylase activity is a novel function of the antifolate drug methotrexate

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used for treating human cancers, and overexpression of histone deacetylase (HDAC) is usually found in tumors. HDAC inhibitors (HDACi) can reactivate tumor suppressor genes and serve as potential anti-cancer drugs. In this study, we found that MTX shared structural similarity with some HDACi and molecular modeling showed that MTX indeed docks into the active site of HDLP, a bacterial homologue of HDAC. Subsequent in vitro assay demonstrated MTX’s inhibition on HDAC activity in human cancer cells. The global acetylation of histone H3 was also induced by MTX. Moreover, MTX inhibited immunoprecipitated HDAC1/2 activity but not their protein levels. This study provides evidence that MTX inhibits HDAC activity.     

Oral Administration of the Pimelic Diphenylamide HDAC Inhibitor HDACi 4b Is Unsuitable for Chronic Inhibition of HDAC Activity in the CNS In Vivo

Histone deacetylase (HDAC) inhibitors have received considerable attention as potential therapeutics for a variety of cancers and neurological disorders. Recent publications on a class of pimelic diphenylamide HDAC inhibitors have highlighted their promise in the treatment of the neurodegenerative diseases Friedreich's ataxia and Huntington's disease, based on efficacy in cell and mouse models. These studies' authors have proposed that the unique action of these compounds compared to hydroxamic acid-based HDAC inhibitors results from their unusual slow-on/slow-off kinetics of binding, preferentially to HDAC3, resulting in a distinctive pharmacological profile and reduced toxicity. Here, we evaluate the HDAC subtype selectivity, cellular activity, absorption, distribution, metabolism and excretion (ADME) properties, as well as the central pharmacodynamic profile of one such compound, HDACi 4b, previously described to show efficacy in vivo in the R6/2 mouse model of Huntington's disease. Based on our data reported here, we conclude that while the in vitro selectivity and binding mode are largely in agreement with previous reports, the physicochemical properties, metabolic and p-glycoprotein (Pgp) substrate liability of HDACi 4b render this compound suboptimal to investigate central Class I HDAC inhibition in vivo in mouse per oral administration. A drug administration regimen using HDACi 4b dissolved in drinking water was used in the previous proof of concept study, casting doubt on the validation of CNS HDAC3 inhibition as a target for the treatment of Huntington's disease. We highlight physicochemical stability and metabolic issues with 4b that are likely intrinsic liabilities of the benzamide chemotype in general.     

Histone deacetylase inhibitors with a primary amide zinc binding group display antitumor activity in xenograft model

Histone deacetylase (HDAC) inhibition causes hyperacetylation of histones leading to differentiation, growth arrest and apoptosis of malignant cells, representing a new strategy in cancer therapy. Many of the known HDAC inhibitors (HDACi) that are in clinical trials possess a hydroxamic acid, that is a strong Zn²⁺ binding group, thereby inhibiting some of the class I and class II isoforms. Herein we describe the identification of a selective class I HDAC inhibitor bearing a primary carboxamide moiety as zinc binding group. This HDACi displays good antiproliferative activity against multiple cancer cell lines, and demonstrates efficacy in a xenograft model comparable to vorinostat.     

Design,synthesis and evaluation of antiproliferative activity of melanoma-targeted histone deacetylase inhibitors

The clinical validation of histone deacetylase inhibition as a cancer therapeutic modality has stimulated interest in the development of new generation of potent and tumor selective histone deacetylase inhibitors (HDACi). With the goal of selective delivery of the HDACi to melanoma cells, we incorporated the benzamide, a high affinity melanin-binding template, into the design of HDACi to generate a new series of compounds 10a-b and 11a-b which display high potency towards HDAC1 and HDAC6. However, these compounds have attenuated antiproliferative activities relative to the untargeted HDACi. An alternative strategy furnished compound 14, a prodrug bearing the benzamide template linked via a labile bond to a hydroxamate-based HDACi. This pro-drug compound showed promising antiproliferative activity and warrant further study.     

Novel uracil-based 2-aminoanilide and 2-aminoanilide-like derivatives: histone deacetylase inhibition and in-cell activities

A novel series of non-hydroxamate HDAC inhibitors (HDACi) showing a uracil group at the left and a 2-aminoanilide/2-aminoanilide-like portion at the right head have been reported. In particular, the new compounds incorporating a 2-aminoanilide moiety behaved as class I-selective HDACi. Compound 8, the most potent and class I-selective, showed weak apoptosis (higher than MS-275) joined to cytodifferentiating activity on U937 cells. Surprisingly, the highest differentiation was observed with 13, through an effect that seems to be unrelated to HDAC inhibition.     

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.     

Interplay between PKCδ and Sp1 on histone deacetylase inhibitor-mediated Epstein-Barr virus reactivation

Epstein-Barr virus (EBV) undergoes latent and lytic replication cycles, and its reactivation from latency to lytic replication is initiated by expression of the two viral immediate-early transactivators, Zta and Rta. In vitro, reactivation of EBV can be induced by anti-immunoglobulin, tetradecanoyl phorbol acetate, and histone deacetylase inhibitor (HDACi). We have discovered that protein kinase C delta (PKCδ) is required specifically for EBV reactivation by HDACi. Overexpression of PKCδ is sufficient to induce the activity of the Zta promoter (Zp) but not of the Rta promoter (Rp). Deletion analysis revealed that the ZID element of Zp is important for PKCδ activation. Moreover, the Sp1 putative sequence on ZID is essential for PKCδ-induced Zp activity, and the physiological binding of Sp1 on ZID has been confirmed. After HDACi treatment, activated PKCδ can phosphorylate Sp1 at serine residues and might result in dissociation of the HDAC2 repressor from ZID. HDACi-mediated HDAC2-Sp1 dissociation can be inhibited by the PKCδ inhibitor, Rotterlin. Furthermore, overexpression of HDAC2 can suppress the HDACi-induced Zp activity. Consequently, we hypothesize that HDACi induces PKCδ activation, causing phosphorylation of Sp1, and that the interplay between PKCδ and Sp1 results in the release of HDAC2 repressor from Zp and initiation of Zta expression.     

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.     

p21waf1/Cip1 partially mediates apoptosis in hepatocellular carcinoma cells

p21waf1/Cip1 (p21) is a tumor suppressor gene involved in apoptosis in many cancer cell types induced by different agents. In spite of concomitant induction of p21 by many anti-cancer drugs, including inhibitors of histone deacetylases (HDACi), its pro-apoptotic action has been debated during the last several years due to a lack of direct evidence regarding the exact role of p21 in apoptosis. With the help of anti-sense p21 expression, we show here that p21 is mediating the apoptotic effects of HDACi 4-phenylbutyrate (4-PB) and Trichostatin A (TSA) on the hepatocellular hepatocarcinoma Hep3B cells. Hep3B cells were transfected by EGFP-p21 anti-sense or sense plasmids, and apoptosis induced by HDACi was assessed by TUNEL assay. The results show that the p21 anti-sense construct prevents apoptosis, induced by HDAC inhibitors in Hep3B cells. The obtained results suggest an important role for p21 in mediating the apoptotic effect of HDACi.     

Exploring bis-(indolyl)methane moiety as an alternative and innovative CAP group in the design of histone deacetylase (HDAC) inhibitors

In order to gather further knowledge about the structural requirements on histone deacetylase inhibitors (HDACi), starting from the schematic model of the common pharmacophore that characterizes this class of molecules (surface recognition CAP group—connection unit—linker region—Zinc Binding Group), we designed and synthesized a series of hydroxamic acids containing a bis-(indolyl)methane moiety. HDAC inhibition profile and antiproliferative activity were evaluated.     

Monitoring histone deacetylase inhibition in vivo: noninvasive magnetic resonance spectroscopy method

Histone deacetylase inhibitors (HDACis) are emerging as promising and selective antitumor agents. However, HDACis can lead to tumor stasis rather than shrinkage, in which case, traditional imaging methods are not adequate to monitor response. Consequently, novel approaches are needed. We have shown in cells that (19)F magnetic resonance spectroscopy (MRS)-detectable levels of the HDAC substrate Boc-Lys-TFA-OH (BLT) are inversely correlated with HDAC activity. We extended our investigations to a tumor xenograft model. Following intraperitoneal injection of BLT, its accumulation within the tumor was monitored by in vivo (19)F MRS. In animals treated with the HDACi suberoylanilide hydroxamic acid (SAHA), tumoral BLT levels were higher by 77% and 132% on days 2 and 7 of treatment compared with pretreatment levels (n = 6; p < .05). In contrast, tumoral BLT levels remained unchanged in control animals and in normal tissue. Thus, (19)F MRS of BLT detected the effect of HDACi treatment as early as day 2 of treatment. Importantly, tumor size confirmed that SAHA treatment leads to inhibition of tumor growth. However, difference in tumor size reached significance only on day 6 of treatment. Thus, this work identifies BLT as a potential molecular imaging agent for the early noninvasive MRS detection of HDAC inhibition in vivo.     

Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation

In primary effusion lymphoma (PEL) cells infected with latent Kaposi''s sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta''s promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.