Emerging approaches for histone deacetylase inhibitor drug discovery.

Review

Emerging approaches for histone deacetylase inhibitor drug discovery 1.

Introduction

2.

HDACis in the treatment of cancer

3.

HDACis for treatment of neurological diseases

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Kainan University on 04/25/15 For personal use only.

4.

HDACis in inflammatory diseases

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HDACis as an antiviral therapy

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Conclusion

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Expert opinion

Clemens Zwergel, Sergio Valente†, Claus Jacob & Antonello Mai* †,

*Sapienza University of Rome, Department of Drug Chemistry and Technologies, Rome, Italy

Introduction: Histone deacetylases (HDACs) are key players in the mediation of gene expression for both cancerous and noncancerous malignancies. Overexpression of these enzymes has been demonstrated in numerous types of cancer with some enzyme isoforms also involved in neurological, inflammatory and viral pathologies. Hence, the development of HDAC inhibitors (HDACis) represents a promising approach for their treatment. Numerous chemical entities have been studied in the recent years and some of them have reached clinical trials. Areas covered: This review summarizes the recent efforts in the drug development of HDACis and their potential application as therapeutic agents in cancerous, neurological, inflammatory and viral diseases. Expert opinion: The development of novel potent and selective HDACis is ongoing. However, increased scientific effort is needed to aid the fight of specific types of cancerous or noncancerous disease with more selective agents required to avoid side effects during therapy. An interesting therapeutic approach is the use of HDACis in combination with other epigenetic target modulators to combine their therapeutic potential for a synergistic effect. Keywords: cancer, epigenetics, histone deacetylases, inhibitors, neurology, small molecules, viral infections Expert Opin. Drug Discov. [Early Online]

1.

Introduction

Nowadays, more and more connections are discovered between genomic and genetic variations and human diseases leading to a greater understanding of the etiology of various diseases. Seeking for ‘personalized medicines’ for the treatment of cancer, neurological disorders, inflammatory diseases and virus infections, this genomic and genetic information is useful in developing new therapeutic strategies with a strong molecular rationale. Epigenetic aberrations, which are often directly resulting in the loss or gain of function of epigenetic regulatory proteins, are an important research field and contribute substantially to the onset and progression of the abovementioned human diseases [1]. In this article, we present the development of histone deacetylase inhibitors (HDACis) in the context of human diseases that are linked to abnormal expression and/or function of HDACs, as well as their emerging clinical importance. Acetylation and deacetylation are very common post-translational modifications and > 1750 proteins can be acetylated at lysine residues in human cells [2]. These modifications have emerged to be key players in modulating the epigenetic indexing system through targeting transcriptionally active chromatin domains. It is a dynamic system actively influenced by two important families of enzymes, histone acetyl transferases (HATs) and HDACs [3]. HATs catalyze the acetylation of lysine 10.1517/17460441.2015.1038236 © 2015 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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Histone deacetylases (HDACs) are key players in mediating the gene expression involved in cancer and noncancerous malignancies. HDACs are overexpressed in a number of forms of cancer with some isoforms also implicated in other pathologies such as neurological and viral pathologies. There is a large amount of experimental proof demonstrating a direct link between antitumor efficacy and apoptosis induction by HDAC inhibitors (HDACis). HDACis also play a role in other ailments such as neurological disorders, inflammatory diseases and viral infections. A deeper knowledge of the molecular and biological roles of distinct HDACs and HDAC-containing complexes is necessary for the strategic development and rational use of HDACis in the future.

This box summarizes key points contained in the article.

residues to neutralize their positive charges subsequently leading to a relaxed chromatin structure, which is more accessible to the transcription machinery. In contrast, HDACs are responsible for the removal of acetyl groups from histones (and other acetyl-lysine-containing proteins), resulting in chromatin condensation and transcriptional repression [4,5]. These deacetylating enzymes are mainly considered as important targets to reverse aberrant epigenetic changes associated with cancer but also with other diseases such as neurological disorders, inflammation and viral infections [6-8]. HDACs are expressed in all eukaryotic cells, and their activity is crucial for cell proliferation, differentiation and homeostasis. Eighteen different HDACs have been identified in humans so far, and they are classified based on their homology to yeast HDACs. Of these 18 enzymes, 11 contain highly conserved deacetylase domains and are zinc-dependent. To these classes belong: class I (HDACs 1, 2, 3 and 8, yeast RPD3 homologs); class IIa (HDACs 4, 5, 7 and 9), class IIb (HDACs 6 and 10 both with two catalytic sites) and class IV (HDAC 11, has conserved residues shared with both class I and class II deacetylases). The whole class II isoforms are yeast HDA1 homologs. All the aforementioned classes differ in structure, enzymatic activity, localization and tissue expression pattern. Class I HDACs, being expressed in all tissues, display significant HDAC activity primarily in the nucleus where they are present in multiprotein complexes with transcription factors and corepressors [4]. In contrast, class IIa HDACs are tissuespecific, endowed with a low enzymatic activity, and they are believed to have low basal activities with acetyl lysines as well as to be efficient processors of specific, to date unknown natural substrates [9]. Class IIb HDACs are regulating protein folding and turnover thus having primarily non-epigenetic functions [10]. Additionally to the already-mentioned ‘classical HDAC classes’, there are class III HDACs described, which are better known as sirtuins (SIRT 1 -- 7). These enzymes 2

possess a structural relation to yeast protein SIRT-2 and are all NAD+-dependent proteases. Their biological features and functions are well reviewed [11,12]. Although HDACis are mainly studied for their anticancer activity, there is growing evidence ascribing these enzymes to play an important role in other ailments such as neurological disorders, inflammatory diseases and viral infections [13,14]. HDACis possess usually the following well-known pharmacophore: a cap group (CAP) capable of interacting with the rim of the catalytic tunnel of the enzyme, a polar connection unit (CU) connecting to a hydrophobic spacer (HS) allowing the molecule to lie into the aforementioned tunnel and a Zn-binding group (ZBG) capable of complexing the Zn2+ at the bottom of the enzyme cavity [15]. To date, numerous HDACis have been synthesized or isolated as natural products, and these agents have varying target specificity, pharmacokinetic properties and activities in laboratory [16] and clinical settings [17]. Consequently, till date, a lot of different CAP, CU, HS and ZBG combinations have been evaluated as HDACis. As HDACis are inhibiting the deacetylating leading to hyperacetylation of histone and non-histone HDAC substrates, they can induce a range of cellular and molecular effects influencing various human diseases; in the present review, we want to discuss their role and therapeutic potential, mainly in cancer; however, other therapeutic approaches of these inhibitors in neurological diseases, infection and inflammation are also presented (Summary Table 1). 2.

HDACis in the treatment of cancer

Mainly, HDAC inhibition is known and studied for its antitumor effects such as cell death. Large experimental proof showed a direct link between antitumor efficacy and apoptosis induction by HDACis [18]. Another possible mechanism of HDACis is the indirect induction of tumor cell death via immunogenic pathway: The expression of cell surface molecules (e.g., calreticulin) and release of soluble factors (e.g., high-mobility group protein 1 and ATP) leads to an increased presentation of tumor antigens by dendritic cells to T cells [19], possibly resulting in immunogenic cell death with increased tumor clearance [20]. There is evidence that an intact host immune system plays a crucial role in vorinostat (1) and panobinostat (2) treatment to induce sustained anticancer responses against solid and liquid malignancies [21]. Another mode of action initialized by HDACis, such as butyrate (3), is based on their ability to induce cell differentiation [22], with promising evidence to contribute to their anticancer effects [23]. Approved drugs and clinical trials So far only a few HDACis have been approved by the FDA: vorinostat (1) (Zolinza; Merck) for the treatment of refractory cutaneous T-cell lymphoma (CTCL) [24], romidepsin (4) (Istodax; Celgene) for the treatment of CTCL and peripheral 2.1

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Emerging approaches for histone deacetylase inhibitor drug discovery

T-cell lymphoma (PTCL) [25], and belinostat (5) (Beleodaq; Spectrum Pharmaceuticals) for the treatment of PTCL [17]. More than 350 clinical trials [26] have been carried out or are ongoing using HDACis against various human diseases focusing on hematological tumors [27], not only as single therapeutic [28] but also in combination with known chemotherapeutic and other targeted agents [29]. Often clinical trials with HDACis are carried out in pretreated patients in a refractory state of their disease and this might be the reason for the poor efficacy that is seen in many trials [30], supporting the abovementioned hypothesis that an intact immune system is potentially crucial for HDACi response in patients [21]. The pan-HDACi givinostat (6) was leading to the induction of histone and tubulin acetylation in NSCLC cell lines. Furthermore, it downregulated the level of thymidylate synthase, a typical overexpressed enzyme in this type of cancer, leading to both apoptosis and autophagy. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells [31]. Nowadays, there are two opposite foci in HDACi drug design: on the one hand, dual- or multi-targeted inhibitors and, on the other hand, the highly selective inhibitor strategy. The former approach has promising potential for the antitumor therapy based on HDACis. The latter method, which is supposed to elucidate the function of individual HDAC and provide candidate inhibitors with fewer side effects, has been now widely accepted in the literature [8]. Drug combinations HDACis have been and are currently studied in combination with a broad range of therapeutic agents with varying degrees of success [32]. One prominent example of a successful empirical testing approach of HDACis in combination is with DNA-damaging chemotherapeutics [33]. For example, the combination of vorinostat (1) with idarubicin and cytarabine in non-pretreated patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) resulted in a remarkable response rate of 85% [30]. Another possibly successful combination is the one with DNA methyltransferase inhibitors (DNMTis) [33]. However, it remains unclear whether the addition of valproic acid (VPA) 7 actually improves the efficacy of DNMTis [34]. To answer this question, a large Phase II trial is currently ongoing assessing the efficacy of 5-azacytidine with and without entinostat (8) for the treatment of MDS, chronic myeloid leukemia and AML [35]. Another promising rational combination involves an HDACi with a lysine (K)-specific demethylase 1 (LSD1) antagonist. The combination of LSD1 antagonist SP2509 and pan-HDACi panobinostat (2) was synergistically lethal against cultured and primary AML blasts, exhibiting no toxicity. This lethal effect may be explained by greater induction of the levels of proapoptotic proteins p27 and Bcl-2-like protein 11 in AML cells exposed to co-treatment with SP2509 and 2.2

compound 2, as compared with each drug alone consequently providing a promising therapy approach against AML [36]. Furthermore, HDACi in combination with hormone therapy is a promising attempt to repress the transcription and function of estrogen receptors and androgen receptors via modified acetylation patterns [37]. Treatment of cultured breast cancer cells with vorinostat (1) leads to downregulation and reversal of tamoxifen- induced stabilization of the estrogen receptors. In the presence of compound 1, tamoxifen induced apoptosis probably mediated through inhibition of HDAC2 [37]. These promising preclinical results [33] led to a Phase II clinical trial of vorinostat (1) and tamoxifen in patients with hormone therapy-resistant breast cancer with an overall response rate of 19% [37]. For the treatment of prostate cancer based on positive preclinical studies [33], the anti-androgen agent bicalutamide is currently being evaluated together with vorinostat (1) and panobinostat (2) [35]. The combination of HDACis with agents targeting apoptosis proteins is very promising in preclinical evaluations. Considering that HDACis are capable of modulating the expression of key apoptosis proteins, there is a strong rationale to combine them with agonists of death receptors or antagonists of pro-survival proteins. For example, sublethal doses of HDACis such as vorinostat (1) or panobinostat (2) can sensitize tumor cells, but not healthy cells, leading to TNF-related apoptosis-inducing ligand-induced apoptosis in syngeneic murine models of breast cancer and adenocarcinomas [38]. Trichostatin A (TSA) (9) exhibits potent and specific inhibition of mammalian HDAC both in vitro and in vivo. For example, in treating human multidrug-resistant osteosarcoma, HosDXR150 in combination with the DNMTi azacytidine indicates that the acquired growth and survival advantage of these cancer cells is inactivated by simultaneous epigenetic inactivation of both p53-independent apoptotic signals and osteoblast differentiation pathways [39]. Unfortunately, TSA (9) has shown poor results in clinical trials, possibly due to its low biodistribution and rapid metabolism [40]. Other encouraging results have been observed in preclinical combination studies of vorinostat (1) with daunorubicin (a topoisomerase II inhibitor) [41], camptothecin (a topoisomerase I inhibitor) [42], bexarotene (a retinoid X receptor agonist) [43], lovastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) [44], pazopanib (multi-kinase inhibitor) [45], as well as panobinostat (2) with MK-1775 (a Wee1 inhibitor) [46] and azacytidine (a DNMTi) [47]. Class and isoform selective HDACis Specific inhibitors of class IIa HDACs emerged just recently. YK-4-272 (10) is an inhibitor of class II HDACs that inhibits their nuclear import [48]. Notably, this compound restricted tumor cell growth in vivo [48], supporting the idea that nuclear class II HDACs might be necessary for, or at least increase, the enzymatic activity of nuclear class I HDACs. It is known in the literature that class IIa HDACs display a much weaker 2.3


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Table 1. HDAC inhibitors studied in cancer and non cancer diseases.


No

Compound

HDAC classes

Cancer

1

SAHA, Vorinostat (Zolinza)

pan

2

Panobinostat (LBH589)

pan

3

Sodium butyrate

pan

CTCL [24] Combination with idarubicin and cytarabine in non-pretreated patients with AML and MDS [30] with tamoxifen in patients with hormone therapy-resistant breast cancer [37] with bicalutamide prostate cancer [35] with daunorubicin (a topoisomerase II inhibitor) [41], with camptothecin (a topoisomerase I inhibitor) [42], with bexarotene (a retinoid X receptor agonist) [43], with lovastatin (a 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitor) [44], with pazopanib (multikinase inhibitor) [45] TRAIL activator breast cancer and adenocarcinomas [38] Combination with LSD1 antagonist SP2509 in AML [36] with bicalutamide prostate cancer [35] with MK-1775 (a Wee1 inhibitor) [46], with azacytidine (a DNMTi) [47] TRAIL activator breast cancer and adenocarcinomas [38] EBV-driven lymphomas [94]

4

FK228, Romidepsin (Istodax)

pan

CTCL PTCL [25]

5

Belinostat (PDX101) Givinostat Valproic acid

pan

PTCL [17]

pan pan

NSCLC and lung cancer stem cells [31] Increased efficacy of DNMTis [34]

6 7

8

Entinostat (MS275)

1,3

MDS and AML [35] Accumulation of reactive oxygen species [64]

9

Trichostatin A

pan

Osteosarcoma HosDXR150 in combination with azacytidine [39]

10 YK-4-272 11 APHA9

class IIa class II

12 RGFP966 13 BG45 14 Rocilinostat (ACY1215)

3 3 6

Blocks tumor growth in mice [48] Breast cancer, not active in erythroid cells [49] CTCL [50] Myeloma cell lines [51] Multiple myeloma in combination with bortezomib [52]

15 1A12

6

16 PCI-34051

8

Neurological diseases Huntington’s disease [69] No mood-related behavioral changes [80]

Inflammatory diseases Colitis in rodent model [86] Sickle cell disease [26] GVHD [26]

Viral infections

HIV infection [90,91] also in combinations [26]

Sickle cell disease [26] GVHD [26]

Huntington’s disease [69]

HIV infection [90,91] also in combinations [26] EBV-driven lymphomas in combination with ganciclovir [95] Acute gout [88] EBV-driven lymphomas in combination with ganciclovir [94] HIV infection mainly in combinations [26] Activation of latent hepatitis B virus and EBV [95] HIV infection [90,91]

Spinal muscular atrophy alone [79] or in combination with levocarnitine Not active in EBV-driven lymphomas colitis in rodent in combination with model [86] ganciclovir [95] Colitis in rodent model [86]

Alzheimer’s [73]

Contact hypersensitivity and GVHD disease [89]

Cancerous heat shock protein 90 chaperone disruptor [54] T-cell leukemia and lymphoma [55]

AML: Acute myeloid leukemia; CTCL: Cutaneous T-cell lymphoma; DNMTi: DNA methyltransferase inhibitor; EBV: Epstein--Barr virus; GVHD: Graft-versus-hostdisease; HDAC: Histone deacetylase; MDS: Myelodysplastic syndrome; PTCL: Peripheral T-cell lymphoma; TRAIL: TNF-related apoptosis-inducing ligand.

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Table 1. HDAC inhibitors studied in cancer and non cancer diseases (continued). No


17 18 19 20

Compound

HDAC classes

Cancer

C149 MRLB-223 3i T247

8 1,2 1 3

T-cell leukemia and lymphoma [55] In vivo antitumor effects [57] AML, U937 [58] Colon cancer growth inhibition [59]

21 CUDC-907

pan

22 CUDC-101

class I/II

23 Tefinostat (CHR-2845) 24 MC2392

pan

Lymphoma and multiple myeloma in combination with phosphoinositide 3-kinase inhibitor [60] Solid tumors tyrosine kinases EGFR and human epidermal growth factor receptor 2 [61] Esterase-sensitive moiety myeloma cells [62] Acute promyelocytic leukemia HDAC-containing complex with the retinoic acid receptor a [65]

25 4b

3

26 Tubastatin A

6

27 RG2833

3

28 Compound 60

1,2

29 BRD6688

2

30 BRD4884

2

31 FRM-0334

Class I and II Pan Class IIa 6 class I

Neurological diseases

Inflammatory diseases

Viral infections

Increased HIV gene expression [59]

Huntington’s disease [68] Neuroprotection [72] Alzheimer’s treatment [73] Friedreich’s ataxia [75]

32 33 34 35

MPT0G009 MC1568 Tubacin Largazole

Mood stabilization and antidepressant effects [80] Alzheimer’s treatment [56] Alzheimer’s treatment [56] Frontotemporal dementia Arthritis [87]

EBV-driven lymphomas in combination with ganciclovir [95]

HIV [92] Influenza virus [93] EBV-driven lymphomas in combination with ganciclovir [95]

AML: Acute myeloid leukemia; CTCL: Cutaneous T-cell lymphoma; DNMTi: DNA methyltransferase inhibitor; EBV: Epstein--Barr virus; GVHD: Graft-versus-hostdisease; HDAC: Histone deacetylase; MDS: Myelodysplastic syndrome; PTCL: Peripheral T-cell lymphoma; TRAIL: TNF-related apoptosis-inducing ligand.

deacetylating activity than class I HDACs when assessed with standard acetyl-lysine assay [9]. Class IIa HDACs, but not class I or class IIb HDACs, can interplay with trifluoroacetyl-lysine substrates indicating that the natural substrate or substrates for class IIa HDACs still need to be discovered rather than assuming a low catalytic activity [9]. APHA9 (11) increased ¡/(¡ + b) globin expression ratios suggesting that the HDAC complex named nuclear remodeling shuttle erythroid composed of HDAC5, erythroid transcription factor, erythroid Krueppel-like factor and protein kinase R present in human erythroid cells (NuRSERY) may regulate globin gene expression. Since exposure to APHA9 did not affect survival rates or p21 activation, NuRSERY may represent a novel, possibly less toxic, target for epigenetic

therapies of hemoglobinopathies and other disorders such as cancer. For example, compound (11) increased p21 content, inducing apoptosis, in breast cancer cells, but did not induce p21 expression or affect survival of erythroid cells. The lack of apoptosis-inducing effects in erythroid cells observed with this compound suggests that class II-selective HDACis may be more suitable for epigenomic therapy of cancer than class I-selective or pan-HDACis [49]. The HDAC3-selective inhibitor RGFP966 (12) inhibits CTCL cell growth in vitro as well as DNA replication. The inhibition of HDAC3 led to a significant reduction in DNA replication fork velocity within the first hour of drug treatment, thus disrupting DNA replication of the rapidly cycling tumor cells, ultimately leading to cell death. Importantly,


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Figure 1. Schematic representation of the chemical structures of compounds 1 -- 12.

these molecular and biological effects were proven via small interfering RNA-mediated knockdown of HDAC3, supporting genetic evidence for the HDAC3-selective effects of this compound (Figure 1) [50]. BG45 (13), another recent HDAC3-selective inhibitor can induce the death in multiple myeloma cell lines with hyperacetylation and hypophosphorylation of signal transducer and activator of transcription 3 (STAT3). Nevertheless, it could not be demonstrated so far that deregulated STAT3 activity was necessary for the activity of the compound [51]. One of the few HDAC6-specific inhibitors available rocilinostat (ACY-1215) (14) is being evaluated clinically in multiple myeloma in combination with bortezomib, after exhibiting promising preclinical results; this combination resulted in protracted endoplasmic reticulum stress and triggered synergistically apoptosis via activation of caspase-3, caspase-8 and caspase-9 and poly (ADP) ribosome polymerase in multiple myleloma [52]. Multiple myeloma cells are producing a large number of misfolded proteins that need to be eliminated through the HDAC6-regulated aggresome and the 6

proteasome [53]. Dual inhibition of these two pathways leads to the accumulation of defective proteins and subsequent tumor cell death associated with proteotoxicity, thus explaining the mechanistic rationale behind the combination of HDAC6 selective inhibitors and proteasome inhibitors in multiple myeloma. HDAC6 is able to inhibit cancerous heat shock protein 90 (Hsp90) chaperone activities by disrupting Hsp90--p23 interactions. Recently, compound 1A12 (15) was identified as a dose-dependent selective HDAC6 inhibitor disrupting the aforementioned interactions in tumor xenografts mice [54]. There are also HDAC8-specific inhibitors such as PCI-34051 (16) and C149 (17) available. For both compounds, decreased proliferation and induction of apoptosis was observed in T-cell leukemia and lymphoma possibly targeting cohesin, a protein complex that regulates the separation of sister chromatids during cell division. However, it should be considered that the physiological properties and functions of HDAC8 are still poorly understood, questioning the rationale use of HDAC8-selective inhibitors in human disease [55].




To date, isoform selective inhibitors of HDAC1 or HDAC2 are a challenge for medicinal chemists due to their isoform 95% similarity within the catalytic binding domain [56]. However, compounds that preferentially inhibit both of these class I HDACs have been described recently. MRLB-223 (18), an HDAC1 and HDAC2 selective inhibitor, has been demonstrated to link histone hyperacetylation with in vivo antitumor effects using the same apoptotic pathways as vorinostat (1) via the intrinsic apoptotic pathway and both function independently of p53 [57]. Notably, the antitumor effects of this compound were comparable to those observed using the pan-HDACi vorinostat (1) in the same experimental settings [57]. Our research group published a 1,3,4-oxadiazole-containing 2-aminoanilide (3i) (19) as a selective HDAC1 inhibitor. In western blot experiments, compound (3i) (19) increased both histone H3 and a-tubulin acetylation and led to p21 induction, more effectively than vorinostat (1). In several AML cell lines, as well as in U937 cells in combination with doxorubicin, compound (19) showed higher antiproliferative effects than vorinostat (1) [58]. Polypharmacological HDACis Polypharmacological HDACis do not inhibit only HDACs but involve at least one other target. T247 (20) displayed potent selective HDAC3 inhibition with a submicromolar IC50 inducing a dose-dependent selective increase of NF-kB acetylation, a known target of HDAC3, in human colon cancer HCT116 cells leading to growth inhibition [59]. For example, CUDC-907 (21), containing a hydroxamic acid and a morpholinopyrimidine to inhibit both HDACs and phosphoinositide 3-kinase (PI3K) [60], is currently being evaluated in Phase I for the treatment of lymphoma and multiple myeloma (NCT01742988). This compound can downregulate and suppress the activation of the SRC/STAT signaling pathway and multiple receptor tyrosine kinases, again presumably because of its HDAC inhibitory activity, as we observed that panobinostat (2) also induced a similar effect exhibiting greater growth inhibition and proapoptotic activity than single-target PI3K or HDACis in both cultured and implanted cancer cells [60]. Another interesting compound in clinical trials for solid tumors is CUDC-101 (22). This compound inhibits, beside HDACs, the tyrosine kinases EGFR and human epidermal growth factor receptor 2 combining a hydroxamic acid with a phenylaminoquinazoline (NCT01702285, NCT01171924, NCT01384799 and NCT00728793) [26,61]. Tefinostat (CHR-2845) (23), studied in Phase I, contains an esterase-sensitive moiety that can be cleaved after uptake into macrophages and monocytes, where the active form of compound (23) is obtained, leading to apoptosis specifically in myeloma cells [62] but not in lymphoid cells due to doselimiting toxicities [63]. Furthermore, entinostat (8) leads to the accumulation of reactive oxygen species and caspase activation in transformed 2.4

but not normal cells, and increased the levels of an important reducing protein, thioredoxin, only in normal cells [64]. In acute promyelocytic leukemia (APL), the promyelocytic leukemia gene is mediated by an HDAC-containing complex with the retinoic acid receptor a. MC2392 (24) binds to the aforementioned moiety and selectively inhibits the HDACs resident in this repressive complex responsible for the transcriptional impairment in APL (Figure 2) [65].

HDACis for treatment of neurological diseases

3.

HDACis are applied to patients in neurology since many years, even before the molecular targets of these drugs were identified. VPA (7) was approved by the FDA in 1978 as an anticonvulsant drug for the treatment of absences as well as partial and generalized seizure disorders. This drug is still in use today as an anticonvulsant and mood stabilizer in the treatment of various neurological disorders such as epilepsy, bipolar imbalances, depression, schizophrenia and migraine, mainly influencing the neurotransmitter system involving GABA [66]. Later, once the role of acetylation and individual HDACs were discovered in a range of neuropsychiatric disorders, a rational, targeted approach is nowadays more and more applied to investigate HDACis in the abovementioned diseases. Furthermore, HDACis display promising activities in several animal models of neurodegenerative diseases, such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, ischemic stroke, amyotrophic lateral sclerosis and spinal muscular atrophy (reviewed in [67]). Mainly, pan-HDACis have been evaluated, but isoform-specific inhibitors might be more effective and less toxic. For instance, the inhibition of HDAC3 alone with compound 4b (25) prevented Huntington’s disease associated eye neurodegeneration in a Drosophila model as well as a mouse model via the reduction of disease-associated genes such as protein phosphatase 1 regulatory subunit 1B, which encodes dopamine- and cAMPregulated neuronal phosphoprotein-32, a marker for medium spiny striatal neurons [68]. In the same paper Jia et al. described that HDAC1 inhibition displayed positive results in Huntington’s disease [68], comparable to the pan-HDACi vorinostat (1) and butyrate (3) [69]. However, selective HDAC1i should be considered as problematic in neuronal tissue as its inactivation leads to double-stranded DNA breaks, resulting in undesired neuronal cell death [70]. Under oxidative stress conditions via glutathione depletion, broad-spectrum hydroxamate-based HDACi can induce neurotoxicity [71], whereas in contrast the HDAC6-specific inhibitor tubastatin A (26) possesses neuroprotective properties [72]. Furthermore, specific inhibition of HDAC6 with tubastatin A (26) or ACY-1215 (14) can decrease high levels of pathogenic tau ameliorating clinical particularities of tauopathies such as Alzheimer’s disease [73]. Both aforementioned compounds (26) and (14) not only promoted tubulin acetylation, controlled by HDAC6, but also reduced production and


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Figure 2. Schematic representation of the chemical structures of compounds 13 -- 24.

facilitated autophagic clearance of Ab and hyperphosphorylated tau. However, despite their promising initial results, these findings require a deeper evaluation, as HDAC6 might have both neurodegenerative and neuroprotective effects [74]. HDAC3 is strongly involved in the pathogenesis of the neurodegenerative disease Friedreich’s ataxia. This disorder is caused by aberrant heterochromatin formation at the frataxin (FXN) gene locus resulting in FXN repression [75]. The HDAC3-specific inhibitor RG2833 (27) increased FXN expression in clinical samples and ameliorated the disease phenotype in a murine model [76] and is currently evaluated in Phase I clinical trials. Spinal muscular atrophy is linked to a loss of the survival of motor neuron 1 (SMN1) gene resulting in neuromuscular degeneration [77]. A promising therapeutic concept is the activation of the nearly identical SMN2. Specific HDACs are important in the expression regulation of SMN2, although suppressing HDAC2 and HDAC6 can enhance appropriate splicing and expression of this protein [78]. The successful transcriptional activation of this gene has been achieved via the treatment with VPA (7) [79]. Regarding this target VPA (7) and levocarnitine are studied in children with spinal 8

muscular atrophy in advanced clinical trial studies (Phase III) (NCT01671384) [26]. As mentioned above, isoform selective inhibitors of HDAC1 or HDAC2 are a challenge, nevertheless, compound 60 (28) was recently discovered quite selectively inhibiting both of these class I HDACs [80]. Compound (28) as HDAC1/2 selective inhibitor is able to induce mood stabilization and display antidepressant effects via elevated levels of acetylated H4K12, thus leading to an increase of the following mood-related proteins alanine-glyoxylate aminotransferase 2-like 1, serum/glucocorticoid regulated kinase 1, sulfo-transferase family 1A phenol-preferring member 1 and TSC22 domain family member 3 [80]. Of particular interest is that vorinostat did not alter the mood-related behavioral effects, as compound (28) did, as well as the changes in gene expression observed in the brains of mice. Having in mind the other previously mentioned HDAC1/2 selective compound MRLB-223 (18) with comparable antitumor effects regarding vorinostat, the true specificity and selectivity of HDACis and the differential effects of small molecules on HDACs in the recombinant and purified forms compared to physiological conditions need to be carefully examined in further studies [57,80].




The very recently published HDAC2 selective inhibitors BRD6688 (29) and BRD4884 (30) could serve not only as a promising Alzheimer’s treatment and lead structures for the development of isoform-selective HDACi but also as useful tools for probing the biological functions and relevance of the different HDAC isoforms [56]. Both compounds increased H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays, in the hippocampus of CK-p25 mice, a model of neurodegenerative disease, and rescued the associated memory deficits of these mice in a cognition behavioral model. FRM-0334 (31) is currently tested in Phase II clinical trials in patients with frontotemporal dementia with granulin mutation (NCT02149160). However, regarding the diverse roles of HDACs, including side effects in neurobiology and neurological disease, the development of pan-HDACis to treat neurological diseases should proceed with caution [81]. 4.

HDACis in inflammatory diseases

HDACis used in rodent models of inflammatory diseases such as arthritis, inflammatory bowel disease, hypertension, septic shock, colitis and graft-versus-host-disease (GVHD) gave encouraging results via enhanced regulatory T (Treg) cell number and function. During these studies, it was becoming more and more obvious that class II-, class IIa- or isoformspecific inhibitors may be best suited for the treatment of inflammatory diseases [82,83]. Pan-HDACis are probably compromising host defense aggravating atherosclerosis as well as chronic obstructive pulmonary disease [84,85] via inhibition of class I HDACs, which are key repressors of proinflammatory cytokines. PanHDACis such as TSA (9) and vorinostat (1), but not class I-specific HDACis such as MS275 (8) prevented development and promoted the resolution of colitis in rodent models via FOXP3 Treg cells modulated through class II-specific HDACis [86]. Recent studies have demonstrated the effectiveness of HDACi MPT0G009 (32) in preclinical models of inflammatory arthritis, highlighting the potential for the clinical development of these agents in this setting. Compound (32) inhibited cytokine secretion and macrophage colonystimulating factor/receptor activator of NF-kB ligandinduced osteoclastogenesis by macrophages. These effects were decreased by the overexpression of HDAC1 and HDAC6 in cells, thus suggesting that these effects were due to the inhibition of its activity [87]. Butyrate (3) as specific inhibitor of class I HDACs decreased the production of IL-1b, IL-6 and IL-8 leading to potent anti-inflammatory effects in the treatment of acute gout [88]. ACY-1215 (14), an HDAC6 inhibitor, prevented the development of contact hypersensitivity and GVHD-like disease in vivo by modulating CD8 T-cell activation and functions [89].

In the context of immune modulation and inflammation, current clinical trials of HDACis are focused on sickle cell disease using pan-HDACi vorinostat (1) or panobinostat (2) (NCT01245179 and NCT01000155) [26], in which inflammation is a major contributor to disease pathology. Another area current importance in clinical evaluation lies in the treatment and/or prevention of GVHD in the context of stem cell transplantation using the same drugs (compounds 1 and 2) mentioned before (NCT00810602, and NCT01111526) [26]. 5.

HDACis as an antiviral therapy

HDACis have recently gained more and more interest in antiviral therapeutic strategies. The problem to fight HIV is the eradication of latent virus reservoirs. The cells infected with integrated latent provirus can elude pharmacological attacks and further hide themselves from the immune system. HIV production is upregulated in vitro by either class I-selective or pan-HDACis [90]. The reactivation of latent virus reservoirs via HDACis such as panobinostat (2), belinostat (5) and vorinostat (1) could be demonstrated in infected cell lines as well as in resting CD4+ T cells from patients under antiretroviral treatment; these preclinical findings were confirmed in a clinical setting in patients with latent HIV infection [90,91]. Treatment with vorinostat (1) resulted in increased cellular acetylation inducing long terminal repeat promoter expression, important for the viral latency, thus elevating HIV RNA production in CD4+ T cells in patients whose viremia was fully suppressed with antiretroviral therapy. Vorinostat (1), panobinostat (2) and romidepsin (4) are currently being tested in combination with various antiretroviral therapies (NCT01680094, NCT01319383, NCT02336074, NCT02092116) [26]. T247 (20), the previously mentioned selective HDAC3 inhibitor, can also activate HDAC3-controlled HIV-1 gene expression in latent HIV-infected cells [59]. Recently it has been described that selective inhibition of HDAC4 with MC1568 (33) might represent a unique strategy for modulating the expression of therapeutic viral vectors, as well as that of integrated HIV-1 proviruses in latent reservoirs without significant cytotoxicity [92]. Recently it has been demonstrated that HDAC6 inhibition via tubacin (34), a domain-specific inhibitor binding to one of the two HDAC6 catalytic domains, influences influenza virus functions by negatively regulating the trafficking of viral components to the site of influenza virus via the HDAC6 substrate, acetylated microtubules [93]. HDACis can be also used as sensitizers in Epstein--Barr virus (EBV)-driven lymphomas that are not responsive to ganciclovir. Resistance to ganciclovir arises when EBV is unable to express the molecular target of ganciclovir, the EBV thymidine kinase. Butyrate (3) was able to re-express the EBV thymidine kinase first in a preclinical setting in vitro enabling the ganciclovir to fight against EVB [94]. Further studies with panobinostat (2), entinostat (8) or largazole (35) instead of butyrate resulted in a more potent


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N

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Figure 3. Schematic representation of the chemical structures of compounds 25 -- 30, 32 -- 35. The structure of compound 31 is unknown.

combination with ganciclovir in vitro [95]. However, till date, no small molecule, including HDACis, has proven to be safe or effective in clinical trials for treatment of EBV-positive cancers (Figure 3). Even if HDACis may be useful in future treatments for eradicating viruses such as HIV, the risk of activating a latent virus in such infected patients when receiving HDACis for another indication should be carefully considered. For example, latent hepatitis B virus and EBV infections were activated in patients with cancer following treatment with romidepsin (4) [95]. 6.

Conclusion

Numerous parthways involving HDACi and serveral transcription factors associated with HDACs have been and are ongoing to be studied for cancer but also non-cancer therapy. In this review, numerous HDACis in preclinical and clinical settings sometimes in combination with other agents have been evaluated and summarized mainly for cancer; however, in recent literature, it is becoming more and more evident 10

that even other diseases such as neurological disorders, inflammation or viral infections might be ameliorated or even cured. 7.

Expert opinion

Nowadays there is much evidence in the literature that alterations to the epigenome could initiate and prolong various human diseases. Having in mind the dynamic nature of epigenetic modifications, in many cases, these alterations may be reversible through targeting of distinct epigenetic enzymes such as HDACs. There is a rapidly growing understanding of specific HDACs in various disease etiologies through the discovery of the molecular and biological alterations via HDAC inhibition in cancer but also non-cancer diseases such as neurological and inflammatory disorders as well as viral infections. Besides the antiproliferative activity, HDACis are also capable of influencing: i) DNA damage, through downregulation of DNA-repair genes as well as acetylation of DNA-repair proteins; ii) angiogenesis, by decreased expression of pro-angiogenic factors; and iii) immunologic response




and neurodegeneration [13]. Despite the evaluation of numerous HDACis in Phase I -- III clinical trials for mainly hematological malignancies such as CTCL, lymphoma and AML, only three of them (vorinostat [1], romidepsin [4] and belinostat [5]) hit the commercial market with the FDA approval as Zolinza, Istodax and Beleodaq, respectively, for the therapy of CTCL as unique type of cancer. In the case of these approved drugs, treatments of other types of cancer were ineffective, thus we believe that there is an urgent need of novel HDACis and novel innovative therapeutic approaches. In order to address this need, HDACis should be evaluated rather in the context of large multiprotein complexes [96,97] than in the inhibition of independent targets. The first rather naı¨ve initial view on the function of HDACs that there is generally a direct relationship between transcriptional activation and histone hyperacetylation at a given gene locus has been replaced by more sophisticated concept describing a complex interplay of different epigenetically active enzymes regulating gene transcription [98,99]. The most known HDACis target multiple HDACs, which makes it challenging to evaluate whether their biological effects including clinical toxicities are a consequence of inhibition of a specific HDAC, the combined inhibition of multiple HDACs and/or impacts on one or more multiprotein complexes in which HDACs act as key enzymatic components [96]. Especially the latter point is an underappreciated factor that requires further investigation. To address the aforementioned problem as well as to overcome the undesired side effects of pan-inhibitors the discovery of isoform-specific HDACis remains to be urgent and necessary. Because HDACs play an important role in multiple mechanisms, they are particularly interesting to apply rational combination strategies involving either conventional cytotoxic agents or other targeted agents [100]. To date, few details are known about the three-dimensional structures for most human HDAC isoforms, making it difficult to develop selective HDACis. Some promising molecules presented in this review are the first ones selectively inhibiting class I HDACs or HDAC1, HDAC2, HDAC6, HDAC9, HDAC8 subtype, mainly studied not only for cancer therapy but also for neurodegenerative and inflammatory diseases. A good example is HDAC3-selective inhibitor T247 (20),

which is, on the one hand, able to lead to apoptosis in colon cancer cells via HDAC3-activated NF-kB and, on the other hand, is able to activate HDAC3-controlled HIV-1 gene expression in latent HIV-infected cells [59]. However, as we stated above, the true specificity and selectivity of HDACis, such as MRLB-223 (17), are sometimes uncertain, thus raising again the question if the inhibition of multiple HDACs and/or of on one or more multiprotein complexes in which HDACs act as key enzymatic components is leading to the desired biological effects. Even if a lot of novel non-hydroxamate scaffolds, including hybrid polypharmacological molecules are being developed, the not so biologically stable and selectively Zn2+ ion chelating hydroxamic acid moiety remains to be the most studied ZBG up to now. Hence we believe that more attention should be paid to discover novel ZBG combined with better stability, potency and selectivity. Overall, it is evident that a better understanding of the underlying mechanisms of action of HDACis is still necessary and that the inhibiton of one specific HDAC isoform may have important influence on the effect of other agents able to alter the epigenome, for example, DNMTi, histone/protein methyltransferases and histone demethylases inhibitors. However, also off-target effects of HDACis should be carefully considered. In our view, a deeper knowledge of the molecular and biological roles of distinct HDACs and HDAC-containing complexes will certainly promote a more strategic development and rational use of HDACis in the years to come.

Declaration of interest This work was supported by the RBFR10ZJQT FIRB grant, the RF-2010-2318330 grant from the Italian Ministry of the Health, the Sapienza Ateneo Project 2013, the IITSapienza Project, the FP7 Projects BLUEPRINT/282510 and A-PARADDISE/602080. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


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Affiliation

Clemens Zwergel1,2, Sergio Valente†1, Claus Jacob2 & Antonello Mai*1,3 †, *Authors for correspondence 1 Sapienza University of Rome, Department of Drug Chemistry and Technologies, Piazzale Aldo Moro 5, 00185 Rome, Italy Tel: +00390649913392; E-mail: [email protected]; [email protected] 2 Saarland State University, Bioorganic Chemistry, Department of Pharmacy, Campus, Building B 2.1., 66123 Saarbruecken, Germany 3 Sapienza University of Roma, Pasteur Institute - Cenci Bolognetti Foundation, Piazzale Aldo Moro 5, 00185 Rome, Italy


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Histone deacetylase inhibitor for NUT midline carcinoma.

Computational approaches for drug discovery.

Histone Deacetylase Inhibitory Approaches for the Management of Osteoarthritis.

Histone deacetylase inhibitor-polymer conjugate nanoparticles for acid-responsive drug delivery.

Histone deacetylase 3 (HDAC 3) as emerging drug target in NF-κB-mediated inflammation.

Epigenetic approaches for bipolar disorder drug discovery.

Attenuation of choroidal neovascularization by histone deacetylase inhibitor.

Histone deacetylase inhibitor romidepsin inhibits de novo HIV-1 infections.

Electrocardiographic effects of class 1 selective histone deacetylase inhibitor romidepsin.

Histone deacetylase inhibitor panobinostat induces calcineurin degradation in multiple myeloma.

Histone lysine methyltransferases as anti-cancer targets for drug discovery.

Tuberculosis drug discovery and emerging targets.

Groebke-Blackburn-Bienaymé multicomponent reaction: emerging chemistry for drug discovery.

Latest approaches for efficient protein production in drug discovery.

Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5).

Proteasome inhibitor carfilzomib interacts synergistically with histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells.

HDACiDB: a database for histone deacetylase inhibitors.

Histone deacetylase activity assay.

The role of butyrate, a histone deacetylase inhibitor in diabetes mellitus: experimental evidence for therapeutic intervention.

Ku70 is essential for histone deacetylase inhibitor trichostatin A-induced apoptosis.

Histone Deacetylase Inhibitor SAHA as Potential Targeted Therapy Agent for Larynx Cancer Cells.

The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis.

Neuroprotective Functions for the Histone Deacetylase SIRT6.

Combining the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: a potential treatment for BCR-ABL-positive leukemia.