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1.  Synthesis and Biological Evaluation of Largazole Analogues with Modified Surface Recognition Cap Groups 
Several largazole analogues with modified surface recognition cap groups were synthesized and their HDAC inhibitory activities were determined. The C7-epimer 12 caused negligible inhibition of HDAC activity, failed to induce global histone 3 (H3) acetylation in the HCT116 colorectal cancer cell line and demonstrated minimal effect on growth. Although previous studies have shown some degree of tolerance of structural changes at C7 position of largazole, these data show the negative effect of conformational change accompanying change of configuration at this position. Similarly, analogue 16a with D-1-naphthylmethyl side chain at C2 too had negligible inhibition of HDAC activity, failed to induce global histone 3 (H3) acetylation in the HCT116 colorectal cancer cell line and demonstrated minimal effect on growth. In contrast, the L-allyl analogue 16b and the L-1-naphthylmethyl analogue 16c were potent HDAC inhibitors, showing robust induction of global H3 acetylation and significant effect on cell growth. The data suggest that even bulky substituents are tolerated at this position, provided the stereochemistry at C2 is retained. With bulky substituents, inversion of configuration at C2 results in loss of inhibitory activity. The activity profiles of 16b and 16c on Class I HDAC1 vs Class II HDAC6 are similar to those of largazole and, taken together with x-ray crystallography information of HDAC8-largazole complex, may suggest that the C2 position of largazole is not a suitable target for structural optimization to achieve isoform selectivity. The results of these studies may guide the synthesis of more potent and selective HDAC inhibitors.
PMCID: PMC4188730  PMID: 25203782
Antitumor agents; biological activity; antiproliferation; histone deacetylase inhibitors; molecular modeling
2.  Total Syntheses of the Histone Deacetylase Inhibitors Largazole and 2-epi-Largazole: Application of N-Heterocyclic Carbene Mediated Acylations in Complex Molecule Synthesis 
The Journal of organic chemistry  2011;76(4):1140-1150.
Details of the evolution of strategies toward convergent assembly of the histone deacetylase inhibiting natural product largazole exploiting γ,δ-unsaturated-α,β-epoxy-aldehydes and a thiazole-thiazoline containing ω-amino-acid are described. The initial N-heterocyclic carbene mediated redox amidation exploying these two types of building blocks representing largazole’s structural domains of distinct biosynthetic origin directly afforded the seco-acid of largazole. This was accomplished without any protecting groups resident upon either thioester bearing epoxy-aldehyde or the tetrapeptide. However, the ineffective production of largazole via the final macrolactonization led to an alternative intramolecular esterification/macrolactamization strategy employing the established two building blocks. This provided largazole along with its C2-epimer via an unexpected inversion of the α-stereocenter at the valine residue. The biological evaluation demonstrated that both largazole and 2-epi-largazole led to dose-dependent increases of acetylation of histone H3, indicating their potencies as class I histone deacetylase selective inhibitiors. Enhanced p21 expression was also induced by largazole and its C2 epimer. In addition, 2-epi-largazole displayed more potent activity than largazole in cell viability assays against PC-3 and LNCaP prostate cancer cell lines.
PMCID: PMC4201586  PMID: 21244075
3.  The Total Synthesis and Biological Mode of Action of Largazole: A Potent Class I Histone Deacetylase (HDAC) Inhibitor 
Journal of the American Chemical Society  2008;130(33):11219-11222.
The efficient total synthesis of the recently described natural substance largazole (1) and it’s active metabolite largazole thiol (2) is described. The synthesis required eight linear steps and proceeded in 37% overall yield. It is demonstrated that largazole is a pro-drug, that is activated by removal of the octanoyl residue from the 3-hydroxy-7-mercaptohept-4-enoic acid moiety to generate the active metabolite largazole thiol (2) which is an extraordinarily potent Class I histone deacetylase inhibitor. Synthetic largazole and the largazole thiol (2) have been evaluated side-by-side with FK228 and SAHA for inhibition of HDACs 1, 2, 3, and 6. Largazole and largazole thiol were further assayed for cytotoxic activity against a panel of chemoresistant melanoma cell lines and it was found that largazole is substantially more cytotoxic than largazole thiol; this difference being attributed to differences in cell permeability of the two substances, respectively.
PMCID: PMC3090445  PMID: 18642817
4.  Structural Basis of the Antiproliferative Activity of Largazole, a Depsipeptide Inhibitor of the Histone Deacetylases 
Journal of the American Chemical Society  2011;133(32):12474-12477.
Largazole is a macrocyclic depsipeptide originally isolated from the marine cyanobacterium Symploca sp., which is indigenous to the warm, blue-green waters of Key Largo, Florida (whence largazole derives its name). Largazole contains an unusual thiazoline-thiazole ring system that rigidifies its macrocyclic skeleton, and it also contains a lipophilic thioester side chain. Hydrolysis of the thioester in vivo yields largazole thiol, which exhibits remarkable antiproliferative effects and is believed to be the most potent inhibitor of the metal-dependent histone deacetylases (HDACs). Here, the 2.14 Å-resolution crystal structure of the HDAC8-largazole thiol complex is the first of an HDAC complexed with a macrocyclic inhibitor and reveals that ideal thiolate-zinc coordination geometry is the key chemical feature responsible for its exceptional affinity and biological activity. Notably, the core structure of largazole is conserved in romidepsin, a depsipeptide natural product formulated as the drug Istodax® recently approved for cancer chemotherapy. Accordingly, the structure of the HDAC8-largazole thiol complex is the first to illustrate the mode of action of a new class of therapeutically important HDAC inhibitors.
PMCID: PMC3162211  PMID: 21790156
5.  Modulation of Activity Profiles for Largazole-Based HDAC Inhibitors through Alteration of Prodrug Properties 
ACS Medicinal Chemistry Letters  2014;5(8):905-910.
Largazole is a potent and class I-selective histone deacetylase (HDAC) inhibitor purified from marine cyanobacteria and was demonstrated to possess antitumor activity. Largazole employs a unique prodrug strategy, via a thioester moiety, to liberate the bioactive species largazole thiol. Here we report alternate prodrug strategies to modulate the pharmacokinetic and pharmacodynamics profiles of new largazole-based compounds. The in vitro effects of largazole analogues on cancer cell proliferation and enzymatic activities of purified HDACs were comparable to the natural product. However, in vitro and in vivo histone hyperacetylation in HCT116 cells and implanted tumors, respectively, showed differences, particularly in the onset of action and oral bioavailability. These results indicate that, by employing a different approach to disguise the “warhead” moiety, the functional consequence of these prodrugs can be significantly modulated. Our data corroborate the role of the pharmacokinetic properties of this class of compounds to elicit the desired and timely functional response.
PMCID: PMC4137384  PMID: 25147612
Largazole; histone deacetylases; antitumor activity; natural products; prodrugs
6.  Largazole Pharmacokinetics in Rats by LC-MS/MS 
Marine Drugs  2014;12(3):1623-1640.
A highly sensitive and specific LC-MS/MS method for the quantitation of largazole thiol, the active species of the marine-derived preclinical histone deacetylase inhibitor, largazole (prodrug), was developed and validated. Largazole thiol was extracted with ethyl acetate from human or rat plasma along with the internal standard, harmine. Samples were separated on an Onyx Monolithic C18 column by a stepwise gradient elution with 0.1% formic acid in methanol and 0.1% aqueous formic acid employing multiple reaction monitoring (MRM) detection. Linear calibration curves were obtained in the range of 12.5–400 ng/mL with 200 µL of human plasma. The overall intra-day precision was from 3.87% to 12.6%, and the inter-day precision was from 7.12% to 9.8%. The accuracy at low, medium and high concentrations ranged from 101.55% to 105.84%. Plasma protein bindings of largazole thiol in human and rat plasma as determined by an ultrafiltration method were 90.13% and 77.14%, respectively. Plasma drug concentrations were measured by this LC-MS/MS method. The pharmacokinetics of largazole thiol in rats was studied following i.v. administration at 10 mg/kg and found to follow a two-compartment model. Largazole thiol was rapidly eliminated from systemic circulation within 2 h. The established LC-MS/MS method is suitable for the analysis of largazole thiol in human plasma, as well.
PMCID: PMC3967229  PMID: 24658499
largazole; LC-MS/MS; pharmacokinetics; protein binding
7.  Evaluation of class I HDAC isoform selectivity of largazole analogues 
Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole-thiazoline and the warhead moiety. In order to elucidate class I-specific structure–activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors.
PMCID: PMC4135083  PMID: 25070421
Largazole; HDAC inhibitor; Class I histone deacetylase; Isoform selectivity; Structure–activity relationship
8.  Synthesis and Conformation-Activity Relationships of the Peptide Isosteres of FK228 and Largazole 
The peptide isosteres (10 and 11) of the naturally occurring and potent histone deacetylase (HDAC) inhibitors FK228 and largazole have been synthesized and evaluated side-by-side with FK228, largazole and SAHA for inhibition of the class I HDACs 1, 2, 3, and 6.
PMCID: PMC2880701  PMID: 19193120
9.  Biological Evaluation of New Largazole Analogues: Alteration of Macrocyclic Scaffold with Click Chemistry 
ACS Medicinal Chemistry Letters  2012;4(1):132-136.
We report the design, synthesis, and biological evaluation of a new series of largazole analogues in which a 4-methylthiazoline moiety was replaced with a triazole and tetrazole ring, respectively. Compound 7 bearing a tetrazole ring was identified to show much better selectivity for HDAC1 over HDAC9 than largazole (10-fold). This work could serve as a foundation for further exploration of selective HDAC inhibitors using a largazole molecular scaffold.
PMCID: PMC4027506  PMID: 24900575
HDAC inhibitor; peptides; macrocycles; largazole; click chemistry
10.  In Vitro and In Vivo Osteogenic Activity of Largazole 
ACS medicinal chemistry letters  2011;2(3):248-251.
Due to their capability of modifying chromatin structure and thereby regulating gene transcription, histone deacetylases (HDACs) have been reported to play important roles in osteogenesis and considered a promising potential therapeutic target for bone diseases, including osteoporosis. We showed that the novel marine-derived HDAC inhibitor largazole exhibits in vitro and in vivo osteogenic activity. Largazole significantly induced the expression of ALP and OPN. The osteogenic activity of largazole was mediated through the increased expression of Runx2 and BMPs. Importantly, largazole showed in vivo bone-forming efficacy in the mouse calvarial bone formation assay and the rabbit calvarial bone fracture healing model. The dual action of largazole to stimulate bone formation and inhibit bone resorption would be a useful feature in drug development for bone-related disorders.
PMCID: PMC3109915  PMID: 21666868
largazole; osteogenic activity; histone deacetylases; Runx2; bone morphogenetic protein
11.  In Vitro and In Vivo Osteogenic Activity of Largazole 
ACS Medicinal Chemistry Letters  2011;2(3):248-251.
Due to their capability of modifying chromatin structure and thereby regulating gene transcription, histone deacetylases (HDACs) have been reported to play important roles in osteogenesis and considered a promising potential therapeutic target for bone diseases, including osteoporosis. We showed that the novel marine-derived HDAC inhibitor largazole exhibits in vitro and in vivo osteogenic activity. Largazole significantly induced the expression of ALP and OPN. The osteogenic activity of largazole was mediated through the increased expression of Runx2 and BMPs. Importantly, largazole showed in vivo bone-forming efficacy in the mouse calvarial bone formation assay and the rabbit calvarial bone fracture healing model. The dual action of largazole to stimulate bone formation and inhibit bone resorption would be a useful feature in drug development for bone-related disorders.
PMCID: PMC3109915  PMID: 21666868
Largazole; osteogenic activity; histone deacetylases; Runx2; bone morphogenetic protein
12.  Glucocorticoids and histone deacetylase inhibitors cooperate to block the invasiveness of basal-like breast cancer cells through novel mechanisms 
Oncogene  2012;32(10):1316-1329.
Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.
PMCID: PMC3773700  PMID: 22543582
E-cadherin; breast cancer; invasion; glucocorticoid; HDAC inhibitor; PAI-1
13.  Enantioselective Total Synthesis of (+)-Largazole, a Potent Inhibitor of Histone Deacetylase 
Organic letters  2008;10(17):3907-3909.
An enantioselective total synthesis of cytotoxic natural product, (+)-largazole (1) is described. It is a potent histone deacetylase inhibitor. Our synthesis is convergent and involves the assembly of thiazole 3-derived carboxylic acid with amino ester 4 followed by cycloamidation of the corresponding amino acid. The synthesis features an efficient cross metathesis, an enzymatic kinetic resolution of a β-hydroxy ester, a selective removal of a Boc-protecting group, a HATU/HOAt-promoted cycloamidation reaction, and synthetic manipulations to a sensitive thioester functional group.
PMCID: PMC2945909  PMID: 18662003
14.  Synthesis and HDAC Inhibitory Activity of Largazole Analogs: Alteration of the Zinc-Binding Domain and Macrocyclic Scaffold 
Organic letters  2009;11(6):1301-1304.
Fourteen analogs of the marine natural product largazole have been prepared and assayed against HDACs 1,2, 3, and 6. Olefin cross-metathesis was used to efficiently access six variants of the side-chain zinc-binding domain, while adaptation of our previously reported modular synthesis allowed probing of the macrocyclic cap group
PMCID: PMC2673910  PMID: 19239241
15.  Polyaminohydroxamic Acids and Polyaminobenzamides as Isoform Selective Histone Deacetylase Inhibitors§ 
Journal of medicinal chemistry  2008;51(8):2447-2456.
A series of polyaminohydroxamic acids (PAHAs) and polyaminobenzamides (PABAs) were synthesized and evaluated as isoform-selective histone deacetylase (HDAC) inhibitors. These analogues contain a polyamine chain to increase affinity for chromatin and facilitate cellular import. Seven PAHAs inhibited HDAC >50% (1 µM), and two PABAs inhibited HDAC >50% (5 µM). Compound 17 increased acetylated α-tubulin in HCT116 colon tumor cells 253-fold but only modestly increased p21waf1 and acetylated histones 3 and 4, suggesting that 17 selectively inhibits HDAC 6. PABA 22 alone minimally increased p21waf1 and acetylated histones 3 and 4 but caused dose-dependent increases in p21waf1 in combination with 0.1 µM 5-azadeoxycytidine. Finally, 22 appeared to be a substrate for the polyamine transport system. None of these compounds were cytotoxic at 100 µM. PAHAs and PABAs exhibit strikingly different cellular effects from SAHA and have the potential for use in combination antitumor therapies with reduced toxicity.
PMCID: PMC3556737  PMID: 18348516
16.  Largazole Arrests Cell Cycle at G1 Phase and Triggers Proteasomal Degradation of E2F1 in Lung Cancer Cells 
ACS Medicinal Chemistry Letters  2013;4(10):921-926.
Aberration in cell cycle has been shown to be a common occurrence in lung cancer, and cell cycle inhibitor represents an effective therapeutic strategy. In this study, we test the effects of a natural macrocyclic depsipeptide largazole on lung cancer cells and report that this compound potently inhibits the proliferation and clonogenic activity of lung cancer cells but not normal bronchial epithelial cells. Largazole arrests cell cycle at G1 phase with up-regulation of the expression of cyclin-dependent kinase inhibitor p21. Interestingly, largazole enhances the E2F1-HDAC1 binding affinity and induces a proteasomal degradation of E2F1, leading to suppression of E2F1 function in lung cancer but not normal bronchial epithelial cells. Because E2F1 is overexpressed in lung cancer tumor samples, these data indicate that largazole is an E2F1-targeting cell cycle inhibitor, which bears therapeutic potentials for this malignant neoplasm.
PMCID: PMC4027503  PMID: 24900585
Lung cancer; cell cycle; largazole; E2F1; degradation
17.  The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells 
Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn++-chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4– but not (R)-2 – caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy.
PMCID: PMC3822689  PMID: 22004558
HDAC inhibitors (HDACi); benzodiazepine (BDZ); acute myeloid leukaemia (AML); cell growth arrest; apoptosis; H2AX; PARP; in vivo toxicity
18.  Structure, Mechanism, and Inhibition of Histone Deacetylases and Related Metalloenzymes 
Metal-dependent histone deacetylases (HDACs) catalyze the hydrolysis of acetyl-L-lysine side chains in histone and non-histone proteins to yield L-lysine and acetate. This chemistry plays a critical role in the regulation of numerous biological processes. Aberrant HDAC activity is implicated in various diseases, and HDACs are validated targets for drug design. Two HDAC inhibitors are currently approved for cancer chemotherapy, and other inhibitors are in clinical trials. To date, X-ray crystal structures are available for four human HDACs (2, 4, 7, 8) and three HDAC-related deacetylases from bacteria (histone deacetylase-like protein, HDLP; histone deacetylase-like amidohydrolase, HDAH; acetylpolyamine amidohydrolase, APAH). Structural comparisons among these enzymes reveal a conserved constellation of active site residues, suggesting a common mechanism for the metal-dependent hydrolysis of acetylated substrates. Structural analyses of HDACs and HDAC-related deacetylases guide the design of tight-binding inhibitors, and future prospects for developing isozyme-specific inhibitors are quite promising.
PMCID: PMC3232309  PMID: 21872466
19.  Loss of Deacetylation Activity of Hdac6 Affects Emotional Behavior in Mice 
PLoS ONE  2012;7(2):e30924.
Acetylation is mediated by acetyltransferases and deacetylases, and occurs not only on histones but also on diverse proteins. Although histone acetylation in chromatin structure and transcription has been well studied, the biological roles of non-histone acetylation remain elusive. Histone deacetylase 6 (Hdac6), a member of the histone deacetylase (HDAC) family, is a unique deacetylase that localizes to cytoplasm and functions in many cellular events by deacetylating non-histone proteins including α-tubulin, Hsp90, and cortactin. Since robust expression of Hdac6 is observed in brain, it would be expected that Hdac6-mediated reversible acetylation plays essential roles in CNS. Here we demonstrate the crucial roles of Hdac6 deacetylase activity in the expression of emotional behavior in mice. We found that Hdac6-deficient mice exhibit hyperactivity, less anxiety, and antidepressant-like behavior in behavioral tests. Moreover, administration of Hdac6-specific inhibitor replicated antidepressant-like behavior in mice. In good agreement with behavioral phenotypes of Hdac6-deficient mice, Hdac6 dominantly localizes to the dorsal and median raphe nuclei, which are involved in emotional behaviors. These findings suggest that HDAC6-mediated reversible acetylation might contribute to maintain proper neuronal activity in serotonergic neurons, and also provide a new therapeutic target for depression.
PMCID: PMC3273475  PMID: 22328923
20.  Design, Synthesis, Biological Evaluation, and Structural Characterization of Potent Histone Deacetylase Inhibitors Based on Cyclic α/β-Tetrapeptide Architectures 
Histone deacetylases (HDACs) are a family of enzymes found in bacteria, fungi, plants, and animals that profoundly affect cellular function by catalyzing the removal of acetyl groups from ε-N-acetylated lysine residues of various protein substrates including histones, transcription factors, α-tubulin, and nuclear importers. Although the precise roles of HDAC isoforms in cellular function are not yet completely understood, inhibition of HDAC activity has emerged as a promising approach for reversing the aberrant epigenetic states associated with cancer and other chronic diseases. Potent new isoform selective HDAC inhibitors would therefore help expand our understanding of the HDAC enzymes and would represent attractive lead compounds for drug design, especially if combined with high resolution structural analyses of such inhibitors to shed light on the three-dimensional pharmacophoric features necessary for the future design of more potent and selective compounds. Here we present structural and functional analyses of a series of β-amino acid-containing HDAC inhibitors inspired by cyclic tetrapeptide natural products. To survey a diverse ensemble of pharmacophoric configurations, we systematically varied the position of the β-amino acid, amino acid chirality, functionalization of the Zn2+-coordinating amino acid side chain, and alkylation of the backbone amide nitrogen atoms around the macrocycle. In many cases, the compounds were a single conformation in solution and exhibited potent activities against a number of HDAC isoforms as well as effective antiproliferative and cytotoxic activities against human tumor cells. High resolution NMR solution structures were determined for a selection of the inhibitors, providing a useful means of correlating detailed structural information with potency. The structure-based approach described here is expected to furnish valuable insights toward the future design of more selective HDAC inhibitors.
PMCID: PMC2751792  PMID: 19239270
beta-amino acids; drug design; cyclic tetrapeptides; histone deacetylase; structure-activity relationship
21.  Potent and Orally Efficacious Bisthiazole-Based Histone Deacetylase Inhibitors 
ACS Medicinal Chemistry Letters  2014;5(6):628-633.
Inspired by the thiazole–thiazoline cap group in natural product largazole, a series of structurally simplified bisthiazole-based histone deacetylase inhibitors were prepared and evaluated. Compound 8f was evaluated in vivo in an experimental autoimmune encephalomyelitis (EAE) model and found to be orally efficacious in ameliorating clinical symptoms of EAE mice.
PMCID: PMC4060935  PMID: 24944733
Histone deacetylase inhibitors; largazole; bisthiazole
22.  Aminoglycoside-induced histone deacetylation and hair cell death in the mouse cochlea 
Journal of Neurochemistry  2009;108(5):1226-1236.
Post-translational modification of histones is an important form of chromatin regulation impacting transcriptional activation. Histone acetyltransferases (HATs), for example, acetylate lysine residues on histone tails enhancing gene transcription, while histone deacetylases (HDACs) remove those acetyl groups and repress gene transcription. Deficient histone acetylation is associated with pathologies, and histone deacetylase inhibitors have been studied in the treatment of cancer and neurodegenerative diseases.
Here we explore histone acetylation in cochlear sensory cells following a challenge with gentamicin, an aminoglycoside antibiotic known to cause loss of auditory hair cells and hearing. The addition of the drug to organotypic cultures of the mouse organ of Corti decreased the acetylation of histone core proteins (H2A Ack5, H2B Ack12, H3 Ack9, and H4 Ack8) followed by a loss of sensory cells. Protein levels of HDAC1, HDAC3 and HDAC4 were increased while the HATs CBP and p300 remained unchanged. We next hypothesized that protecting histone acetylation should prevent cell death and tested the effects of HDAC-inhibitors on the actions of gentamicin. Co-treatment with trichostatin-A maintained near-normal levels of acetylation of histone core proteins in cochlear outer hair cells and attenuated gentamicin-induced cell death. The addition of sodium butyrate also rescued hair cells from damage by gentamicin. The results are consistent with an involvement of deficient histone acetylation in aminoglycoside-induced hair cell death by and point to the potential value of HDAC-inhibitors in protection from the side effects of these drugs.
PMCID: PMC3341988  PMID: 19141081
23.  The Synthesis and Evaluation of N1-(4-(2-[18F]-fluoroethyl)phenyl)-N8-hydroxyoctanediamide ([18F]-FESAHA), A PET Radiotracer Designed for the Delineation of Histone Deacetylase Expression in Cancer 
Nuclear medicine and biology  2011;38(5):683-696.
Given the significant utility of suberoylanilide hydroxamic acid (SAHA) in chemotherapeutic protocols, a PET tracer that mimics the histone deacetylase (HDAC) inhibition of SAHA could be a valuable tool in the diagnosis, treatment planning, and treatment monitoring of cancer. Here, we describe the synthesis, characterization, and evaluation of N1-(4-(2-[18F]-fluoroethyl)phenyl)-N8-hydroxyoctanediamide ([18F]-FESAHA), a PET tracer designed for the delineation of HDAC expression in cancer.
FESAHA was synthesized and biologically characterized in vivo and in vitro. [18F]-FESAHA was then synthesized in high radiochemical purity, and the logP and serum stability of the radiotracer were determined. In vitro cellular uptake experiments and acute biodistribution and small animal PET studies were performed with [18F]-FESAHA in mice bearing LNCaP xenografts.
[18F]-FESAHA was synthesized in high radiochemical purity via an innovative one-pot procedure. Enzymatic inhibition assays illustrated that FESAHA is a potent HDAC inhibitor, with IC50 values from 3 nM to 1.7 μM against the eleven HDAC subtypes. Cell proliferation experiments revealed that the cytostatic properties of FESAHA very closely resemble those of SAHA in both LNCaP cells and PC-3 cells. Acute biodistribution and PET imaging experiments revealed tumor uptake of [18F]-FESAHA and substantially higher values in the small intestine, kidneys, liver, and bone.
The significant non-tumor background uptake of [18F]-FESAHA presents a substantial obstacle to the use of the radiotracer as an HDAC expression imaging agent. The study at hand, however, does present a number of lessons critical to both the synthesis of hydroxamic acid containing PET radiotracers and imaging agents aimed at delineating HDAC expression.
PMCID: PMC3145497  PMID: 21718944
PET; Histone deacetylase; Hydroxamic acid; SAHA; [18F]-FAHA; [18F]-FESAHA
24.  Structural Basis for the Inhibition of Histone Deacetylase 8 (HDAC8), a Key Epigenetic Player in the Blood Fluke Schistosoma mansoni 
PLoS Pathogens  2013;9(9):e1003645.
The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.
Author Summary
Schistosomiasis, a neglected parasitic disease caused by flatworms of the genus Schistosoma, is responsible for hundreds of thousands of deaths yearly. Its treatment currently depends on a single drug, praziquantel, with reports of drug-resistant parasites. Human epigenetic enzymes, in particular histone deacetylases (HDACs), are predominantly attractive inhibitory targets for anti-cancer therapies. Validated scaffolds against these enzymes could also be used as leads in the search for novel specific drugs against schistosomiasis. In our study, we show that Schistosoma mansoni histone deacetylase 8 (smHDAC8) is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity and is therefore a relevant target for drug discovery. The determination of the atomic structures of smHDAC8 in complex with generic HDAC inhibitors revealed that the architecture of the smHDAC8 active site pocket differed significantly from its human counterparts and provided a framework for the development of inhibitors selectively interfering with schistosome epigenetic mechanisms. In agreement, this information enabled us to identify several small-molecule scaffolds that possess specific inhibitory effects on smHDAC8 and cause mortality in schistosomes. Our results provide the proof of concept that targeting epigenetic enzymes is a valid approach to treat neglected diseases caused by eukaryotic pathogens.
PMCID: PMC3784479  PMID: 24086136
25.  Synthesis and HDAC Inhibitory Activity of Isosteric Thiazoline-Oxazole Largazole Analogs 
Bioorganic & medicinal chemistry letters  2013;23(21):10.1016/j.bmcl.2013.06.012.
The synthesis of an isosteric analog of the natural product and HDAC inhibitor largazole is described. The sulfur atom in the thizaole ring of the natural product has been replaced with an oxygen atom, constituting an oxazole ring. The biochemical activity and cytotoxicity of this species is described.
PMCID: PMC3859309  PMID: 24035339

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