Suberohydroxamic Acid Research Articles

Metabolomic Strategy to Characterize the Profile of Secondary Metabolites in Aspergillus aculeatus DL1011 Regulated by Chemical Epigenetic Agents.

Chemical epigenetic regulation (CER) is an effective method to activate the silent pathway of fungal secondary metabolite synthesis. However, conventional methods for CER study are laborious and time-consuming. In the meantime, the overall profile of the secondary metabolites in the fungi treated by the CER reagent is not well characterized. In this study, suberohydroxamic acid (SBHA), a histone deacetylase inhibitor, was added to a culture of Aspergillus aculeatus DL1011 and a new strategy based on LC-MS/MS analysis integrated with various metabolomic tools (MetaboAnalyst, MS-DIAL, SIRIUS and GNPS) was developed to characterize the profile of induced metabolites. As a result, 13.6%, 29.5% and 27.2% of metabolites were identified as newly biosynthesized, increasing and decreasing in abundance by CER, respectively. The structures of the 18 newly induced secondary metabolites were further identified by the new strategy to demonstrate that 72.2% of them (1 novel compound and 12 known compounds) were first discovered in A. aculeatus upon SBHA treatment. The accuracy of the new approach was confirmed by purification and NMR data analysis of major newly biosynthesized secondary metabolites. The bioassay showed that the newly biosynthesized compounds, roseopurpurin analogues, showed selective activities against DPPH scavenging, cytotoxicity and SHP1 inhibition. Our research demonstrated that CER was beneficial for changing the secondary metabolic profile of fungi and was an effective means of increasing the diversity of active metabolites. Our work also supplied a metabolomic strategy to characterize the profile changes and determine the newly induced compounds in the secondary metabolites of fungi treated with the chemical epigenetic regulator.

Exploring the chemical diversity of phytopathogenic fungi infecting edible fruits

Two fungi, Fusarium guttiforme and Colletotrichum horii, were cultured under different conditions to obtain fourteen compounds. The axenic cultures of F. guttiforme and C. horii in potato dextrose broth (PDB) medium yielded fusaric acid (1), 9,10-dehydrofusaric acid (2), and tyrosol, whereas their co-cultivation produced fusarinol (5), a fusaric acid complex with magnesium (3), 9,10-dehydrofusaric acid complex with magnesium (4), and 5-butyl-5-(hydroxymethyl) dihydrofuranone (9). Upon changing the medium from PDB to Czapek, different compounds (uracil, p-hydroxy acetophenone, and cyclo(L-Leu-L-Pro) were obtained. Fusaric acid (1) was biotransformed into fusarinol (5) by C. horii, suggesting a detoxification process, and three other compounds were obtained: 7-hydroxyfusarinol (7), 9,10-dehydrofusarinol (6), and fusarinyl acetate (8). Epigenetic modulation of suberohydroxamic acid against F. guttiforme afforded gibepyrone B (10). These compounds were subjected to a papain inhibition enzymatic assay; the highest inhibitory activity was displayed by the two magnesium complexes, at 56 and 54% inhibition, respectively.

The epigenetic impact of suberohydroxamic acid and 5‑Aza‑2'‑deoxycytidine on DNMT3B expression in myeloma cell lines differing in IL‑6 expression.

Gene inactivation of the cyclin-dependent kinase inhibitors p16INK4a, p15INK4b and p21WAF is frequently mediated by promoter gene methylation, whereas histone deacetylases (HDACs) control gene expression through their ability to deacetylate proteins. The effect of suberohydroxamic acid (SBHA) and 5-Aza-2′-deoxycytidine (Decitabine) (DAC) treatments on the transcription of CDKN2A, CDKN2B and CDKN1A genes, and their effects on molecular biological behavior were examined in two myeloma cell lines, RPMI8226 and U266, which differ in p53-functionality and IL-6 expression. In both tested myeloma cell lines, a non-methylated state of the CDKN2B gene promoter region was detected with normal gene expression, and the same level of p15INK4b protein was detected by immunocytochemical staining. Furthermore, in myeloma cells treated with SBHA and DAC alone, the expression of both p15INK4b and p21WAF was significantly upregulated in RPMI8226 cells (p53-functional, without IL-6 expression), whereas in the U266 cell line (p53 deleted, expressing IL-6) only p21WAF expression was significantly increased. Moreover, the analysis revealed that treatment with DAC induced DNMT3B enhancement in U266 cells. In conclusion, in myeloma cells with IL-6 expression, significantly increased DNMT3B expression indicated the tumorigenic consequences of 5-Aza-2′deoxycytidine treatment, which requires careful use in diseases involving epigenetic dysregulation, such as multiple myeloma (MM).

Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation

Latency reversing agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-κB and induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and synergize with LRAs to reactivate latent HIV-1. This study investigates whether a panel of epigenetic modifiers, including HDACi, could dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal. We screened an epigenetic modifier library for compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo. We identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-α, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when combined with Ingenol-3,20-dibenzoate. Combining SBHA and Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when combined with Ingenol-3,20-dibenzoate suggests SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

GM101 in Combination with Histone Deacetylase Inhibitor Enhances Anti-Tumor Effects in Desmoplastic Microenvironment.

Oncolytic adenoviruses (oAds) have been evaluated in numerous clinical trials due to their promising attributes as cancer therapeutics. However, the therapeutic efficacy of oAds was limited due to variable coxsackie and adenovirus receptor (CAR) expression levels and the dense extracellular matrix (ECM) of heterogenic clinical tumors. To overcome these limitations, our present report investigated the therapeutic efficacy of combining GM101, an oAd with excellent tumor ECM degrading properties, and histone deacetylase inhibitor (HDACi). Four different HDACi (suberohydroxamic acid (SBHA), MS-275, trichostatin A (TSA), and valproic acid) candidates in combination with replication-incompetent and GFP-expressing Ad (dAd/GFP) revealed that SBHA and MS-275 exerted more potent enhancement in Ad transduction efficacy than TSA or valproic acid. Further characterization revealed that SBHA and MS-275 effectively upregulated CAR expression in cancer cells, improved the binding of Ad with cancer cell membranes, and led to dynamin 2- and clathrin-mediated endocytosis of Ad. The combination of GM101 with HDACi induced superior cancer cell killing effects compared to any of the monotherapies, without any additional cytotoxicity in normal cell lines. Further, GM101+SBHA and GM101+MS-275 induced more potent antitumor efficacy than any monotherapy in U343 xenograft tumor model. Potent antitumor efficacy was achieved via the combination of GM101 with HDACi, inducing necrotic and apoptotic cancer cell death, inhibiting cancer cell proliferation, degrading ECM in tumor tissue, and thus exerting the highest level of virus dispersion and accumulation. Collectively, these data demonstrate that the combination of GM101 and HDACi can enhance intratumoral dispersion and accumulation of oAd through multifaced mechanisms, making it a promising strategy to address the challenges toward successful clinical development of oAd.

Chemical epigenetic manipulation triggers the production of sesquiterpenes from the deep-sea derived Eutypella fungus

Chemical epigenetic manipulation of a deep-sea-derived Eutypella sp. fungus by the co-treatment with a histonedeacetylase inhibitor (suberohydroxamic acid, SBHA) and a DNA methyltransferase inhibitor (5-azacytidine, 5-Aza), resulted in the activation of a sesquiterpene-related biosynthetic gene cluster. Chromatographic separation of the elicitor-treated cultures led the isolation of 21 sesquiterpenes, including 17 undescribed compounds, eutypeterpenes A-Q. Their structures were identified by the extensive analysis of the spectroscopic data, including the single-crystal X-ray diffraction, chemical conversion, and the calculated NMR and ECD data for configurational assignments. Eutypeterpene A is a first bergamotene-type sesquiterpene incorporated with a dioxolanone unit, and eutypeterpenes O-Q with a cyclopentane ring represent an undescribed subtype of sesquiterpenes. The bioassay results showed that most compounds exert inhibitory effects against the lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages, and eutypeterpene N is the most active. This study demonstrates that the epigenetic manipulation is an effective approach to trigger the production of cryptic metabolites from deep-sea derived fungus. The significant inhibition against LPS-induced NO production in vitro suggests eutypeterpenes to be potential for the development as anti-inflammatory agents.

Epigenetic Manipulation Induces the Production of Coumarin‐Type Secondary Metabolite from Arthrobotrys foliicola

AbstractPursuing epigenetic manipulation approach in fungi led to the isolation of an unusual coumarin metabolite from fungi. An addition of the histone deacetylase inhibitor, suberohydroxamic acid (SBHA), to the culture medium of Arthrobotrys foliicola induced production of the coumarin‐type secondary metabolite represented by a single intensive peak in the HPLC profile of the ethyl acetate extract. The compound which was identified as 4‐ethyl‐7‐hydroxy‐8‐methyl‐2H‐chromen‐2‐one (1) was isolated from nature for the first time. Moreover, the investigation on the remaining part of the HPLC profile led to the separation of 6‐ethyl‐2,4‐dihydroxy‐3‐methylbenzaldehyde (2) and ten 2,5‐diketopiperazine compounds (3–12). The structures of isolates were deduced by their mass and NMR spectroscopic data. The coumarin‐type secondary metabolite (1) with peculiar smell induced by epigenetic stimulation is found for the first time in the Arthrobotrys species and the family Orbiliaceae. We evaluated 1 for the cytotoxic, anti‐inflammatory, anti‐allergic and nematocidal activities, however, it was found inactive.

Eremophilane-type sesquiterpenoids in a deep-sea fungus Eutypella sp. activated by chemical epigenetic manipulation

Cultivation of a deep-sea-derived fungus Eutypella sp. MCCC 3A00281 by a chemical epigenetic manipulation using suberohydroxamic acid (SBHA), a histone deacetylase inhibitor (HDI), resulted in the significant change of the metabolite profile. Chromatographic application of the extended metabolites led to the isolation of a total of 30 eremophilane-type sesquiterpenoids, of which 26 were identified as new entities namely eutyperemophilanes A-Z (1–26). Their structures were established by extensive analyses of the 1D and 2D NMR, and HRESIMS data, as well as the modified Mosher's method, X-ray crystallography, and ECD data for the assignment of their absolute configurations. Most eremophilanes from the fungal strain showed a trans-fusion of rings A and B, uncommonly found from nature. All compounds were evaluated for the inhibitory effects toward the nitric oxide (NO) production that was induced by lipopolysaccharide (LPS) in RAW 264.7 macrophage cells, while compounds 9 and 10 exerted significant inhibition.

A kinetic study of the oxidation of hydroxamic acids by compounds I and II of horseradish peroxidase: Effect of transition metal ions

Oxidation of hydroxamic acids (HXs) generates HNO, and it is not clear whether it is formed also in the presence of metal ions. The kinetics of the oxidation of HXs, such as acetohydroxamic acid, suberohydroxamic acid, and suberoylanilide hydroxamic acid (SAHA), by compounds I and II of horseradish peroxidase (HRP) at pH 7.0 and 25 °C have been studied using rapid-mixing stopped-flow. The kinetics of these reactions were compared to those observed in the presence of Cu(ClO4)2, NiSO4, or ZnSO4. The rates decrease upon increasing [CuII] at constant [HXs], and no oxidation of HX occurs when [HX]/[CuII] ≈ 2, implying that HX oxidation in the presence of CuII proceeds through the free ligand since the predominant complex is CuX2. In the case of NiII, the oxidation rate decreases upon increasing the ratio [NiII]/[HX] beyond 1, where the predominant complex is NiIIX+, implying that its oxidation is feasible. The effect of ZnII could be studied only on the rate of HXs oxidation by compound II demonstrating similar behavior to that of NiII. HXs were also oxidized catalytically by HRP/H2O2 at pH 7.0, demonstrating that metal ions facilitate the formation of HNO while hardly affecting its yield and the extent of HX oxidation.

Epigenetic Modifier Based Enhancement of Piperine Production in Endophytic Diaporthe sp. PF20

Biodiversity and metabolite richness of endophytic fungi are highly fascinating. Some of them have even been reported to have the shared biosynthetic basis for the production of plant specific metabolites. However, only limited reports are there on enhancement of production of plant specific metabolites from endophytic fungi. In the study, endophytic Diaporthe sp. PF20 from Piper nigrum L. was identified to have the ability to produce piperine by liquid chromatography tandem mass spectroscopy. The isolate PF20 was further subjected to epigenetic treatment along with previously characterized piperine producing Colletotrichum sp. and Mycosphaerella sp. from the same plant. Very interestingly, use of histone deacetylase inhibitor suberohydroxamic acid enhanced the piperine production in PF20. Here, the epigenetic modulator mediated enhancement of phytochemical biosynthetic potential of endophytic fungi is novel in its approach. Hence the results of the study open up new avenues to maintain the biosynthetic competency of endophytic fungi, which is highly challenging. Even though piperine production has previously been reported from endophytic fungi, epigenetic modulator mediated multiplexing of this property as observed for PF20 is highly attractive. This is because, only this organism was found to be susceptible to epigenetic modulation based piperine enhancement among the selected isolates.

Nitrogen Dioxide Reaction with Nitroxide Radical Derived from Hydroxamic Acids: The Intermediacy of Acyl Nitroso and Nitroxyl (HNO).

Hydroxamic acids (RC(O)NHOH) form a class of compounds that display interesting chemical and biological properties The chemistry of RC(O)NHOH) is associated with one- and two-electron oxidations forming the respective nitroxide radical (RC(O)NHO•) and acyl nitroso (RC(O)N═O), respectively, which are relatively unstable species. In the present study, the kinetics and mechanism of the •NO2 reaction with nitroxide radicals derived from acetohydroxamic acid, suberohydroxamic acid, benzohydroxamic acid, and suberoylanilide hydroxamic acid have been studied in alkaline solutions. Ionizing radiation was used to generate about equal yields of these radicals, demonstrating that the oxidation of the transient nitroxide radical by •NO2 produces HNO and nitrite at about equal yields. The rate constant of •NO2 reaction with the nitroxide radical derived from acetohydroxamic acid has been determined to be (2.5 ± 0.5) × 109 M-1 s-1. This reaction forms a transient intermediate absorbing at 314 nm, which decays via a first-order reaction whose rate increases upon increasing the pH or the hydroxamic acid concentration. Transient intermediates absorbing around 314 nm are also formed during the oxidation of hydroxamic acids by H2O2 catalyzed by horseradish peroxidase. It is shown that HNO is formed during the decomposition of these intermediates, and therefore, they are assigned to acyl nitroso compounds. This study provides for the first time a direct spectrophotometric detection of acyl nitroso compounds in aqueous solutions allowing the study of their chemistry and reaction kinetics.

Identification and Biological Evaluation of Secondary Metabolites from Marine Derived Fungi-Aspergillus sp. SCSIOW3, Cultivated in the Presence of Epigenetic Modifying Agents.

Chemical epigenetic manipulation was applied to a deep marine-derived fungus, Aspergillus sp. SCSIOW3, resulting in significant changes of the secondary metabolites. One new diphenylether-O-glycoside (diorcinol 3-O-α-D-ribofuranoside), along with seven known compounds, were isolated from the culture treated with a combination of histone deacetylase inhibitor (suberohydroxamic acid) and DNA methyltransferase inhibitor (5-azacytidine). Compounds 2 and 4 exhibited significant biomembrane protective effect of erythrocytes. 2 also showed algicidal activity against Chattonella marina, a bloom forming alga responsible for large scale fish deaths.

Antimicrobial activity and acetylcholinesterase inhibition by extracts from chromatin modulated fungi.

Major health challenges as the increasing number of cases of infections by antibiotic multiresistant microorganisms and cases of Alzheimer's disease have led to searching new control drugs. The present study aims to verify a new way of obtaining bioactive extracts from filamentous fungi with potential antimicrobial and acetylcholinesterase inhibitory activities, using epigenetic modulation to promote the expression of genes commonly silenced. For such finality, five filamentous fungal species (Talaromyces funiculosus, Talaromyces islandicus, Talaromyces minioluteus, Talaromyces pinophilus, Penicillium janthinellum) were grown or not with DNA methyltransferases inhibitors (procainamide or hydralazine) and/or a histone deacetylase inhibitor (suberohydroxamic acid). Extracts from T. islandicus cultured or not with hydralazine inhibited Listeria monocytogenes growth in 57.66 ± 5.98% and 15.38 ± 1.99%, respectively. Increment in inhibition of acetylcholinesterase activity was observed for the extract from P. janthinellum grown with procainamide (100%), when compared to the control extract (39.62 ± 3.76%). Similarly, inhibition of acetylcholinesterase activity increased from 20.91 ± 3.90% (control) to 92.20 ± 3.72% when the tested extract was obtained from T. pinophilus under a combination of suberohydroxamic acid and procainamide. Concluding, increases in antimicrobial activity and acetylcholinesterase inhibition were observed when fungal extracts in the presence of DNA methyltransferases and/or histone deacetylase modulators were tested.

HTP Nutraceutical Screening for Histone Deacetylase Inhibitors and Effects of HDACis on Tumor-suppressing miRNAs by Trichostatin A and Grapeseed (Vitis vinifera) in HeLa cells.

Aggressive tumor malignancies are a consequence of delayed diagnosis, epigenetic/phenotype changes and chemo-radiation resistance. Histone deacetylases (HDACs) are a major epigenetic regulator of transcriptional repression, which are highly overexpressed in advanced malignancy. While original chemotherapy drugs were modeled after phytochemicals elucidated by botanical screenings, HDAC inhibitors (HDACi) such as apicidin, trichostatin A (TSA) and butyrate were discovered as products of fungus and microbes, in particular, gut microbiota. Therefore, a persistent question remains as to the inherent existence of HDACis in raw undigested dietary plant material. In this study, we conduct a high-throughput (HTP) screening of ~1,600 non-fermented commonly used nutraceuticals (spices, herbs, teas, vegetables, fruits, seeds, rinds etc.) at (<600 μg/ml) and food-based polyphenolics (<240 μg/ml) for evidence of HDAC activity inhibition in nuclear HeLa cell lysates. Human HDAC kinetic validation was performed using a standard fluorometric activity assay, followed by an enzymatic-linked immuno-captured ELISA. Both methods were verified using HDACi panel drugs: TSA, apicidin, suberohydroxamic acid, M344, CL-994, valproic acid and sodium phenylbutyrate. The HTP screening was then conducted, followed by a study comparing biological effects of HDACis in HeLa cells, including analysis of whole-transcriptome non-coding RNAs using Affymetrix miRNA 4.1-panel arrays. The HTP screening results confirmed 44/1600 as potential HDACis to which 31 were further eliminated as false-positives. Methodological challenges/concerns are addressed regarding plant product false-positives that arise from the signal reduction of commercial lysine development reagents. Only 13 HDACis were found having an IC50 under <200 μg/ml: Grapeseed extract (Vitis vinifera), Great burnet root (Sanguisorba Officinalis), Babul (Acacia arabica), Chinese gallnut (Melaphis chinensis), Konaberry extract (Coffea arabica), Uva Ursi (Arctostaphylos uva ursi), Green tea (Camellia sinensis), Meadowsweet (Filipendula ulmaria), Sassafras (Sassafras officinale), Turkey rhubarb (Rheum palmatum), epigallocatechin gallate (EGCG), gossypol and gallic acid. Next, we investigate the biological consequence of HDACi panel drugs in HeLa cells, where the data suggest predominant effects are anti-mitotic rather than cytotoxic. Lastly, differential effects of TSA vs. GSE at sub-lethal concentrations tested on HeLa cells show 6,631 miRNAs expressed in resting cells, 35 significantly up-regulated (TSA) and 81 up-regulated (GSE), with several miRNAs overlapping in the upward direction by both GSE and TSA (e.g. hsa-miR-23b-5p, hsa-miR-27b-5p, hsa-miR-1180-3p, hsa-miR-6880-5p and hsa-mir-943). Using DIANA miRNA online tools, it was determined that GSE and TSA simultaneously cause overexpression of similar miRNAs predicted to destroy the following influential oncogenes: NFkB, NRAS, KRAS, HRAS, MYC, TGFBR1, E2F1, E2F2, BCL21, CDKN1A, CDK6, HIF1a, and VEGFA. The data from this study show that plant- based HDACis are relatively rare, and can elicit a similar pattern to TSA in up-regulating miRNAs involved with tumor suppression of HeLa cervical carcinoma.

Histone Deacetylase Inhibitors Reduce the Number of Herpes Simplex Virus-1 Genomes Initiating Expression in Individual Cells

Although many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACi’s). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid, Valporic Acid, and Suberoylanilide Hydroxamic Acid. We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the gene expression from the viral genomes. Different cell types (HFF, Vero, and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (promyelocytic leukemia and ATRX), which may explain the lower number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in differences in the number of genomes that initiate expression.

Eremophilane Sesquiterpenes from a Deep Marine-Derived Fungus, Aspergillus sp. SCSIOW2, Cultivated in the Presence of Epigenetic Modifying Agents.

Chemical epigenetic manipulation was applied to a deep marine-derived fungus, Aspergillus sp. SCSIOW2, resulting in significant changes of the secondary metabolites. Three new eremophilane-type sesquiterpenes, dihydrobipolaroxin B (2), dihydrobipolaroxin C (3), and dihydrobipolaroxin D (4), along with one known analogue, dihydrobipolaroxin (1), were isolated from the culture treated with a combination of histone deacetylase inhibitor (suberohydroxamic acid) and DNA methyltransferase inhibitor (5-azacytidine). 1–4 were not produced in the untreated cultures. 2 and 3 might be artificial because 1 could form 2 and 3 spontaneously in water by intracellular acetalization reaction. The absolute configurations of 1 and 2 were assigned based on ECD spectroscopy combined with time-dependent density functional theory calculations. All four compounds exhibited moderate nitric oxide inhibitory activities without cytotoxic effects.

Structural comparison of suberanilohydroxamic acid (SAHA) and other zinc-enzyme inhibitors bound to a monomeric zinc species

Histone Deacetylase (HDAC) and similar proteins contain an active Zn–OH group and have been targets for inhibitors. Known inhibitors such as suberanilohydroxamic acid (SAHA), suberohydroxamic acid (SBHA), valproic acid, and 8-hydroxyquinoline (8-HQ) derivatives act on these enzymes by binding to the zinc center. To examine how these drugs interacted with zinc centers and to examine their structural differences, a homogeneous zinc complex was synthesized and reacted with SAHA, SBHA, valproic acid, and 8-HQ to form stable adducts, which were characterized by spectroscopy and X-ray crystallography. A comparison of their structures with known complexes and enzyme active sites bound to drugs was performed in order to understand how these drugs interact with their targets.

Defeating EpCAM+ liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma

Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma.

A Soft-Drug Histone Deacetylase Inhibitor for Cutaneous T-Cell Lymphoma.

Cutaneous T-Cell Lymphoma (CTCL) comprises a group of related lymphoproliferative disorders characterized by the presence of malignant lymphocytes in the skin. Most patients with CTCL enjoy a normal life expectancy, though all experience chronic morbidity due to the symptomatic and cosmetic manifestations of epidermotropism. There is no curative therapeutic option for patients with CTCL and consequently topical approaches have become a core component of standard care. Recently, activity against advanced CTCL has been demonstrated by a new class of targeted agents: histone deacetylase inhibitors (HDACi). One HDACi has been approved by the FDA for use in advanced disease (vorinostat; SAHA; Merck Research Laboratories, Rahway, NJ). Dose-limiting toxicities with parenteral HDAC inhibitors include fatigue, diarrhea, nausea and myelosuppression. The poor tolerability of the currently available, nonselective small molecules render most patients with this indolent disease unlikely to benefit from this pharmacologic class. We therefore conceived of reverse pro-drug HDAC inhibitors to allow high dose-intensity at the site of disease and presystemic metabolism to mitigate side effects and reduce exposure to genotoxic agents. Suberohydroxamic acid phenyl ester (SHAPE) is a first-in-class soft-drug HDACi. SHAPE is a soluble, scalable analog of SAHA possessing an isosteric, isoelectronic ester bond at a site predicted by molecular modeling not to impact target recognition features. Serum metabolism was confirmed using a real-time absorbance assay capable of monitoring SHAPE degradation to suberic acid hydroxamate and phenol. We have determined that the rapid hydrolysis of SHAPE in serum is due to the catalytic activities of butyrylcholinesterase and paraoxonase. Cellular inhibition of Class I and II HDACs was confirmed using high content imaging; effects on histone and tubulin acetylation comparable to SAHA are witnessed at concentrations above 20 micromolar. Active site binding to HDAC isoforms was determined using purified protein and a novel, miniaturized fluorescence polarization assay we designed to permit instantaneous kinetic measurements of labile compounds. Tolerability studies of topical SHAPE were performed on shaved immunocompetent mice and on immunodeficient animals bearing human skin xenografts. Histologic evidence of inflammation or damage was not visualized. We next performed experiments testing efficacy against CTCL in vivo. Using cell lines established from neoplastic, skin-homing lymphocytes arising from an interleukin-7 transgenic murine model of CTCL, we developed a transplantation model in syngeneic animals with rapid, homogeneous disease onset compatible with studies of investigational agents. Daily administration of SHAPE (50 mg/mL) for two weeks resulted in clearance of skin-homing lymphocytes, compared to vehicle treated controls. For use in murine model studies and anticipating human clinical trials, we identified acetylation response biomarkers using mass spectrometry and developed immunohistochemical protocols with acetylation state-specific antibodies. In sum, we report the design, characterization and translational studies supporting the clinical development of a novel, soft-drug HDAC inhibitor in CTCL.

Design and Characterization of a Novel, Reverse Prodrug Histone Deacetylase Inhibitor for Cutaneous T-Cell Lymphoma.

Cutaneous T-Cell Lymphoma (CTCL) comprises a group of related lymphoproliferative disorders characterized by the presence of malignant lymphocytes in the skin. The most common variant is Mycosis Fungoides (MF), which affects approximately 500 patients per year in the United States. Though most patients with MF enjoy a normal life expectancy, they experience chronic morbidity due to the symptomatic and cosmetic manifestations of epidermotropism: erythematous patches, elevated plaques, alopecia, and cellulitis. Many patients will experience progressive disease with lymph node infiltration, tumor and a leukemic phase termed the Sezary Syndrome. The lack of a survival benefit offered by clinical trials of combination chemotherapy and radiation supports the current standard of initial topical therapy for most patients with CTCL. Topical or skin-directed approaches widely offered include: nitrogen mustards (meclorethamine or carmustine), corticosteroids, oral psoralen with UVA radiation, UVB phototherapy and electron beam irradiation. Recent, early phase clinical studies of histone deacetylase inhibitors (HDACi) have illustrated a remarkable activity among patients with advanced CTCL. The structural dissimilarity of these ligands supports an on-target antineoplastic effect. Unfortunately, the frequently severe side effects associated with the systemic delivery of these nonselective ligands may limit their downstream clinical utility in the majority of patients with this disease, even with topical administration. We therefore devised a chemical strategy to achieve high dose-intensity at the site of cutaneous disease following topical administration with limited systemic exposure. Suberohydroxamic acid phenyl ester (SHAPE) is a first-in-class reverse prodrug inhibitor of histone deacetylases. Between the aliphatic chain and aromatic capping element of this canonical HDACi, we positioned an ester bond so as to promote presystemic metabolism by serum esterases. Preclinical studies of this ligand demonstrate potent activity against nuclear and cytosolic HDAC proteins. Doses required to achieve maximal histone hyperacetylation in human cancer cell tissue culture are comparable to vorinostat (SAHA; Merck & Co., Rahway, NJ) and MS-275 (Berlex Pharmaceuticals, Montville, NJ). These studies support the stability of SHAPE in the intracellular environment amid cellular esterases. The rapid metabolism (t-½ less than 5 minutes) by serum esterases has been established biochemically using continuous spectrophotometry, and is mediated by both butyrylcholinesterase and paraoxonase. Tolerability and preliminary activity have recently been demonstrated in an IL-7 transgenic mouse model of CTCL supporting clinical development as a therapeutic strategy in humans. Application to premalignant, neoplastic and inflammatory conditions of the oropharynx is being explored. Further studies characterizing the impact of SHAPE on T-cell function are also ongoing, to assess utility in conditions such as psoriasis and cutaneous graft-versus-host disease.