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CONTENTSEDITORIAL: Targeting the Bromome: are we there yet? Future Medicinal Chemistry Vol. 8 Issue 13

REVIEW: Targeting histone deacetylase 8 as a therapeutic approach to cancer and neurodegenerative diseases Future Medicinal Chemistry Vol. 8 Issue 13

REVIEW: Recently discovered EZH2 and EHMT2 (G9a) inhibitors Future Medicinal Chemistry Vol. 8 Issue 13

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Editorial

Targeting the Bromome: are we there yet?

Susanne MüllerStructural Genomics Consortium, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford, OX3 7FZ, UK Tel.: +49 (0)69 798 42714 Fax: + 49 (0)69 798 763 42501 [email protected]

“There are still many worthwhile targets in the bromodomain family; the knowledge and tools acquired in

recent years should help fill in the missing pieces.”

SPECIAL FOCUS y Epigenetic drug discovery

1529Future Med. Chem. (2016) 8(13), 1529–1532 ISSN 1756-891910.4155/fmc-2016-0136 © 2016 Future Science Ltd

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First draft submitted: 1 July 2016; Accepted for publication: 8 July 2016; Published online: 31 August 2016

Keywords: bromodomains • chemical probes • chromatin • epigenetics • open access • target validation

The histone code is a central part of epi-genetic regulation. Post-translational modi-fications, in particular, of the histone tails play an important part in gene regulation, controlling the accessibility to the packaged DNA. Enzymes add or remove specific mod-ifications in response to external stimuli or physiological processes and so-called reader proteins bind to and interpret the modifi-cations of the histone code. In recent years, the role of many of these chromatin pro-teins has been elucidated by the generation of high-quality, well-validated chemical tool compounds ‘probes’; most of them are freely available to the scientific community [1]. Particularly, successful has been the genera-tion of inhibitors to different protein lysine methyltransferases and to a family of histone reader domains, the bromodomains (BRDs), which preferentially bind to acetylated lysine residues, although some BRDs also may r ecognize propionyl or butyryl marks [2].

BRDs have made a remarkable career as drug targets. In less than 6 years, after publi-cation of the first potent inhibitors of the bro-modomain and extra-terminal (BET) family (BRD2, BRD3, BRD4 and BRDT) of BRDs in December 2010, about >16 different BET inhibitors have or are being tested in human clinical trials, mostly in oncology. In addi-tion, about >600 articles have been published elucidating the role of BET family members in biology and disease. The unrestricted availability of probe molecules has had a

major impact on this success story, but, in part, also the central role that BET proteins play in biology- and pathology-regulating linage-specific genes via binding to promoter and enhancer regions certainly contributed to the success story. Nevertheless, with the help of the available probe molecules, this crucial function in gene regulation could be interrogated both on the molecular level and in the whole organism.

Another contributing factor to the success in inhibitor development for this family is the fact that BRDs contain a central deep, largely hydrophobic, acetyl lysine binding pocket, which represents an attractive site for the development of small, cell active mol-ecules. Also other BRDs have successfully been targeted thanks to this highly drug-gable pocket. A conserved asparagine residue located in this binding cavity is essential for acetyl-lysine recognition and serves as an anchor point for almost all available inhibi-tors by forming a hydrogen bond that mim-ics the binding mode of acetylated lysine. However, some BRDs contain a tyrosine, threonine or glutamate residue instead of the otherwise conserved asparagine [3], but nei-ther probes nor recognition sequences against nonasparagine containing BRDs have been reported yet.

Currently, a large number of inhibitors targeting almost all branches of the BRD phylogenetic tree have been described. In characterizing these inhibitors, it has become

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“The human proteome encodes 61 b romodomains, which are present in 46 diverse

nuclear and cytoplasmic proteins.”

1530 Future Med. Chem. (2016) 8(13) future science group

Editorial Müller

apparent that selectivity over the BET, BRD is of vital importance due to the many biological functions of the BET family. The successful targeting of other BRD-containing proteins has elucidated important roles of this protein class in biology and their potential as drug targets. The CBP/EP300 inhibitor I-CBP112 has shown promising results in leukemic cell lines and multiple myeloma [4,5]. Two other CBP inhibitors, SGC-CBP30 and PF-CBP1, demonstrated immu-nomodulatory functions in different models. SGC-CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines in cells derived from ankylosing spondylitis and psoriatic arthritic patients and PF-CBP1 reduced levels of IL-6, IL-1β and IFN-γ in an lipopolysaccharide (LPS) stimulated mouse mac-rophage cell line. Inhibitors disclosed by Constellation Pharmaceuticals (MA, USA) have extended the func-tions of CBP/p300 BRDs to altering the human regu-latory T-cell function and therefore show a potential in immune oncology (reviewed in [6]).

Similarly, promising results have been reported for several BRD9/7 as well as BRD9 selective inhibitors based on different scaffolds. Together these probes point toward an important role for BRD9/7 in oncol-ogy and in inflammatory diseases regulating cytokine expression in type 2 helper T cells as well as LPS-stimulated human monocyte cell lines [1,6]. Before the generation of specific BRD inhibitors, BRD9 has been an underexplored target, but cellular and in vivo stud-ies using a xenograft model of human AML now show the potential of BRD9/7 inhibitors in treatment of this disease [7].

For other bona fide BRD-containing cancer targets the generated inhibitors revealed unexpected out-comes. BRG1/BRM are part of the SWI/SNF chro-matin remodeling complex, and have been implicated in diverse cancers such as hepatocellular carcinoma, myeloma and lung cancer as well as inflammatory diseases [8,9]. However, studies with specific inhibi-tors showed that the BRD is not the relevant domain in these diseases [10] [S Müller, unpublished data], but rather plays a role during development and differen-tiation processes. The inhibitor PFI-3 targeting the BRD of BRG1 and BRM as well as the fifth domain in polybromo (PB1) induces embryonic stem (ES) cell and trophoblast cell differentiation and has also been reported to attenuate adipocyte and myoblast differ-entiation [11,12]. However, the molecule did not inhibit proliferation in any of the cell lines investigated, but it seems that inhibition of the ATPase domain inhib-

iting the helicase activity of BRG1 might be the rel-evant target for the development of an effective strat-egy in breast cancer by regulating gene expression of ABC transporters, which are upregulated in response to treatment with chemotherapeutic drugs [10]. An alternative to targeting the catalytic domains of BRD-containing targets such as BRG1, BRM or ATAD2, another BRD-containing protein linked to many dif-ferent cancer types, might be protag strategies, ‘mark-ing’ a protein for degradation, which makes use of the relative ease to target BRD even if inhibition of the BRD does not result in the desired outcome. Initial results are looking promising, but applicability to the wider class of BRDs has not yet been described [13].

There are now inhibitors for most of the branches of the phylogenetic tree of BRDs available. Inhibitors for BA2A/B have been described as well as for the BRPF family members BRPF1B and BRD1, but no biological effects have been reported yet. Several dual inhibitors, for example, targeting TRIM24 and BRPF1B as well as BPTF and BRD4 have been reported providing start-ing points for selective inhibitors to explore the bio-logical activities of these proteins (for review see [14]). Also BRD1/TAF1 dual inhibitors have recently been generated [15] as well as TAF1/BRD4 inhibitors. TAF1 thereby was found to synergize with BRD4 to control proliferation of cancer cells, making TAF1 an a ttractive epigenetic target in cancers driven by MYC [16].

The human proteome encodes 61 BRDs, which are present in 46 diverse nuclear and cytoplasmic pro-teins [3]. So far for >20 small molecules inhibitors are available, in particular for those targets for which lit-erature information points toward interesting disease links or biological functions. Some of the BRD-con-taining proteins have been associated with biological roles that may trigger the development of specific tool compounds although they may not be obvious drug targets, for example, CECR2 is a protein, which is involved in neural tube closure [17], but the currently available inhibitor is not suitable to explore this in an in vivo setting [15]. BAZ1B has been implicated in neu-rodevelopment and its haploinsufficiency is a likely contributor to the neurological phenotypes in Wil-liams syndrome, a genetic condition that is character-ized by medical problems, including cardiovascular disease, developmental delays and learning disabili-ties. BAZ1B is also involved in regulation of reward behaviors and is upregulated in a specific brain area in response to cocaine and social defeat stress [18]. Learn-ing more about this target via chemical probes may open up new therapeutic avenues for the treatment of cocaine- and stress-related disorders. Another example of an underexplored target is BRD8; siRNA-mediated knockdown of BRD8 has been reported to induce cell

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Targeting the Bromome: are we there yet? Editorial

death or growth delay in colorectal cancer cell lines, and surviving BRD8-knockdown cells were particu-larly sensitive to spindle poisons and a proteasome inhibitor. Targeting BRD8 would therefore potentially improve therapy’s outcome against aggressive/meta-static colorectal cancers [19]. Similarly, for PCAF and the related GCN5L2 no selective inhibitors have been described to explore their potential in inflammatory diseases and oncology [9].

Some of these unexplored targets contain atypical BRDs, in which the conserved asparagine is replaced by another amino acid. The second BRDs of the double BRD-containing proteins BRWD3, WDR9 (BRWD1) and PHIP all contain a threonine instead of the conserved asparagine in the BRD pocket. Mem-bers of the SP100 subfamily the first BRD of PB1 as well as ZMYND11 contain a BRD with a tyrosine instead of the asparagine [3]. Recent structural analy-ses revealed that frequently these BRDs do not bind to chromatin per se, but still contribute to binding by sta-bilizing neighboring domains to attach to chromatin. A frequent arrangement is a PHD–BRD unit found, for example, in members of the SP100 and TRIM family. The PHD-Bromo cassette of SP100C has been shown to bind as a unit to unmethylated H3K4me0 via the PHD domain, but the structural integrity of the PHD domain is enabled by the BRD [20]. Simi-larly, in ZMYND11 association with chromatin is mediated not only by the isolated BRD but also by the PWWP domain to the H3.3K36me3 mark. How-ever, the unit of PHD-BRD-PWWP is responsible for maximum binding and deletion of the PHD or the Bromo domain resulted in a loss of ZMYND11 bind-ing to its target genes [21]. Interestingly, fluorescence recovery after photobleaching (FRAP) experiments have shown that mutation of the tyrosine to phenyl-alanine residue leads to dissociation of ZMYND11 from chromatin in cells with hyperacetylated chro-matin, in accordance with classical BRDs, indicat-ing that targeting this atypical BRD might lead to

displacement of ZMYND11 from chromatin [22]. No potent inhibitors have been generated for any of these targets although some, such as PHIP(2), a potential melanoma target, have been identified as being drug-gable [23]. Together these data indicate that nonclassi-cal BRDs may also indirectly provide a good starting point to target a protein even if they are not the main chromatin i nteraction modules.

In the past 6 years, much has been learned how to design specific inhibitors against BRDs and many BRD-specific libraries are now available in different academic laboratories as well as in industry. It is impor-tant to use this knowledge, and we have now a unique opportunity to learn about a whole family and develop inhibitors also for the less obvious targets or proteins that don’t seem seem attractive with the current knowl-edge. There are still many worthwhile targets in the BRD family; the knowledge and tools acquired in recent years should help fill in the missing pieces.

AcknowledgementsThe author would like to thank S Knapp for carefully reading

the manuscript. The authors apologize to all their colleagues

whose important work could not be directly cited.

Financial & competing interests disclosureThe SGC is a registered charity (No. 1097737) that receives

funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,

Canada Foundation for Innovation, Eshelman Institute for In-

novation, Genome Canada, Innovative Medicines Initiative

(EU/EFPIA; ULTRA-DD Grant No. 115766), Janssen, Merck &

Co., Novartis Pharma AG, Ontario Ministry of Economic Devel-

opment and Innovation, Pfizer, São Paulo Research Foundation

– FAPESP, Takeda, and Wellcome Trust (106169/ZZ14/Z). The

author has no other relevant affiliations or financial involve-

ment with any organization or entity with a financial inter-

est in or financial conflict with the subject matter or m aterials

d iscussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

References1 Brown PJ, Müller S. Open access chemical probes for

epigenetic targets. Future Med. Chem. 7(14), 1901–1917 (2015).

2 Flynn EM, Huang OW, Poy F et al. A subset of human bromodomains recognizes butyryllysine and crotonyllysine histone peptide modifications. Structure 23(10), 1801–1814 (2015).

3 Filippakopoulos P, Picaud S, Mangos M et al. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149(1), 214–231 (2012).

4 Picaud S, Fedorov O, Thanasopoulou A et al. Generation of a selective small molecule inhibitor of the CBP/p300 bromodomain for leukemia therapy. Cancer Res. 75(23), 5106–5119 (2015).

5 Conery AR, Centore RC, Neiss A et al. Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. eLife 5, pii: e10483 (2016).

6 Theodoulou NH, Tomkinson NC, Prinjha RK, Humphreys PG. Clinical progress and pharmacology of small molecule bromodomain inhibitors. Curr. Opin. Chem. Biol. 33, 58–66 (2016).


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7 Martin LJ, Koegl M, Bader G et al. Structure-based design of an in vivo active selective BRD9 inhibitor. J. Med. Chem. 59(10), 4462–4475 (2016).

8 Barbieri I, Cannizzaro E, Dawson MA. Bromodomains as therapeutic targets in cancer. Brief. Funct. Genomics 12(3), 219–230 (2013).

9 Muller S, Filippakopoulos P, Knapp S. Bromodomains as therapeutic targets. Expert Rev. Mol. Med. 13, e29 (2011).

10 Wu Q, Sharma S, Cui H et al. Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells. Oncotarget doi:10.18632/oncotarget.8384 (2016) (Epub ahead of print).

11 Fedorov O, Castex J, Tallant C et al. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance. Sci. Adv. 1(10), e1500723 (2015).

12 Gerstenberger BS, Trzupek JD, Tallant C et al. Identification of a chemical probe for family VIII bromodomains through optimization of a fragment hit. J. Med. Chem. 59(10), 4800–4811 (2016).

13 Winter GE, Buckley DL, Paulk J et al. drug development. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348(6241), 1376–1381 (2015).

14 Theodoulou NH, Tomkinson NC, Prinjha RK, Humphreys PG. Progress in the development of non-bet bromodomain chemical probes. ChemMedChem 11(5), 477–487 (2016).

15 SGC: Chemical Probes. www.thesgc.org/chemical-probes

16 Sdelci S, Lardeau CH, Tallant C et al. Mapping the chemical chromatin reactivation landscape identifies BRD4-TAF1 cross-talk. Nat. Chem. Biol. 12(7), 504–510 (2016).

17 Fairbridge NA, Dawe CE, Niri FH, Kooistra MK, King-Jones K, Mcdermid HE. Cecr2 mutations causing exencephaly trigger misregulation of mesenchymal/ectodermal transcription factors. Birth Defects Res. A Clin. Mol. Teratol. 88(8), 619–625 (2010).

18 Sun H, Martin JA, Werner CT et al. BAZ1B in nucleus accumbens regulates reward-related behaviors in response to distinct emotional stimuli. J. Neurosci. 36(14), 3954–3961 (2016).

19 Yamada HY, Rao CV. BRD8 is a potential chemosensitizing target for spindle poisons in colorectal cancer therapy. Int. J. Oncol. 35(5), 1101–1109 (2009).

20 Zhang X, Zhao D, Xiong X, He Z, Li H. Multifaceted histone H3 methylation and phosphorylation readout by the plant homeodomain finger of human nuclear antigen Sp100C. J. Biol. Chem. 291(24), 12786–12798 (2016).

21 Harter MR, Liu CD, Shen CL et al. BS69/ZMYND11 C-terminal domains bind and inhibit EBNA2. PLoS Pathog. 12(2), e1005414 (2016).

22 Philpott M, Rogers CM, Yapp C et al. Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching. Epigenetics Chromatin 7, 14 (2014).

23 Vidler LR, Brown N, Knapp S, Hoelder S. Druggability analysis and structural classification of bromodomain acetyl-lysine binding sites. J. Med. Chem. 55(17), 7346–7359 (2012).

www.thesgc.org/chemical-probes


Review

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Targeting histone deacetylase 8 as a therapeutic approach to cancer and neurodegenerative diseases

Alokta Chakrabarti1, Jelena Melesina2, Fiona R Kolbinger3, Ina Oehme3, Johanna Senger1, Olaf Witt3,4, Wolfgang Sippl2 & Manfred Jung*,1,5

1Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 2Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany 3Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Centre (DKFZ), Heidelberg, Germany 4Department of Pediatric Oncology, Hematology & Immunology, University Hospital Heidelberg, Heidelberg, Germany 5German Cancer Consortium (DKTK), Freiburg, Germany *Author for correspondence: Tel: +49 761 203 4896 Fax: +49 761 203 6321 [email protected]



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Histone deacetylase 8 (HDAC8), a unique class I zinc-dependent HDAC, is an emerging target in cancer and other diseases. Its substrate repertoire extends beyond histones to many nonhistone proteins. Besides being a deacetylase, HDAC8 also mediates signaling via scaffolding functions. Aberrant expression or deregulated interactions with transcription factors are critical in HDAC8-dependent cancers. Many potent HDAC8-selective inhibitors with cellular activity and anticancer effects have been reported. We present HDAC8 as a druggable target and discuss inhibitors of different chemical scaffolds with cellular effects. Furthermore, we review HDAC8 activators that revert activity of mutant enzymes. Isotype-selective HDAC8 targeting in patients with HDAC8-relevant cancers is challenging, however, is promising to avoid adverse side effects as observed with pan-HDAC inhibitors.

First draft submitted: 1 June 2016; Accepted for publication: 19 July 2016; Published online: 30 August 2016

Keywords: cancer • druggable • HDAC8 • hydroxamic acid • inhibitor • SMC3 • stem cell • T-cell • therapy

The past decade has witnessed an increased attention to the development of targeted can-cer therapies. The vast understanding of the molecular dynamics in cancer cells and their difference in normal cells has triggered thor-ough investigations of ‘cancer-specific molec-ular targets’ and treatment strategies [1]. Besides compounds that target specific onco-genes, small molecules, which influence gene transcription, are currently in the limelight as new modalities for cancer therapy. Examples for drugs of this category are histone deacet-ylase (HDAC) inhibitors, such as vorinostat suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor to be approved for clinical use by US FDA for the treatment of refractory cutaneous T-cell lymphoma [2].

HDACs remove acetate from acety-lated ε-amino groups of lysines in histones. Removal of acetyl groups causes global condensation of chromatin and suppres-sion of gene expression [3] and HDACs play

an important role in epigenetic regulation. Such epigenetic modifiers, called ‘erasers’ [3], often occur in multiprotein complexes [4]. HDACs are also termed lysine deacetylases (KDACs), since these enzymes also remove acetyl groups from lysines of numerous nuclear and cytosolic proteins, affecting both gene transcription and cellular signaling [5,6]. Disturbances in HDAC normal expression or activity result in aberrant cellular func-tions [7], thus, associating them with a mul-titude of diseases [8]. Therefore, HDACs are emerging as stimulating molecular targets for t herapeutic intervention, including cancer.

Four classes of HDACs are known, classes I–IV that constitute 18 different isotypes which are either zinc (classical HDACs: I, II and IV) or nicotinamide adenine dinucleo-tide (NAD, Sirtuins: III) dependent [9]. All the FDA-approved HDAC inhibitors are broad-spectrum HDAC inhibitors, hitting more than one of the isotypes. Because of



Review Chakrabarti, Melesina, Kolbinger et al.

the associated adverse side effects of pan inhibitors, recently, the focus has shifted to specific isotypes. HDAC8, a class I isotype, is gaining importance due to its prominent role in specific cancer subtypes as T-cell lymphoma, childhood neuroblastoma and other ail-ments as X-linked intellectual disability and p arasitic infections [10–13].

HDAC8 is a Zn2+-dependent class I HDAC, identi-fied almost 15 years ago as a 42 kDa protein contain-ing 377 amino acids (aa) [14–16]. Found both in nucleus and cytoplasm [17,18], many of the cellular deacety-lation targets of HDAC8 identified so far are known to localize into the nucleus (e.g., AT-rich interactive domain-containing protein 1A, estrogen receptor alpha, hEST1B and structural maintenance of chro-mosome 3 [SMC3]) [19–22]. It is an X-linked protein in human [14] and had diverged early from other class I members implying significant functional specializa-tion [9]. Indeed, the independence of cofactors for activity and the absence of the protein binding domain reflect specific characteristics of HDAC8 not present in other members of this class [14–16]. Some active site fea-tures such as the highly flexible L1 loop, the conserved aspartate 101 and a regulation by serine 39 phosphory-lation add to the discrete functional specialization of HDAC8 [23,24]. Hence, mutations to HDAC8 active site lead to loss of activity and are for example, linked with Cornelia de Lange syndrome (CdLS) [19,25,26].

One fundamental epigenetic role of HDAC8 is the control of the skull morphogenesis. Haberland et al. showed that deletion of HDAC8 leads to perinatal lethality in mice due to skull instability [27]. Further fascinating roles of HDAC8 are still in discovery. Whether in bone differentiation [28] or repression of interleukin β [29] or toxin-induced resistance of mac-rophages [30], HDAC8 reveals novel functions in each case. HDAC8 has been implicated in viral infec-tions [31] and in parasitic disease models HDAC8 inhi-bition reduced infection load [13,32]. Very distinctively, HDAC8 is correlated to T-cell lymphoma, childhood neuroblastoma and gastric cancer [33,34]. This review aims to discuss the therapeutic intervention of HDAC8 in cancer and the available inhibitors for isotype spe-cific targeting in HDAC8 relevant ailments. A short section on HDAC8 activators is also presented.

Deacetylase activity & interaction partnersAlthough histones are viewed as the classical HDAC substrates, there is ample proof on nonhistone sub-strates [3,6]. For HDAC8, there is conflicting evidence concerning histones as bonafide in vivo deacetylation substrates [35]. While there is data that hyperacety-lation results as a consequence of HDAC8 inhibitor treatment [36] or overexpression leads to low acetyla-

tion [15,37], sophisticated MS analyses have failed to detect histones as HDAC8 substrates [22,38]. There is a possibility that HDAC8 might prefer deacetylation of only very particular sites, which could be masked by global acetylation patterns [39] or likely be depen-dent on the cell type [40]. However, in vitro, deacety-lation of histone variants like H2A/H2B, H3 and H4 by HDAC8 has been observed [14,16]. Even short peptides derived from histones are excellent in vitro substrates and knowledge on deacetylation sites in such peptides has contributed to the understanding of sequence specificity for HDAC8 deacetylation sites, for example, deacetylation occurs at lysines 14, 16, 20 on histone H4-derived peptides [14,16,41] and K(Ac)RHR is a p referred motif in this histone subtype [42].

Besides histones, a large number of nonhistone can-didates have been recognized as substrates or interac-tion partners of HDAC8. SMC3 [19], ERRα [20] and p53 [43] have been shown by RNAi or pharmacological inhibition of HDAC8 to be direct cellular substrates with clear concentration-dependent hyperacetylation effects. Mass spectrometric analysis of cellular lysates after treatment with HDAC8 inhibitors has estab-lished several novel proteins as retinoic acid-induced 1, zinc-finger Ran binding domain-containing protein 2, AT-rich interactive domain-containing protein 1A, nuclear receptor coactivator 3 and thyroid hormone receptor-associated protein 3 as HDAC8 deacetylation targets [22]. Very recently, four new members have joined the known substrates: peroxiredoxin 6, pho-shoglycerate mutase 1, high mobility group protein B1 and Parkinson protein 7 [38]. Peptide arrays have revealed an even broader range of acceptable range of substrates, indicating further physiological substrates to be discovered [44]. Other proteins, such as inv(16) fusion protein [45], CREB [46], DEC1 [47], Hsp20 [48], human ever-shorter telomeres 1B (hEST1B) [21] and α-actin [18] have been found to be associated with HDAC8. However, it is unclear whether these proteins are direct acetylation targets or form a part of com-plex in which HDAC8 acts as a scaffold. For instance, HDAC8 coimmunoprecipitated with both CREB and protein phosphatase 1; ectopic expression of HDAC8 decreased CREB activity [46]. Similarly, HDAC8 colo-calized and immunoprecipitated with smooth muscle myosin heavy chain and possibly connects to inv(16) fusion protein via this domain [45]. In this case, phar-macological inhibition with trichostatin A affected transcriptional activities of the inv(16) fusion pro-tein [45]. Both for CREB and inv(16), such evidence suggests HDAC8 can interact with these proteins as deacetylation substrate or as members of a cooperative complex. More clues for scaffolding function come from interactions with α-actin [18]. Colocalization of


Targeting HDAC8 as a therapeutic approach to cancer & neurodegenerative diseases Review

HDAC8 with α-actin and subsequent knockdown effects in human smooth muscle cells (including phe-notypic changes such as smaller cells with loss of con-tractibility or spreading) without changes in global α-actin acetylation points toward a scaffolding behav-ior of HDAC8 rather than deacetylase activity in those cases [49]. Furthermore, DNA-ChIP analysis demon-strated a colocalization of HDAC8 with the transcrip-tion factor DEC1 [47]. Modulation of HDAC8 expres-sion affected DEC1- and DEC1-regulated TAp73 transcription factor [47] implying a recruiting role of DEC-1 with HDAC8, but, it remains unclear whether HDCA8 catalyzes the deacetylation of DEC1. In a cel-lular context, dissection of deacetylation and scaffold-ing functions for HDAC8 is difficult. Most probably, both functionalities are intertwined and studies with catalytically dead mutants could provide mechanistic clues about the mystery behind HDAC8 physiology. However, the likelihood that many HDAC8 substrates could be shared by other HDACs complicates such mechanistic approaches. Moreover, nonepigenetic roles of HDAC8 are also possible. Recent studies demon-strating an ability of HDAC8 to control Notch stabil-ity [50] or binding to miRNA miR-216b [34] point to an ever-increasing complexity of the role of HDAC8 in cells.

HDAC8 structure & inhibitorsAssociation of HDAC8 with a large number of diverse proteins (stated previously) leaves no doubt that it mediates crucial signaling events in human pathophys-iology. Considering the wide range of interaction part-ners, HDAC8 is a key enzyme in cancer, CdLS, virus and parasite infection (see review [51]), pointing out its great potential as a therapeutic target. This raises the question whether we can create drug molecules to selectively inhibit the enzyme and envisage a cure for the subset of diseases that depend on HDAC8 activ-ity. For this purpose, a closer look at HDAC8 crystal structure sheds light on the druggability of this HDAC isotype.

Currently (end of 2015) 50 x-ray structures of human HDACs and six structures of Schistosoma man-soni HDAC8 are stored in the Protein Data Bank, giving structural insight into zinc-dependent HDACs (Table 1). This information allows the comparison between HDAC8 and other HDAC isotypes. Structur-ally, HDAC8 has conserved features similar to those of other representatives of the family. First of all, it shares a common architecture of the HDAC catalytic domain, the core of which consists of eight-stranded β-sheets surrounded by α-helices. These secondary structure elements are connected by loops [35,52], seven of which form the substrate binding pocket with the

zinc ion at the bottom [53], where all cocrystallized inhibitors bind [52] (Figure 1A). Second, the binding pocket of HDAC8 has conserved residues [52], which are observed among all currently solved crystal struc-tures of HDACs and adopt highly similar side chain conformations. In particular, the zinc ion coordi-nating residues (corresponding to D178, D267 and H180 in hHDAC8), two histidines, coordinating the hydroxamic acid of SAHA in hHDAC8 PDB ID 1T69 (H142 and H143) and phenylalanine forming the wall of the lysine binding channel (corresponding to F208 in hHDAC8) are all conserved [53] (Figure 1B). The structural similarities of HDACs explain their prefer-ences to similar ligands and pose a major challenge to the design of HDAC8-selective compounds.

Fortunately, HDAC8 exhibits a number of unique characteristics allowing the development of selective inhibitors. This makes HDAC8 an attractive target for structure-based drug design. One particularly interest-ing feature in this regard is the formation of the foot pocket (also called acetate release channel). This sub-pocket is observed in class I HDACs (HDAC1, 2, 3 and 8), but not in class II isotypes (HDAC4 and 7) x-ray structures. Its presence or absence is caused by a different conformation of the loop L3 and HDAC-class specific differences in the aa composition in these two classes of HDACs, as shown for HDAC8 and HDAC7 in Figure 1C. This binding pocket shape dif-ference explains why class I HDACs are able to accom-modate ligands with large scaffolds occupying the foot pocket (like substituted ortho-anilinobenzamides or aa derivatives), while class IIa HDACs prefer compact groups, filling the small cavity near the zinc ion (tri-fluoromethylketones, trifluoromethyloxadiazoles) [54]. In all x-ray structures of HDAC4 and HDAC7 the entrance to the foot pocket is closed due to alterna-tive conformation of the loop L3 site, preceding the conserved histidines discussed previously (H142 and H143 in hHDAC8) (Figure 1C). This part of the loop is formed by the same residues (glycine and two prolines) in all class IIa HDACs, suggesting that probably none of them form the foot pocket. Of note, S. mansoni HDAC8 has structural differences, allowing for spe-cies-selective HDAC8 inhibitors [13,32]. Comparison of the gatekeeper residues, opening the foot pocket in human class I HDACs, shows that HDAC8 stands out from the other representatives. Instead of the leucine residue (corresponding to L144 in hHDAC2) observed in HDAC1, 2 and 3, HDAC8 has a bulkier trypto-phan residue (W141). This aa substitution changes the shape of the pocket (Figure 1D) and causes preferences to d ifferent ligands.

Another characteristic structural feature of HDAC8 is the presence of the side pocket. Both human and S. man-

Figure 1. Histone deacetylase 8 crystal structures. Similarities (A, B) and differences (C–E) between HDAC8 and other histone deacetylases. (A & B) Superimposed all currently available 56 x-ray structures of human and parasitic HDACs (hHDAC1, hHDAC2, hHDAC3, hHDAC8, smHDAC8, hHDAC4 and hHDAC7). The zinc ion (orange ball) and the ligand (SAHA, gray sticks) are shown for hHDAC8 PDB ID 1T69 to define the binding pocket. (A) Common catalytic domain in all HDACs, built by conserved β-sheet (yellow ribbons) surrounded by α-helices (red ribbons) and flexible loops. Loops (or their parts), forming the binding pocket, are colored (L1 – cyan, L2 – turquoise, L3 – blue, L4 – lime, L5 – dark green, L6 – magenta, L7 – deep purple) and the others are white. (B) Conserved binding pocket residues (dark yellow sticks) are adopting similar side chain conformations in different HDACs. The numbers of residues are given only for hHDAC8 for clarity. The loops, bearing the conserved residues are shown as ribbons and colored in the same way as in (A): L3 – blue, L4 – lime, L5 – dark green and L6 – magenta. (C–E) Differences between HDAC8 (cyan residues, L3 and surface of the binding pocket, turquoise ligand) and other HDAC isotypes (white residues, L3 and surface of the binding pocket, gray ligand). Zinc ions are shown as orange ball. Only residues, changing the binding pocket shape dramatically are shown for clarity. (C) Comparison of hHDAC8 (PDB ID 3SFH) and hHDAC7 (PDB ID 3ZNS) – foot pocket in hHDAC8 is opened by the movement of the gatekeeper residue W141, while in hHDAC4 it is closed because of the different orientation of the L3. (D) Comparison of hHDAC8 (PDB ID 3SFH) and hHDAC2 (PDB ID 4LY1) – foot pocket is opened in both isotypes and the orientation of L3 is the same, but the gatekeeper residue in hHDAC8 (W141) is bigger than in hHDAC2 (L144), which restricts the space in this area. (E) Comparison of hHDAC8 (PDB ID 2V5X) and hHDAC2 (PDB ID 4LXZ) – side pocket is observed in hHDAC8, but is closed in hHDAC2, which is caused by a closer proximity of the gatekeeper residues in hHDAC2 (F155 and L276) than in hHDAC8 (F152 and M274). HDAC: Histone deacetylase; SAHA: Suberoylanilide hydroxamic acid.

H142

D178

D267

F208L3

L5

L1

L7

L6L4

L3

L3 L3

L6

L4

Zn2+



soni HDAC8 demonstrate opening of this subpocket, while in other class I HDACs (hHDAC1–3) it is not observed [53]. This explains the preference of HDAC1–3 to rod-shaped SAHA-like ligands with extended alkyl chain linkers, while HDAC8 is more likely inhibited by compounds with bulky linkers (e.g., phenylene spacers), which do not fit well to the narrow binding pocket of the HDAC1–3 isotypes [10,55,56]. A comparison between x-ray structures of hHDAC8 and hHDAC2 shows the

structural reason of the side pocket formation. Due to a different conformation of the loop L6, the gatekeeper residues in hHDAC8 (F152 and M274) are placed more apart than in hHDAC2 (F155 and L276), causing the formation of the cavity (Figure 1E). To conclude, the comparison of available crystal structures of different HDACs indicates that HDAC8 has a number of unique features discriminating it from other isotypes and mak-ing it a druggable target with the possibility to create



potent and isotype-selective inhibitors.

Role of HDAC8 in oncogenesisHDAC8 appears to be an attractive anticancer target, since in some tumor entities HDAC8 seems to play an explicit tumor relevant role (Table 2). Specifically, HDAC8 is expressed in colon, breast, lung, hepatocel-lular carcinoma, gastric cancer, pancreas tumor tissue, metastatic melanoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) as well as in child-hood tumors of the nervous system, such as neuro-blastoma (Figure 2) [33,43,57–59]. Although HDAC8 is also expressed in corresponding normal tissue, it is by trend higher expressed in cancer tissues [43,57] and displays a significant upregulation in aggressive stage 4 neuroblastomas [58]. In invasive breast tumor cells, HDAC8 is among the three HDAC family members that are upregulated and driving invasiveness [60]. Vice versa, the RNAi-mediated knockdown of HDAC8 expression in human lung, colon, leukemia, gastric adenocarcinoma and cervical cancer cell lines inhib-its tumor cell proliferation [33,61,62]. Upregulation of HDAC8 promotes proliferation and inhibits apoptosis in hepatocellular carcinoma [43] and gastric cancer [33]. HDAC8 expression itself can be regulated by the lipo-genic transcription factor sterol regulatory element binding protein-1 and the SOX-family of transcrip-tion factors. SREBP-1 links lipid metabolism, insulin resistance and cancer development and has been shown to directly upregulate HDAC8 in models of nonalco-holic fatty liver disease-associated hepatocellular car-cinoma [40]. Microarray analyses of adult T-cell leuke-mia/lymphoma revealed that HDAC8 expression in these tumor cells is regulated by SOX4, which directly activates the HDAC8 promoter [62]. SOX4 is a tran-scription factor, which is required for B-lymphocyte development [63], but also during development of the sympathetic nervous system [64,65].

So far, several mechanisms of action are described for HDAC8. In colon cancer, for example, the Bcl-2-mod-ifying factor (BMF) has been identified to be a direct target gene of HDAC8 repression and HDAC8 dere-cruitment is sufficient to activate the target gene [73]. Furthermore, STAT3 associates and cooperates with HDAC8 to repress BMF transcription [73]. BMF has an important function in the execution of apoptosis triggered by the HDAC inhibitory metabolite meth-ylselenopyruvate, which is a competitive inhibitor of HDAC8 [73,74]. In addition, HDAC8 has been impli-cated in contributing to tumorigenesis by regulating telomerase activity [21]. In AML, the inv(16) fusion protein associates with HDAC8 in order to repress the transcription of AML1-regulated genes [45]. Sev-eral studies describe a link between HDAC8 and the tumor suppressor p53. The interaction of inv(16) with HDAC8, for example, causes HDAC8-mediated deacetylation and inactivation of p53 in AML leuke-mia stem cells which promotes tumor cell transfor-mation [69]. Yan et al. discovered that the knockdown of HDAC8 results in repression of HoxA5 and con-sequently to decreased HoxA5-dependent expression of wild-type as well as mutant p53 [82]. p53 expres-sion can be rescued by enforced expression of HoxA5. Conversely, the ectopic expression of HDAC8 enables enhanced transcription of p53 [82]. As HDAC8 is required for p53 expression, regardless whether wild-type or mutated, inactivation of HDAC8 may be more effective in tumor cells harboring a p53 mutation. Results with the HDAC8 activator TM-2–51 illustrate another level of complexity. The treatment of p53 wild-type SH-SY5Y neuroblastoma cells with the activator induces apoptosis in these cells, whereas BE(2)-C neu-roblastoma cells, harboring mutated p53 [83] respond well to HDAC8 inhibitor treatment with growth arrest and differentiation [77]. This supports the hypoth-esis that activation of HDAC8 might be profitable

Table 1. Resolved crystal structures of histone deacetylases from human and Schistosoma mansoni.

Organism HDAC PDB ID Number of x-ray structures

Human hHDAC1 4BKX 1

hHDAC2 3MAX, 4LXZ, 4LY1 3

hHDAC3 4A69 1

hHDAC8 1T64, 1T67, 1T69, 1VKG, 1W22, 2V5W, 2V5X, 3EW8, 3EWF, 3EZP, 3EZT, 3F06, 3F07, 3F0R, 3MZ3, 3MZ4, 3MZ6, 3MZ7, 3RQD, 3SFF, 3SFH, 4QA0, 4QA1, 4QA2, 4QA3, 4QA4, 4QA5, 4QA6, 4QA7, 4RN0, 4RN1, 4RN2

32

hHDAC4 2VQJ, 2VQM, 2VQO, 2VQQ, 2VQV, 2VQW, 4CBT, 4CBY 8

hHDAC7 3C0Y, 3C0Z, 3C10, 3ZNR, 3ZNS 5

Schistosoma mansoni smHDAC8 4BZ5, 4BZ6, 4BZ7, 4BZ8, 4BZ9, 4CQF 6

HDAC: Histone deacetylases; PDB: Protein database.

Figure 2. Histone deacetylase 8 expression in normal and tumor cells. R2 MegaSampler [66] with HDAC8 probe 223345_at. (A) HDAC8 expression of normal B cells (N1–2, blue), leukocytes (N3, blue) and expression in AML (1–8, green) and ALL (1–5, orange). (B) Normal (blue) colon (N1) expression compared with expression in colon cancer (T Colon 1–15, red). (C) Normal neural crest (N1, blue) expression compared with expression in neuroblastoma (1–4). (D) Normal neural crest (N1, blue) expression compared with expression in neuroblastoma INSS stage 4 and stage 4s samples of the Versteeg (1), Delattre (2), Hiyama (3) and Latowska (4) cohort. ALL: Acute lymphocytic leukemia; AML: Acute myeloid leukemia; HDAC8: Histone deacetylase 8.

0

1

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4

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6

7

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2 21 3 5 7 1 5

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1 41 5 7 9 11 152 3 6 8 10 141312

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under p53 wild-type conditions, whereas inhibition of HDAC8 is more reasonable in tumors carrying a mutant p53 gene. However, further studies are neces-sary to fully unravel the mechanistic link, since some tumor entities do not follow that model. For example, knockdown of HDAC8 expression elevates the expres-sion and acetylation of p53 in hepatocellular carcinoma cells, resulting in decreased cell proliferation and acti-vation of apoptosis [40,43]. Additionally, further mecha-nistic studies are necessary to fully understand the link of the p53–HDAC8–HoxA5 axis, initially described for adult cancer cells [82], in childhood neuroblastoma. Besides the regulation of p53, HDAC8 is also involved in the regulation of another transcription factor of the p53 family, p73. This transcription factor also plays a key role in many biological processes, such as neu-ronal development and tumorigenesis [84]. The trans-

activating isoform TAp73 has prodifferentiating roles and induces cell cycle arrest as well as apoptosis. In contrast, the aminoterminal truncated ΔNp73 isoform has prosurvival functions and favors oncogenic trans-formation [85]. HDAC8 is required for the transcrip-tion factor DEC1 to enhance TAp73 expression [47]. DEC1 interacts with HDAC8 and recruits HDAC8 to the TAp73, but not the ΔNp73, promoter [47]. DEC1 in turn, is a target of the p53 family [86].

HDAC8 inhibitors as anticancer therapeuticsThe specific involvement of HDAC8 in cancers such as leukemia and childhood neuroblastoma indicates great therapeutic potential. Especially in neuroblas-toma and T-cell lymphoma, both entities with a clear correlation of HDAC8 activity with the disease, spe-cific targeting of HDAC8 might present a rationale to



Table 2. Histone deacetylase 8 expression in cancer.

Tumor entity Trend of expression (compared with normal tissue)

Source and experimental setting Mode of mechanism Ref.

T-cell lymphoma Increased (protein) Primary ATL cells† SOX4 regulates HDAC8 [62]

Jurkat‡, HuT78‡, HSB-2‡, Molt-4‡, HH‡, MT4‡

PLCγ1, indirect effects on p21, p53, SOCS1/3

[10,67]

Myeloid leukemia Increased (mRNA) AML† – [68]

32D-CM (AML)‡ p53, CBFβ-SMMHC fusion protein

[69]

K562 (CML)‡, HEL (AML)‡ – [70]

Lung cancer Expressed (mRNA) NSCLC† – [71]

A549‡ – [10,57,61,72]

H1299‡, CL1–5‡ – [72]

Colon cancer Increased (protein) HCT116‡ STAT3, BMF [10,61,73–74]

RKO‡ – [10]

SW480‡, SW620‡ – [57]

HT29‡ – [73,74]

Cervical cancer Expressed HeLa‡ SMC3 [10,61,67]

Glioma Expressed (mRNA) Low- and high-grade gliomas† – [75]

U87‡ – [10]

Breast cancer Increased (mRNA) Breast cancer† – [76]

MCF-7‡ – [10,50,57,60]

MDA-MB-231‡ Notch1 (indirectly), Fbwx7 [50,57,60]

SUM-159‡, T47D‡, BT474‡, HCC1937‡, SKBR3‡, MDA-MB-468‡, ZR7530‡

– [50]

Ovarian carcinoma Expressed (protein) Ovarian cancer† – [57]

Ovcar-3‡ – [10]

SKOV3‡ – [57]

Gastric carcinoma Increased (protein) Gastric cancer† – [33]

SGC7901‡, MKN45‡, MKN28‡, BGC823‡, AGS‡, GC9811‡, NCI-N87‡

miR216b targets HDAC8 [33,34]

Neuroblastoma Increased in INSS stage 4 (mRNA, protein)

Neuroblastoma† – [58]

BE(2)-C‡ CREB, NTRK1 (indirectly) [58,77]

SH-SY5Y‡, Kelly‡, NGP‡, SH-EP‡, WAC2‡, SK-N-BE(2)‡, IMR-32‡, SK-N-AS‡

– [58,77]

NB2§ – [58]

Medulloblastoma Expressed (protein) Medulloblastoma† [78]

UW-228–2‡, DAOY‡, ONS76‡ – [77]

Malignant peripheral nerve sheath tumor

Expressed (protein) S462† PRDX6, PGAM1, HMGB1, PARK7/DJ-1

[38]

ST88†, STS26T†, MPNST727†, MPNST642†, MPNST6IEPVI (murine)†

– [38]

†In vivo patient tissue samples. ‡In vitro established cell lines.§ In vitro short-term cell culture.AML: Acute myeloid leukemia; ATL: Adult T-cell leukemia/lymphoma; CML: Chronic myeloid leukemia; HDAC8: Histone deacetylase 8; NSCLC: Non-small-cell lung cancer; SMC3: Structural maintenance of chromosome 3.



efficacious treatment with limited off-target effects. The FDA-approved broad-spectrum HDAC inhibi-tor vorinostat (SAHA) which is used to treat refrac-tory cutaneous T-cell lymphoma, is a weaker inhibi-tor of HDAC8 (micromolar range) than of HDACs 1–3 (nanomolar range). Similarly, trichostatin A completely inhibits HDAC8 activity only at 5 μM, though in Molt-4 acute lymphoblastic leukemia cells it attenuates HDAC8 expression [87]. Besides the known clinical side effects of SAHA (e.g. leukope-nia, thrombopenia, fatigue, diarrhea), this inhibitor has very recently been reported to promote epithelial mesenchymal transition and cell motility in triple negative breast cancer cell culture. This negative side effect was reported to be dependent on HDAC8 [88]. In terms of unspecific side effects, the targeting of one single enzyme seems to be superior to broad spec-trum HDAC inhibition when applied in an appro-priate tumor entity that displays oncogenic depen-dency on that particular HDAC family member. In neuroblastoma for example, HDAC8 expression cor-

relates with the aggressive tumor stage 4 and thus, with poor outcome. Selective HDAC8 inhibition in neuroblastoma cell lines induces signs of differentia-tion, such as the outgrowth of neurofilament-positive neurite-like structures [58]. Of note, the targeting of other HDAC family members in this tumor entity affects completely different processes (e.g., apopto-sis or autophagy) [89–93]. Consistent with the obser-vation that neural crest-cell-derived neuroblastoma displays oncogenic HDAC8 dependency, HDAC8 controls patterning of the skull in cranial neural crest cells of mice. Consequently, deletion of the gene for HDAC8 in mice leads to perinatal (P1) lethality due to skull instability [27]. Hence, HDAC8 is proposed as a potential new drug target for differentiation therapy by the usage of selective HDAC8 inhibitors to avoid unspecific side effects [11,94].

Hand in hand with the findings of a tumor relevant expression and mode of action of HDAC8, the develop-ment and usage of selective HDAC8 inhibitors proceeds. Though, specific inhibitor design is challenging due to

Tumor entity Trend of expression (compared with normal tissue)

Source and experimental setting Mode of mechanism Ref.

Hepatocellular carcinoma

Increased (protein)

Hepatocellular carcinoma§ [40,43]

HepG2† p53, β-catenin [40,43]

Bel-7404† p53 (Lys382), β-catenin [40,43]

PLC5† p53, β-catenin [40]

SMMC-7721, Hep3B, HCCLM3† – [43]

Prostate cancer Decreased (protein) Malignant and nonmalignant prostate tissues§

[57,79]

LNCap†, PC-3†, DU145† – [57]

Pancreatic cancer Expressed (protein) MiaPaCa-2†, PANC-1† – [57]

Melanoma Expressed (protein); independent marker of prolonged survival in BRAF-mutated stage 4 melanoma

Melanoma§ – [59]

Urothelial cancer Increased (mRNA) VM-CUB1†, RT-112†, SW-1710†, 639-V†, UM-UC-3†

p21 (indirectly) [80]

Uterine cancer Expressed (protein), specific marker of smooth muscle cell differentiation

Leiomyomas§, highly cellular leiomyomas§, epitheloid smooth muscle cell tumors§, leiomyosarcomas§, endometrial stromal tumors§

– [70,81]

†In vivo patient tissue samples. ‡In vitro established cell lines.§ In vitro short-term cell culture.AML: Acute myeloid leukemia; ATL: Adult T-cell leukemia/lymphoma; CML: Chronic myeloid leukemia; HDAC8: Histone deacetylase 8; NSCLC: Non-small-cell lung cancer; SMC3: Structural maintenance of chromosome 3.

Table 2. Histone deacetylase 8 expression in cancer (cont.).



the flexible structure of this epigenetic modulator, the unique binding pocket of HDAC8 allows the design of HDAC8-selective inhibitors (Figure 3 & Table 3). So far several specific inhibitors have been reported. Lead structures were identified by either structure-based design, for example, linkerless hydroxamic acids (IC

50

0.3 μM, >100-fold selectivity over HDAC1 and 6) [55] or from high-throughput screens like the cyclic thiourea SB379278A (IC

50 0.5 μM) [95] (Figure 3). However, it

remains open which isotype-specific chemical scaffolds would gain preference in follow-up advanced preclini-cal/clinical studies and whether such inhibitors would really be superior to pan-HDAC inhibitors in clinical trials, either for efficacy or toxicity. Within the scope of this review, we discuss the rationale for design of HDAC8-specific inhibitors and present a synopsis of some isotype specific inhibitors of HDAC8 with diverse chemical backbones targeting different mecha-nistic pathways. For cancer related processes relevant HDAC8 modulators are represented and summarized a dditionally in Figure 4.

Design of HDAC8-selective inhibitorsThe structural determinants of HDAC8 specificity as discussed above can be addressed by selective inhibi-tors. For instance, the HDAC8 foot pocket was suc-cessfully targeted with two selective small-molecule aa derivatives (PDB ID 3SFF and 3SFH) (Figure 3) [99]. This is a rare example of HDAC inhibitors without a strong chelating function such as hydroxamate, thiol or benzamide. Furthermore, as shown by molecular docking studies, dual HDAC8- and HDAC6-selective meta-substituted benzhydroxamic acids (Figure 3) also take advantage of the specific binding pocket shape of these HDAC isotypes by occupying their side pocket [56].

Due to limited structural information, rational drug design is in many cases is supported by molecular mod-eling studies. Such computational methods as QSAR modeling [100] and virtual screening [101] have been exploited to search for novel HDAC8 inhibitors. In order to explain structure–activity relationships and to guide the optimization of hit compounds, molecular docking studies are readily used [32,56,72,96]. Molecular dynamics simulations help to understand dynamic and mechanis-tic aspects of HDAC8 behavior [102–105]. Finally, homol-ogy modeling of parasitic HDAC8 isotypes sheds light on druggability of those targets and requirements for their inhibitors [53]. It should be noted, however, that one of the main challenges for molecular modeling is the high flexibility of the enzymes, which makes the predic-tion of the HDACs structure and the binding mode of their inhibitors tricky. A recent analysis of the flexibil-ity of the S. mansoni HDAC8 binding pocket based on

its crystal structures demonstrates that the shape of the pocket undergoes dramatic changes due to induced fit effects caused by binding of various ligands [53].

Another useful tool in drug design is the struc-ture–activity relationship analysis of already-known inhibitors. This approach can be adopted to under-stand pharmacophoric features of molecules, which are necessary to gain HDAC8 selectivity. The struc-tural diversity of known small-molecule HDAC8-selective inhibitors can be seen in Figure 3. Since all cocrystallized HDAC inhibitors bind to the zinc ion, it is obvious that this interaction is very important to specifically inhibit those enzymes in most cases. Therefore, it is comfortable to classify the HDAC8 inhibitors by the zinc-binding group: hydroxamic acids [10,55,56,67,72,96,98,106–109], aa derivatives [99] and other compounds [95,110]. The hydroxamic acid is a very common and universal metal chelating group, which can effectively bind to any HDAC isotype and to some other metalloenzymes (carbonic anhydrase, matrix metalloproteinase, urease, lipoxygenase, etc.) [111]. Nev-ertheless, by adjusting the linker of the inhibitor and further substituents it is possible to achieve not only protein family [112], but also isotype selectivity among hydroxamates. From the structures of known HDAC8-selective hydroxamate-based inhibitors it can be con-cluded that meta-substituted benzhydroxamic acids and hydroxamic acids with ortho-substituted cinnamic acid linker are HDAC8 selective. Sometimes they are also equally active on HDAC6 isotype, but in general this Γ-shape allows gaining selectivity against all other HDAC isotypes [53]. In case of parasubstituted benzhy-droxamic acids [113,114] and hydroxamic acids with para- and meta-substituted cinnamic acid linker (panobino-stat, belinostat) [115], they usually do not show HDAC8 selectivity with rare exceptions [55,107]. The selectivity of SB379278A [95], an HDAC8-inhibitory azetidi-none [110] and β-methylselenopyruvate [74] pr obably depends on their unusual zinc-binding groups.

HDAC8 inhibitorsPCI34051PCI34051 (Figure 3 & Table 3) is probably the most widely used HDAC8-specific inhibitor in research. Based on an indole linker, this hydroxamic acid inhib-ited HDAC8 with an IC

50 of 10 nM and was >200-

fold selective over HDACs 1/6 and >1000-fold over HDACs 2/3/10 [10]. While in seven different solid tumor cell lines PCI34051 did not show any cyto-toxic activity (except Ovcar-3 with GI

50 6 μM), in

T-cell-derived tumor cell lines Jurkat (derived from a T-cell leukemia), HuT78 (derived from peripheral T-cell lymphoma), HSB-2 and Molt-4 (both derived from T-ALL) PCI34051 induced cell death with GI

50


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ll cy

cle

arre

st a

cco

mp

anie

d

by

incr

ease

d le

vels

of

p21

WA

F1/C

IP1 ,

up

reg

ula

tio

n o

f Tr

k-A

p

rote

in, c

ellu

lar

dif

fere

nti

atio

n t

ow

ard

a n

euro

nal

p

hen

oty

pe.

In x

eno

gra

ft m

ou

se m

od

el, d

elay

ed

neu

rob

last

om

a tu

mo

r g

row

th a

nd

incr

ease

d c

ellu

lar

dif

fere

nti

atio

n. S

yner

gis

tic

effe

ct w

ith

13-

cis

reti

no

ic

acid

. No

det

ecta

ble

his

ton

e o

r tu

bu

lin a

cety

lati

on

[77]

BMF:

Bcl

-2-m

odif

ying

fac

tor;

HD

AC

8: H

isto

ne d

eace

tyla

se 8

; ND

: Not

det

erm

ined

; SM

C3

: Str

uctu

ral m

aint

enan

ce o

f ch

rom

osom

e 3.


Targeting HDAC8 as a therapeutic approach to cancer & neurodegenerative diseases ReviewTa

ble

3. H

isto

ne

dea

cety

lase

8 s

elec

tive

inh

ibit

ors

an

d a

ctiv

ato

r (c

on

t.).

Inh

ibit

or

Cel

l-fr

ee

IC50

val

ues

Can

cer

typ

eEf

fect

sR

ef.

PCI-

480

00

/ PC

I-48

012

(str

uct

ura

lly s

imila

r to

PC

I-34

051)

(co

nt.

)

Mal

ign

ant

per

iph

eral

ner

ve

shea

th t

um

ors

Ab

rog

atio

n o

f ce

ll g

row

th a

nd

clo

no

gen

ic p

rolif

erat

ion

in

hu

man

/mu

rin

e ce

ll lin

es. S

-ph

ase

cell

cycl

e ar

rest

, ca

spas

e-3

-dep

end

ent

apo

pto

sis.

No

det

ecta

ble

his

ton

e o

r tu

bu

lin a

cety

lati

on

. Sig

nifi

can

t d

ecre

ases

in b

oth

tu

mo

r vo

lum

e an

d t

um

or

wei

gh

t in

mu

rin

e-d

eriv

ed x

eno

gra

ft

mo

use

mo

del

[38]

NC

C14

9

H NO

H

ON

NN

S

70 n

MT-

cell

lym

ph

om

aG

row

th in

hib

itio

n. N

o e

ffec

ts o

n a

ffec

t th

e g

row

th o

f h

ealt

hy

do

no

r p

erip

her

al b

loo

d m

on

on

ucl

ear

cells

[67,

96]

Neu

rob

last

om

aG

row

th in

hib

itio

n[9

6]

Cer

vica

l can

cer

Do

se-d

epen

den

t ac

etyl

atio

n o

f SM

C3

in H

eLa

cells

w

ith

ou

t a

maj

or

incr

ease

in a

cety

late

d H

4/H

3K9.

A

cety

lati

on

of

tub

ulin

[67,

96]

NC

C14

9-d

eriv

ed c

om

po

un

d 9

H NO

H

O

N SS

150

nM

T-ce

ll ly

mp

ho

ma

Stro

ng

gro

wth

inh

ibit

ion

[67]

Cer

vica

l can

cer

Do

se-d

epen

den

t ac

etyl

atio

n o

f SM

C3

in H

eLa

cells

w

ith

ou

t a

maj

or

incr

ease

in a

cety

late

d H

3K9

or

tub

ulin

[67]

Co

mp

ou

nd

22d

O

N H

O

OH

27.2

nM

Acu

te m

yelo

id

leu

kem

iaR

esto

rati

on

of

p53

ace

tyla

tio

n. D

ose

-dep

end

ent

sele

ctiv

e ap

op

tosi

s o

f C

D34

+ le

uke

mic

ste

m c

ells

an

d p

rog

enit

or

cells

. In

du

ces

acet

ylat

ion

of

SMC

3 an

d a

bro

gat

es a

cute

m

yelo

id le

uke

mia

pro

pag

atio

n in

mic

e

[69]

Lun

g c

ance

rA

nti

pro

lifer

ativ

e ac

tivi

ty in

hu

man

lun

g c

ance

r ce

ll lin

es,

no

sig

nifi

can

t cy

toto

xici

ty in

no

rmal

lun

g c

ells

[72]

BMF:

Bcl

-2-m

odif

ying

fac

tor;

HD

AC

8: H

isto

ne d

eace

tyla

se 8

; ND

: Not

det

erm

ined

; SM

C3

: Str

uctu

ral m

aint

enan

ce o

f ch

rom

osom

e 3.



Inh

ibit

or

Cel

l-fr

ee

IC50

val

ues

Can

cer

typ

eEf

fect

sR

ef.

Aze

tid

ino

ne

1b

N H

O

NO

S

ND

Neu

rob

last

om

aSl

igh

t ef

fect

on

pro

lifer

atio

n, n

o in

du

ctio

n o

f d

iffe

ren

tiat

ion

[97]

Aze

tid

ino

ne

-der

ived

ret

ino

id h

ybri

ds

2E, 2

Z

H N

NH

OO

H N

NH

Cp

d 2E

Cp

d 2Z

OO

ND

Neu

rob

last

om

aSl

igh

t ef

fect

on

pro

lifer

atio

n, m

od

erat

e in

du

ctio

n o

f d

iffe

ren

tiat

ion

, in

du

ctio

n o

f βI

II tu

bu

lin a

s an

ear

ly

mar

ker

of

neu

ron

al d

iffe

ren

tiat

ion

[97]

Co

mp

ou

nd

32

(wit

h 2

-R-a

min

ote

tral

in a

s a

linke

r)

H N

N

N

N

N H

HO

O

80 n

MN

euro

bla

sto

ma

Mo

der

ate

gro

wth

inh

ibit

ion

. Tim

e-d

epen

den

t in

du

ctio

n

of

cell

dif

fere

nti

atio

n a

nd

ind

uct

ion

of

Trk-

A p

rote

in.

No

det

ecta

ble

his

ton

e H

3 ac

etyl

atio

n o

r p

21 b

ut

stro

ng

tu

bu

lin a

cety

lati

on

(p

oin

tin

g t

o H

DA

C6

inh

ibit

ion

as

wel

l)

[98]

Cer

vica

l can

cer

In H

eLa

cells

, no

ind

uct

ion

of

p21

bu

t st

ron

g in

crea

se in

tu

bu

lin a

cety

lati

on

[98]

Mu

ltip

le m

yelo

ma

Mo

der

ate

gro

wth

inh

ibit

ion

[98]

β-m

eth

yl-s

elen

op

yru

vate

O

O

O

Se

20 μ

MC

olo

n c

ance

rIn

du

ctio

n o

f p

21, d

ose

-dep

end

ent

G1/

G2

arre

st, H

3K9

hyp

erac

etyl

atio

n, i

ncr

ease

d e

xpre

ssio

n o

f B

MF.

Cas

pas

e-

3-d

epen

den

t ap

op

tosi

s (a

lso

blo

cks

HD

AC

1, h

ence

HD

AC

8 d

epen

den

ce n

ot

clea

r)

[74]

BMF:

Bcl

-2-m

odif

ying

fac

tor;

HD

AC

8: H

isto

ne d

eace

tyla

se 8

; ND

: Not

det

erm

ined

; SM

C3

: Str

uctu

ral m

aint

enan

ce o

f ch

rom

osom

e 3.

Tab

le 3

. His

ton

e d

eace

tyla

se 8

sel

ecti

ve in

hib

ito

rs a

nd

act

ivat

or

(co

nt.

).



Inh

ibit

or

Cel

l-fr

ee

IC50

val

ues

Can

cer

typ

eEf

fect

sR

ef.

JAH

A-P

IPδ H

N

O

O

NH

NN NH

O

N

NH

O

N

O

H N

NO

HN

N

N NH

O N

NH

O

NN

H

O

N

N HN

O

HN

HN

O

O

NH

OO

H

130

nM

No

ntr

ansf

orm

ed

mo

use

en

do

thel

ial

fib

rob

last

s

Pro

mo

te s

pec

ific

his

ton

e H

3 h

yper

acet

ylat

ion

of

HD

AC

8-

spec

ific

ho

meo

bo

x tr

ansc

rip

tio

n f

acto

rs O

tx2

and

Lh

x1,

ind

uct

ion

of

plu

rip

ote

nt

gen

es li

ke O

ct3

/4, N

ano

g, C

dh1

[39]

TM-2

–51

N H

O

N H

S

ND

(a

ctiv

ato

r o

f H

DA

C8

)

Neu

rob

last

om

aIn

du

ctio

n o

f g

row

th in

hib

itio

n a

nd

ap

op

tosi

s. M

od

erat

e in

crea

se in

p53

exp

ress

ion

an

d e

nh

ance

d e

xpre

ssio

n

of

p21

, eff

ects

on

ly o

bse

rved

in p

53 w

ild-t

ype

neu

rob

last

om

a ce

lls (

SH-S

Y5Y

)

[83]

BMF:

Bcl

-2-m

odif

ying

fac

tor;

HD

AC

8: H

isto

ne d

eace

tyla

se 8

; ND

: Not

det

erm

ined

; SM

C3

: Str

uctu

ral m

aint

enan

ce o

f ch

rom

osom

e 3.

Tab

le 3

. His

ton

e d

eace

tyla

se 8

sel

ecti

ve in

hib

ito

rs a

nd

act

ivat

or

(co

nt.

).



2.4–4 μM [10]. Cell toxicity was specific for T-cell-derived tumors as no toxicity was observed with tumors of B-cell/monocytic/myeloid/myeloma lineage origin. Moreover, in resting or stimulated T cells PCI34051 did not cause any cell death. Interestingly, the specific-ity of PCI34051 toxicity to Jurkat and HuT78 cells was not accompanied by histone or tubulin acetylation [10]. AML leukemia stem cells can respond quite differently to HDAC8 inhibition. For example, PCI34051 treat-ment has shown moderate effects in inv(16) AML by elimination of inv(16+) AML CD34+ leukemic stem cells through reactivation of p53 via restoration of p53 acetylation in these cells [69]. A detailed analysis of the PCI34051-specific T-cell lymphoma effect con-cluded caspase-3-dependent apoptosis as a mechanism of cell death without cleavage of the proapoptotic Bid protein [10]. This relied on PLCγ1-dependent calcium mobilization and cytochrome C release [10]. A quick dose-dependent increment in intracellular calcium lev-els was monitored at concentrations similar to those causing PLCγ1-triggered apoptosis [10] without the requirement of T-cell receptor signaling. However, it was not clear whether PLCγ1 was itself a direct tar-get of HDAC8 or other adaptor proteins (of T-cell si gnaling pathway) that bind to activate it.

PCI34051 has lately been tested in preclinical neuroblastoma models. Treatment of neuroblastoma-derived cell lines with PCI34051 or stable variants PCI48000/PCI48012 (all at 4 μM) led to significant reduction in cell numbers, increase in cell-cycle inhibi-tor p21WAF1/CIP1 and stimulation of cellular differ-entiation by upregulation of Trk-A protein followed by formation of neurofilament-positive outgrowths [77]. Application of the more stable variant PCI48012 (plasma half-life 1 h) to neuroblastoma xenograft mouse models at the maximum tolerable dose of 40 mg/kg/day critically delayed neuroblastoma tumor growth and increased cellular differentiation and apop-tosis as detected by caspase-3 staining of tumor sam-ples [77]. Moreover, treatment of BE(2)-C xenografted NMRI nude mice with PCI48012 and 13-cis retinoic acid, a clinically approved drug currently applied as part of the standard treatment combination during the maintenance phase of the treatment protocol for high-risk neuroblastoma, had a synergistic effect in retard-ing tumor growth and enhancing cell death [77]. The enhancement effect by PCI34051-mediated HDAC8 inhibition and retinoic acid was attributed to CREB, a protein that has been implicated in both HDAC8 and retinoic acid signaling pathways. Of note, this com-pound combination was well tolerated by the animals and no hyperacetylation of tubulin or histone H4 in peripheral blood mononuclear cells was observed as nonspecific effects [77]. From a clinical perspective,

combination of HDAC8 inhibition with retinoic acid treatment might be a promising strategy in the mainte-nance treatment of high-risk neuroblastoma.

PCI34051 and variants have also found application in malignant peripheral nerve sheath tumor cells [38]. Both in human (e.g., S462, STS26T, MPNST642, MPNST724) and in murine-derived cell lines (MPNST6IEPVI), PCI34051 and PCI48012 abol-ished cell growth and potently reduced cell viability as well as clonogenic growth capacity [38]. While an S-phase cell cycle arrest was reported in both human and murine-derived cell lines, these effects were not accompanied by tubulin nor histone (H3/H4) hyper-acetylation [38]. In the MPNST6IEPVI mouse model, treatment with PCI48012 significantly diminished tumor growth and volume [38]. By using PCI34051, new target proteins in this cancer type has also been identified [38].

In addition to above-mentioned cancer types, PCI34051 has been shown to be effective in nonalco-holic fatty liver disease-associated hepatocellular car-cinoma. In such cancers, HDAC8-stimulated insulin resistance and β-catenin activation [40]. Treatment of HDAC8-overexpressing cell lines BEL-7404 and PLC5 with PCI34051-induced cell death, G2-M arrest, apoptosis, H4 acetylation and increased expression of p53 [40]. In PLC5 cells, an additional increase in insu-lin sensitivity was also observed followed by a concom-itant decrease in β-catenin and its downstream target CCND1 [40]. Given the application of PCI34051 and its variants in a broad spectrum of malignancies, these compounds are definitely promising leads for further preclinical evaluation.

NCC149This inhibitor (Figure 3 & Table 3) was identified using a rapid screen of a 151-triazole compound library generated using click chemistry [96]. The alkyne, containing a zinc-binding group, was clicked using Cu (I)-catalyzed azide-alkyne cycloaddition reac-tion to an azide forming triazoles as a linker [96]. In vitro tests revealed a low IC

50 of NCC149 (70 nM)

against HDAC8 with a very high selectivity over major HDACs (nuclear extract: IC

50 54μM, HDAC1: IC

50

38μM, HDAC2 >100 μM, HDAC4: IC50

: 44 μM, HDAC6: IC

50 44 μM). In cellulo, NCC149 induced

dose-dependent acetylation of SMC3 without any sig-nificant acetylation changes in histone H4 indicating a selective cellular inhibition of HDAC8 [96]. This com-pound also exhibited cell-type specific growth inhibi-tion with strong effects in Jurkat, HH, MT2, MT4, NB-1 and LA-N-1 but not in healthy donor peripheral blood mononuclear cells (IC

50 >100 μM). Docking of

NCC149 to HDAC8 indicates that the triazole ring



interacts with the methylene group of HDAC8 Phe152 through hydrophobic interactions. Moreover, this tri-azole ring seems to be pivotal for the appropriate ori-entation of both the hydroxamate and Phe152 binding phenylthiomethyl group contributing to potency [96]. Hence, further derivatives of NCC149 with vari-ous ring systems were designed to increase potency against HDAC8. Compound 11 (Figure 3 & Table 3), a reversely oriented triazole, was found to be even more potent on HDAC8 with an IC

50 of 53 nM maintain-

ing the high selectivity profile of NCC149 [67]. How-ever, inside cells compound 11 was found to acety-late both SMC3 and α-tubulin in a dose-dependent manner without major effects on H3K9. In contrast, a thiazole derivative exhibiting a higher IC

50 value of

150 nM (80–100-fold selectivity over other HDACs), compound 9 (Figure 3 & Table 3), was more selective in cells in acetylating SMC3 compared with α-tubulin or H3K9 [67]. This indicated that compound 9, though less potent on HDAC8 enzyme in vitro, could specifi-cally inhibit HDAC8 inside cells compared with other cellular HDACs. In parity with these observations, compound 9 also showed higher growth inhibition in T-cell lymphoma cell lines (Jurkat, HH, MT4 and HuT78) compared with NCC149 [67]. This might hint to the possibility that a compact binding of HDAC8 with thiazole improves the selectivity of compound 9 over other cellular HDACs and also renders it more active inside cells.

Ortho-aryl N-hydroxycinnamidesThis series was generated using computer guided docking studies and knowledge-based design [72]. Huang et al. generated a chimeric inhibitor with the benzyl moiety of PCI34051 into a core N-hydroxycin-namide of different chain length derived from LBH-589 [72]. An ortho-phenyl N-hydroxycinnamide, com-pound 22a (Figure 3 & Table 3) with a short linker, was identified as a lead compound with similar potency as PCI34051. Further docking studies with 22a led to the insight that inclusion of additional hydrophobic groups could impart 22a better binding capacity to HDAC8. Two derivatives designed on this principle showed excellent HDAC8 inhibitory activities. Com-pound 22b (Figure 3 & Table 3) with a para-bromo group exhibited an IC

50 of 5 nM (tenfold more potent than

PCI34051, 13-fold more potent than 22a) and 22d (Figure 3 & Table 3) with a biphenyl warhead exhibited an IC

50 of 27 nM (twofold more potent than PCI34051,

threefold more potent than 22a), both compounds being selective over other major HDACs [72]. However, only 22d showed moderate growth inhibition with lung cancer cell lines. Specifically, in cell line CL1–5 with elevated HDAC8 level, 22d showed similar GI

50 (7 μM)

compared with SAHA (6.2 μM) [72]. These effects were stronger compared with PCI34051 (GI

50 > 10 μM). 22d

was nontoxic to IMR-90 fibroblasts indicating a prefer-ence toward cancer cells [72]. Moreover, this compound could also enhance chemosensitivity of resistant AML cells by specifically restoring p53 acetylation [69]. Addi-tionally, in inv(16+) AML CD34+ leukemia stem cells, 22d-induced apoptosis [69]. In vivo, in a mouse xenograft AML model, 22d significantly reduced engraftment size, AML burden and abolished leukemia inducing capacity of such leukemia stem cells [69]. Furthermore, the modulation of 22d activity with a concomitant mod-ulation of p53 or HDAC8 levels underlined a possibil-ity of using this compound in combination therapy [69]. Hence, 22d is a promising lead structure to follow-up for further (pre)clinically active derivatives.

AzetidinoneAn azetidinone inhibitor was designed based on the modular structure of the pan-inhibitor SAHA, belonging to the class of monocyclic-β lactams with an azetidin-2-one ring as the zinc binding moiety [110]. Modification of the nitrogen substituents in the azeti-din-2-one ring led to the identification of HDAC8-selective azetidinone 1b (IC

50 4.53 μM, other HDACs

>1000 μM), revealing an N-thiomethyl-β-lactam as a novel zinc chelating group (Figure 3) [110]. Replacement of N-thiomethyl with N–H or N–OH resulted in complete loss of activity signifying the prerequisite of this group for the inhibition. Computational analysis of 1b indicated that the N-thiomethyl-azetidin-2-one interacted with Trp141 of HDAC8 via the sulfur atom thus contributing to the specific activity [110]. This compound also showed moderate cytotoxicity (IC

50

10–28 μM) in SH-SY5Y undifferentiated neuroblas-toma cell line [97]. A further extension of this com-pound as hybrids with retinoic acid (Figure 3 & Table 3) reduced cytotoxic potency (IC

50 30–90 μM range),

nevertheless, imparted neural-like moderate differenti-ation capability by induction of βIII-tubulin [97] which again hints as using such compounds in combination therapy for neuroblastoma. However, these hybrid compounds were neither tested for HDAC8 inhibition nor for their specificity over other HDACs. There-fore, further follow-up studies are needed to define the s pecific mode of action.

Aminotetralin-based dual inhibitorsPhenotypic screens of acetylated tubulin and p21 induc-tion were employed to identify the tetrahydroisoquino-line containing lead compound 12 (Figure 3 & Table 3) as dual inhibitors of HDAC8/HDAC6 [98]. This potent inhibitor (HDAC8: IC

50 30 nM; HDAC6: IC

50 50 nM)

exhibited high selectivity over other HDACs (>20 μM)

NH

O

OH

HN

OH

ON

N NS

HN

OH

O

N

SS

HN

OH

O

N

N NS

HN

O

O

NH

N N

NHO

N

NHO

N

O

HN

N O

HN

N

N

NHO

N

NHO

NNH

O

N

N

HNO

HN

HN

O

O

NHO OH

NH

OH

O

O

NH

OH

O

O

Br

NH

OH

O

O

NO

NO2

NH

O

HO

HN

NH

O

HO

N

N

N

O

N O

HN OH

N

O

Cl

NH2 N

O

F

F

ClN

O

NH2

Cl

N

NH

NH

SO

O

O

Se

NH

O

NOS

HN

ONHO

HN

ONHO

HN

NH

OH

O

O

NH

OH

O O

N

NH

NH

O S

HDAC8-selective inhibitors

Hydroxamic acids Amino acids

Other compounds

Cpd 22alinkerless inhibitor

NCC149

Cpd 9

Cpd 11

NCC149 and derivatives

Cpd 22b

Cpd 12

Cpd 32

Aminotetralin derivatives

PCI34051

PCI48000

JAHA-PIP δ

PCI48012

SAHA

Pan inhibitors HDAC8 activator

TSA

Ortho-aryl N-hydroxycinnamide derivatives

Cpd 22d

Cpd 3

Cpd 4

Amino acid derivatives

SB379278A

Azetidinone

Azetidinone and derivatives

Cpd 2E

Cpd 2Z

β-Methylselenopyruvate

TM-2-51



Figure 3. Inhibitors and activator of histone deacetylase 8 (see facing page). HDAC8-specific inhibitors of different zinc-binding groups (green text), pan inhibitors and a thiourea-based activator of HDA8 are shown in the figure. Cpd: Compound; HDAC8: Histone deacetylase 8.

Figure 4. The hallmarks of pharmacological histone deacetylase 8 modulation. Depicted are the major cancer relevant processes targeted by selective HDAC8 modulators (inhibitors and one activator) according to studies using various cancer cell lines. Additional mechanistic information is presented for cancer hallmarks in dark gray boxes if available. Dotted arrows indicate discrepant results for different cell lines, suggesting cell line and context dependency of the respective finding. Note that the figure displays only relationships that have been experimentally addressed by the cited references. It is of course possible that additional hallmarks are influenced by a certain compound complementary to those indicated in the figure. HDAC8: Histone deacetylase 8.

Genomicstability

Cytoskeletalremodeling

Cell cyclearrest

Cellulardifferentiation

Reduction ofinvasive andmetastatic

abilities

Embryonicdetermination

of cell fate

Reduction ofproliferativesignalling

Apoptosis

NCC149-derived

compound 9

Compound32 (with 2-R-aminotretalinas a linker)

Compound 22d

Azetidinone-derivedretinoic hybrids 2E, 2Z

β-methylseleno-pyruvate

PCI-34051

JAHA-PIPδ

PCI-48000/PCI-48012

TM-2-51[HDAC8activator]

NCC149

Via SMC3acetylation

Via tubulinacetylation

Via p21upregulation



while inside cells, induced 70% tubulin acetylation, slight p21 induction and was moderately toxic (IC

50

3.45 μM) [98]. However, this inhibitor had a low metabolic stability in microsomal metabolite assays due to hydrolysis of the hydroxamic acid group and N–C cleavage on the tetrahydroisoquinoline moi-ety [98]. Moreover, the poor solubility (>10 μg/ml) of the inhibitor is problematic. In order to overcome these issues, new aminotetralin derivatives were designed to mimic the 2, 7-disubstituted tetrahydroisoquino-line linker. Compound 32 (Figure 3 & Table 3) with 2-R-aminotetralin as a linker showed the most potent inhibition with both HDAC8 (IC

50 80 nM)/HDAC6

(IC50

50 nM) and less on other HDAC isotypes (>30 μM) [98]. In cellulo, it led to hyperacetylated tubu-lin with an EC

50 of 1.64 μM but failed to induce p21

expression in HeLa cells [98]. In NCI-H929 (multiple myeloma) and BE(2)-C (neuroblastoma) cells, inhibi-tor 32 imparted moderate toxicity with GI

50 values of

7.7 μM and 5.4 μM, respectively [98]. Nevertheless, 32 was moderately metabolically stable and soluble at 59 μg/ml. Interestingly, 32 stimulated time-dependent differentiation such as neurite growth in BE(2)-C cells treated at only 2 μM [98]. Also, in these cells 32 did not induce HDAC6 expression, p21 induction and H3 acetylation up to 10 μM; instead the cell differentia-tion marker TrkA and acetylated tubulin levels were significantly elevated [98]. With the emerging role of HDAC8 and HDAC6 in cancer such dual inhibi-tors can be of advantage for the treatment of cancers affected by aberrations of both enzymes. Given the fact that 32 did not induce HDAC6 expression in neuro-

Figure 5. Crystal structure of histone deacetylase 8 in complex with an amino acid derivative (PDB ID3SFF) showing the addressing of the foot pocket.

Foot

Zn2+

pocket

PDB ID 3SFF Cpd. 4



blastoma and even stimulated differentiation, this class of compounds can be further optimized for HDAC8 specificity and would be interesting c andidates to test in preclinical animal models.

β-Methylselenopyruvateβ-Methylselenopyruvate (Figure 3 & Table 3) is an α-keto-acid metabolite of natural organoselenium containing compounds like methylselenocysteine (generated by transamination), identified as a micro-molar inhibitor of HDAC8 (IC

50 20 μM) [74]. Molec-

ular modeling suggested that this compound docked in an energetically favored orientation with the α-carbonyl group and one of the carboxylate oxygen atoms interacting with the zinc of HDAC8 [74]. In colon cancer cells HCT116 and HT29, this inhibitor induced histone H3 hyperacetylation, a dose-depen-dent G2 (10 μM) or G1 (2 μM) cell cycle arrest and activation of caspases, indicative for apoptosis. An analysis of cell cycle regulator p21 revealed increased expression upon inhibitor treatment, hyperacety-lation of histone H3K9 and K18 levels at P21WAF1 promoter as observed with chromatin immunopre-cipitation experiments [74]. Further investigation of the apoptotic mechanism identified BMF as the key proapoptotic protein being regulated downstream by HDAC8 [73]. Additionally, this study also pro-posed a model stating that inhibition of HDAC8 by β-methylselenopyruvate in colon cancer cells led

to dislodging of the HDAC8/STAT3 complex from the BMF promoter, recruiting P300 with the conse-quence of an elevated BMF level and activation of apoptosis [73]. Further in vivo studies will be needed to validate this theory in a physiological context. However, this inhibitor also inhibits HDAC1 and the specificity related to HDAC8-mediated down-stream effects on BMF should also be proven with a specific HDAC8i as PCI34051.

JAHA-PIPδThis particular inhibitor is a hybrid of ferrocene-based SAHA analogues called Jay Amin hydroxamic acid (JAHA) with N-methylpyrrole (Py)-N-methylimid-azole (Im) polyamides (PIP) [39,116]. PIPs are synthetic transcriptional modulators that mimic transcription factors by recognizing and binding to specific nucleo-tide sequences in the minor groove of the DNA [117]. The particular analog, JAHA-PIPδ (Figure 3 & Table 3) (HDAC8: IC

50 130 nM) is a hybrid of SAHA without

a capping phenyl ring with an improved PIP called PIPδ with eightfold selectivity over HDAC2 (IC

50 1.12

μM) and fourfold over HDAC1 (IC50

0.49 μM) [39]. Notably, JAHA-PIPδ enhanced endogenous acetyla-tion of histone H3 in the promoter region of pluripo-tent genes as Oct3/4, Nanog, Cdh1 and Rex1 suggest-ing a role of HDAC8 in regulating pluripotency [39]. Even more strikingly, ChIP analysis revealed this analog stimulated HDAC8-specific homeobox tran-

Figure 6. Docking pose of the highly selective histone deacetylase 8 inhibitor PCI34051 addressing the side pocket of histone deacetylase 8 (PDB ID 2VX5).

PCI34051 Hydrophobicside pocketHDAC8



scription factors Otx2 and Lhx1 promoter hyperacet-ylation, thus, consequently increasing the expression of these genes. HDAC8-mediated repression of Otx2 and Lhx1 is important in cell fate determination of neuronal cranial cells; while Otx2 participates in plu-ripotency decision, Lhx-1 determines the mesodermal differentiation [27]. This finding strongly supports the theory that HDAC8 might prefer specific gene regions in histone H3 for deacetylation. Also, the fact that these genes are activated in a somatic cell line like mouse embryonic fibroblasts (MEF), in which both Otx2 and Lhx-1 are not usually expressed, sug-gests that JAHA-PIPδ could act as modulator of cell fate. Especially, in neuroblastoma, which is linked to defects in proper sympathetic neuron maturation, the potency of this inhibitor to change cell fate might be of special interest in order to revert the neuroblastic cell type into differentiated cells. However, this assump-tion remains to be elucidated, since cell culture stud-ies addressing this point with the use of JAHA-PIPδ have not been reported so far.

HDAC8 activatorsCancerRecently, Sing et al. identified TM-2–51 ( Fi gure 3 & Table 3) as a selective activator of H DAC8. The basic structure is an N-acylthiourea derivative N-(phenylcarbamothioyl) benzamide [118]. The pres-

ence of an amide bond is essential to maintain the activating properties. Similarly, the presence of an aro-matic ring connecting the other nitrogen of the thio-urea also contributes to HDAC8 activation. Docking indicates clustering of this molecule at the vicinity of the active site pocket [118]. Testing TM-2–51 in p53 wild-type SH-SY5Y and p53 mutant BE(2)-C human neuroblastoma cell lines yielded surprising results with only SH-SY5Y cells showing decreased proliferation and increased apoptosis. Treatment also correlated well with the moderate increase in p53 level and enhanced elevation in expression of the downstream target p21 in SH-SY5Y cells [83]. Such effects were absent in BE(2)-C cell line. Although, HDAC8 regulates wild-type and mutant p53 gene expression [82] and TM-2–51 can upregulate p53 pro-tein levels for both conditions, mutant p53 is more susceptible to degradation. This points to a prefer-ential use of TM-2–51 in p53 wild-type cancers. In line, the authors propose TM-2–51 as an adjuvant to restore wild-type p53 levels in patients treated with pan HDAC inhibitors as SAHA [83].

Future CdLS therapy?Cornelia de Lange syndrome or CdLS is a broad range of genetic disorders caused by compromised function of cohesin, hence grouped as a cohesin-opathy [119]. This syndrome manifests heteroge-



neous phenotypes affecting multiple organs with intellectual disability, distinctive facial features, growth retardation and limb malformation being the predominant symptoms [119]. The main causes of this disease are traced to genetic mutations in proteins comprising the cohesin complex, includ-ing SMC3 [120]. Along with other component pro-teins, SMC3 forms a clutch to hold the sister chro-matids together during cell cycle progression [121]. Recently, HDAC8 has been identified as a deacet-ylase for SMC3 primarily deacetylating conserved lysines 105, 106 in humans [19]. This deacetylation of SMC3 is important to segregate the sister chro-matids and replenish the SMC3 pool for another cell cycle event [122]. Loss of HDAC8 deacety-lation activity leads to accumulation of acetylated SMC3 with decreased affinity toward chromatids ultimately effecting gene transcription [123]. Cur-rently, several mutations in HDAC8 are linked to CdLS [19], mainly being missense and de novo aber-rations [26]. Together with SMC3/SMC1A/RAD21, HDAC8 mutations account for 5% of the patients showing a heterogeneous CdLS phenotype [26].

A careful analysis of representative missense muta-tions revealed loss of HDAC8 activity and thermal instability in all cases [25,124]. Furthermore, this loss of activity was related to the occurrence of mutations in the vicinity of the active site and consequent struc-tural changes [25]. It is also correlated to the severity of the disease or phenotype, with the most distant mutation causing less severe symptoms. Interestingly, for most mutations (that exhibited residual HDAC8 activity) enzyme activity could be rescued by treat-ment with TM-2–51 in a dose-dependent manner, except for the mutation located nearest to the active site (100% inactive mutant) [25,124]. In cells express-ing wild-type HDAC8 enzyme, TM-2–51 increased activity up to 12-fold at 10 μM concentration [118]. This activator is thought to bind at two distinct sites of HDAC8 in a positive cooperative manner (as indicated from molecular docking studies), change enzyme kinetics (e.g. reduce Km of the substrate and markedly increase catalytic rate) in the presence of an inhibitor/substrate, and rescue catalysis [83]. Hence, the use of small-molecule activators might be used in CdLS patients with HDAC8 mutations to restore the activity of the mutant enzymes or to increase the sensitivity of HDAC8 toward the native substrates. However, the dependency on the muta-tion site might have a significant impact on the ther-apeutic outcome. Even though the present symptoms might not be completely cured, HDAC8 activators might help in slowing down the progression of the disease.

Future perspectiveUntil now, four HDAC inhibitors are approved by the FDA: SAHA and romidepsin for cutaneous T-cell lymphoma, belinostat for peripheral T-cell lymphoma and very recently panobinostat (LBH589) for multiple myeloma. All these drugs have a broadband inhibition profile, moderately selective for one HDAC class but not for a particular isotype. Side effects under therapy vary from nausea, fatigue, fever (pyrexia), low red blood cells (anemia), vomiting to cardiac toxicity, ventricular arrhythmia, leukopenia and thrombocytopenia. With respect to such harmful effects, isotype selective HDAC inhibitors might be advantageous to widen the therapeu-tic window by reducing nonspecific targeting of other HDACs and associated molecular signaling pathways. It remains to be seen, whether for example, the HDAC inhibitor chidamide, that was newly approved in China and is selective for HDAC1–3 will already provide an optimized activity/toxicity relationship [125].

To create selective HDAC8 inhibitors computa-tional approaches have shown promising results by designing HDAC8 selective compounds, for example, based on an amino acid scaffold (Compounds 3 and 4 in Figure 3). The x-ray structures (PDB ID 3SFF and 3SFH) show that the amino and carbonyl group of the inhibitor is able to coordinate to the zinc ion whereas the terminal phenyl group is addressing the foot pocket (Figure 5). As consequence these inhibitors are highly selective for HDAC8. In addition, addressing the hydrophobic side pocket of HDAC8, for example, by inhibitor PCI34051 (Figure 3) results in a highly selective HDAC8 inhibitor. A good shape comple-mentarity between an aromatic substituent and the side pocket is achieved (Figure 6). A similar type of interaction can be observed for example, for the link-erless HDAC8 inhibitors (Figure 3).There is strong evidence that HDAC8 is one of the causative factors for T-cell lymphoma and, hence, isotype-selective HDAC8 inhibitors present a rationale for therapies with less toxicity. Similarly, neuroblastoma-specific and selective HDAC8 inhibitors show ample promise for an improved treatment regime. As HDAC8 inhibi-tors can also target leukemia stem cells [69], more potent compounds can be optimized for an enhanced killing of such cells. Moreover, the possibility to com-bine HDAC8 inhibitors with the current treatment modalities [77] might enhance therapy in HDAC8-related cancers. HDAC8 is linked with Rad51 and a lack of radiosensitization is seen upon HDAC8 down-regulation [126] which seems to point rather toward combination chemotherapeutic regimes as the route to follow. p53 modulators like leptomycin B could also offer a synergistic therapeutic effect [127]. Alterna-tively, small interfering RNAs or miRNAs are prom-



ising in selective HDAC8 inhibition [34]. The role of HDAC8 activators in tumors harboring wild-type p53 is also worth mentioning [83]. Due to its tissue-specific expression HDAC8-expression levels could be deter-mined as a diagnostic marker for tumors as already shown for smooth muscle tumors [81]. Nevertheless, redundancy of HDACs, conserved active sites and shared protein substrates pose tremendous challenges toward isotype-specific targeting in patients. Also, in research there is a lack of preclinical animal models with a well-described dependence on HDAC8 activ-ity. Development of new models will bring in new per-spectives toward isotype selective cancer treatment. Notably, there is evidence for roles of HDAC8 in can-cer progression which are not fully characterized yet and that need to be investigated further [50].

Besides cancer, HDAC8 inhibitors are attractive candidates for influenza therapy, where an explicit role of HDAC8 has been deciphered in late-penetrating viruses [31]. Moreover, HDAC8 inhibitors are cur-rently under investigation in helminth worm infec-tions [13,32,101]. Furthermore, new exciting results of HDAC8 activators in reversing mutant HDAC8

enzymes might open new doors for CdLS disease man-agement. In conclusion, the field will witness a boom of research in the future years with many novel roles of HDAC8 yet to be discovered.

Financial & competing interests disclosureA Chakrabarti, J Melesina, W Sippl and M Jung receive fund-

ing from the European Union’s Seventh Framework Pro-

gramme for research, technological development and demon-

stration under grant agreement numbers 241865 (SEtTReND)

and 602080 (A-ParaDDisE). M Jung (Ju295/13–1), W Sippl

(Si868/13–1), O Witt (W1461/4–1) and I Oehme (Oe542/2–1)

are also funded by the Deutsche Forschungsgemeinschaft

(DFG). Additionally, I Oehme is supported by H.W. & J Hector

foundation #M71. FR Kolbinger is supported by the Deutsche

Krebshilfe (DKH) with a Mildred Scheel doctoral scholarship

(number 112065). The authors have no other relevant affilia-

tions 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.


manuscript.

Executive summary

• Evolutionarily diverged from class I, histone deacetylase 8 (HDAC8) is a unique deacetylase delineating class I from class II HDACs.

Deacetylase activity & interaction partners• While in vitro histones and histone-derived peptides are well deacetylated, conflicting evidences from cellular

studies render histones as controversial substrates.• Though not dependent on any cofactors for activity, HDAC8 associates with many important proteins/protein

complexes either to deacetylate relevant targets or acting as a scaffold for signaling events.HDAC8 structure & inhibitors• Intrinsic flexible structure of HDAC8 and its similarity to other HDACs makes it a challenging target, however,

the unique features of the HDAC8 binding pocket architecture allows the design of HDAC8-selective inhibitors.

Role of HDAC8 in oncogenesis• HDAC8 is intricately involved in tumorigenesis and in addition to other tumors is a relevant target in T-cell

lymphoma/leukemia and in neuroblastoma.HDAC8 inhibitors as anticancer therapeutics• Various isotype-selective inhibitors are identified from different methodologies and are effective in cell

culture/mouse models. Dual inhibitors are also reported to be beneficial.HDAC8 activators• HDAC8 activators are indicated to be used for the treatment of cancers with wild-type p53. Moreover,

restoring of HDAC8 catalytic activity in cells with enzyme mutants by such activators may provide a promising strategy for disease progression management in CdLS patients.

Future perspective• Selective targeting of HDAC8 in patients is a major challenge, however, as it offers potential for reduced side

effects as compared with pan-HDAC inhibitors.• Future perspective therapy could expand beyond cancer to viral and parasitic treatments.

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part of

Review

Recently discovered EZH2 and EHMT2 (G9a) inhibitors

Uttara Soumyanarayanan1 & Brian W Dymock*,1

1Department of Pharmacy, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore *Author for correspondence: [email protected]



Future Med. Chem.

Review 2016/08/308

13

2016

Methyltransferase enzymes are promising epigenetic oncotargets. Recent efforts toward the development of inhibitors of two methyltransferases, EZH2 and G9a, as potential anticancer therapies are reviewed with a focus on the structure–activity relationships of compounds published from 2012. Benzamide-substituted 2-pyridones are still by far the most popular selective EZH2 inhibitor class but alternative classes are now being reported. There are now three EZH2 inhibitors in clinical development with the first responses in lymphoma patients with tazemetostat. Potent inhibitors of G9a are also published but no examples have yet reached the clinic. Dual blockage of EZH2–G9a is exemplified by one series of compounds. We conclude this review by presenting the three clinical stage compounds with the first clinical response data.

First draft submitted: 9 May 2016; Accepted for publication: 7 June 2016; Published online: 22 August 2016

Keywords: 2-pyridones • dual inhibitors • enhancer of zeste homolog 2 • EZH2 • inhibitors • epigenetics • G9a inhibitors • HKMT • oncology • SET domain • tazemetostat

Genomic instructions for the growth and development of living organisms are encoded in DNA which is present inside the nucleus of eukaryotic cells. Approximately 2 m of DNA is tightly packaged being wrapped around alkaline proteins known as histones, which together form a core unit called nucleosomes that play an important role in gene regulation. Epigenetics describes how cells control gene expression through modifications of DNA that do not involve a change in DNA sequence. Specific proteins involved in epigenetics are controlled through post-translational modi-fications, chemical reactions used by cells to control gene expression, involving transfer of specific functionalities to proteins like his-tones, thereby rendering them functional or nonfunctional. Amino acid residues present on histone tails undergo a variety of post-transla-tional modifications such as methylation, acet-ylation, phosphorylation or ubiquitylation, which, in turn, regulate gene expression [1,2]. The role of histone acetylation in chromatin

folding has been attributed to charge differ-ences, although the exact mechanisms by which histone methylation affects downstream processes remain undefined. The site of meth-ylation is dependent on the protein (histones or nonhistones) as well as the specificity of the methyltransferase (MT) enzyme. Methylation of histone proteins primarily occurs only at two amino acid residues: lysine (K) and argi-nine (R), methylation being more common on the lysine residues on the tails of histone 3 and histone 4. These chemical modifications are catalyzed by a group of enzymes called histone methyltransferases (HMTs) – more specifi-cally histone lysine/arginine methyltransfer-ases (HKMT/HRMT) – depending on the amino acid r esidue they methylate [3,4].

Most HKMTs (except DOT1) conserve a region of 110–130 amino acids known as the SET domain, which together with preceding (Pre-SET) and following (Post-SET) amino acids are essential for the catalytic activity [4,5]. SET domain-containing HKMTs that use


Figure 1. Histone methyltransferase target specificities of mammalian SET domain-containing proteins. Adapted with permission from [6] © Elsevier (2013).

Me

Me

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Me

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H3K36 KMTsSET2, NSD1, NSD2,

NSD3, ASH1L, SETMAR

H3K9 KMTsSUV39H1, SUV39H2, G9aGLP, SETDB1, SETDB2,

PRDM3, PRDM16

H3K4 KMTsSET1A, SET1B, MLL1,

MLL2, MLL3, MLL4

H3K27 KMTsEZH1, EZH2

H4K20 KMTsSUV420H1, SUV420H2,

SETD8

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H4 tail


Review Soumyanarayanan & Dymock

S-adenosyl methionine (SAM) to methylate their spe-cific targets, comprise of over 40 members and have been classified in Figure 1 [6]. Cofactor (methyl donor) SAM and substrate histone bind to their respective pockets on the HKMT enzyme with surfaces facing opposite to each other. Transfer of a methyl group from the cofactor SAM to lysine residues present on the histone (H3 and H4) leads to the formation of monomethylated lysines, H3K9Me1 or dimethylated lysines, H3K9Me2, with S-adenosyl- l -homocysteine (SAH) as the byproduct (Figure 2) [7]. Different enzymes in the MT family are specific to methylating only certain lysine residues (K4, K9, K27 or K36); the specificity also includes the capa-bility to either mono-, di- or tri-methylate the substrate lysine (K) residues on the histones (3 and 4). The resul-tant products of the MT catalysis, for example, H3K9Me (monomethylation of lysine 9, histone 3), H3K9Me2 and H3K27, are associated with the activation and repression of certain genes. Some of the SET domain-containing enzymes are also known to methylate other protein targets which re gulate signaling pathways [6].

HKMTs have been classified according to the spe-cific lysine residues that they modify such as H3K9 or H3K27. Functional aberrations in HKMTs, in addi-tion to abnormal levels of methylated lysine residues, have been associated with a variety of cancers [8]. In this review, we discuss two of the most studied MT oncotargets, EZH2 and G9a (EHMT2).

EZH2 MT, together with of a group of four pro-teins, forms the Polycomb repressive complex 2 (PRC2) and mediates gene repression via trimeth-ylation of H3K27 [9,10]. EZH2 has been found to be especially upregulated in breast and prostate carci-noma, while mutations on Y641 and A677, residing in the EZH2 catalytic domain, are implicated in B-cell lymphoma [11].

G9a (EHMT2) is another MT that causes transcrip-tional silencing, acting via mono- and di-methylation of H3K9, by forming a complex with GLP [12,13]. Apart from the catalytic SET domain, MTs contain an additional characteristic domain called the ‘ankyrin repeats’, which facilitate protein–protein interactions and are known to bind to the methylated lysine prod-ucts [14]. G9a is implicated in a multitude of cancers such as breast, lung and melanoma, among others, by promoting tumor growth, affecting cell cycle or metabolism pathways [15]. Evidence is emerging, sup-porting dual blockade of both EZH2 and G9a with the first such inhibitors being recently reported. Sev-eral EZH2 inhibitors have recently been admitted into clinical trials. In this review, we highlight progress in drug discovery of both EZH2 and G9a inhibitors with a focus on the latest progress reported in the past 3 years. Both peer-reviewed literature and published pat-ent applications have been included to give the reader as full a picture as possible of this exciting field.

EZH2 inhibitors2-Pyridone based EZH2 inhibitorsIn 2012, a high throughput screening assay by Epi-zyme (MA, USA) led to the development of an excit-ing small-molecule EZH2 inhibitor EPZ005678 (Figure 3) [16]; one of the first containing the 2-pyri-done moiety, which, combined with a benzamide linkage, has become an accepted standard ‘war head’ for the majority of reported EZH2 inhibitors. Dis-playing a potency of 54 nM against PRC2 complex, compound 1 served as a starting point for the develop-ment novel chemotypes for EZH2 inhibition. Enzyme kinetic studies revealed EPZ005678 to be an SAM competitive inhibitor; its effects on cell growth and H3K27Me3 inhibition being much more prominent

Figure 2. The methylation reaction [5].

S-adenosyl methionine

OS+Me

HO OH

NH3+

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N

NN

NH2

-O2C

OS

HO OH

-O2CNH3

+

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Lysine methyltransferaseenzyme

S-adenosyl homocysteine


Review

in lymphoma cell lines bearing the EZH2 A677G mutation than with lymphomas associated with e levated EZH2 wild-type (WT) [16].

The most recent publication by Epizyme on develop-ment of EZH2 inhibitors illustrates the ironic connec-tion between chemical and biological methyl groups: introduction of methyls in the chemical structures is to remove the methylation biomarker H3K27Me3 by whopping five orders of magnitude [17]. The critical 2-pyridone moiety was connected to fused bicyclics such as indazoles or azaindazoles. Focus on position-6 led to the identification of a phenylmethyl morpholine as crucial to enhancing EZH2 inhibitory activities in vitro. On the other hand, the critical 2-pyridone moi-ety did not tolerate introduction of methyl groups at the 5-position. The indazole nitrogen tolerated a wide vari-ety of substituted rings, although ionizable free amines like piperidine were less well accommodated. Discon-necting the indazole and thus opening the cycle led to EPZ-6438 (tazemetostat, 2) (Figure 3) that exhibited tenfold better potency in inhibiting H3K27Me3 and is currently being studied in clinical trials as a poten-tial therapy for hematological malignancies (see later d iscussion on clinical stage compounds).

Knutson et al. studied the biological effects of EPZ-6438 in great detail as a potential therapy for non-Hodgkin’s lymphoma (NHL) [18]. EPZ-6438 was found to be most effective against lymphomas characterized by EZH2 mutations. Without signifi-cantly affecting other methylation sites, EPZ-6438 eliminates H3K27Me3 at a concentration of 0.1 μM inducing cell death via apoptosis. This was further corroborated with an in vivo tumor xenograft mouse model that demonstrated its ability to remove the H3K27Me3 marker with only 7 days of treatment (160 mg/kg, three-times a day [t.i.d.]), and complete ablation of the tumor in 28 days. In a further patent application (WO2015195848), Epizyme has claimed the use of EPZ-6438 in NHL including among others Diffuse Large B-cell Lymphoma (DLBCL) and follic-ular lymphoma [19]. This patent discloses very promis-ing clinical data (see Table 1).

Zhang et al. reported analogs of EPZ-6438 by fusing

the 2-pyridone with cyclohexane and further modifying the benzamide substitution pattern [20]. The compound, SKLB-1049 (3, Figure 3) although 30-fold less potent than its parent molecule EPZ-6438, exhibited nanomo-lar potencies against three EZH2 forms (WT, Y641N and A677G). While SKLB-1049 could eliminate the methylation mark H3K27Me3 in lymphoma cell lines (SU-DHL-6 and Pfeiffer) at 5 μM, the doses required to produce phenotypic changes (cell killing) were three-times higher. SKLB-1049 ranked higher than EPZ-6438 with regard to solubility in all the solvents tested; importantly, water solubility being 60-times more.

Epizyme continued their research on EPZ-6438-based chemotypes keeping the core benzamide intact, further modifying other substituents in order to achieve a balance of potency and other physicochemical param-eters [17]. This led to the identification of a very potent molecule (EZH2 IC

50 <3 nM), EPZ-011989 (4) which

replaces one of the biaryl rings with acetylene thus retaining the orientation of the attached N-methyl mor-pholine group. The in vitro H3K27Me3 inhibition in WSU-DLBCL-2 cells was further validated by pharma-codynamics results in a Karpass-422 mouse xenograft model, which showed significant reduction of the bio-marker after 7 days of oral administration at 250 mg/kg. Complete disappearance of tumor was observed on the 22nd day of continuous dosing.

Subsequent to the discovery of EPZ005678, Konze et al. modified benzoylpyrazoles primarily by modifying N-cyclopentyl to N-isopropyl and replacing the 6-position phenyl with pyridine resulting in a series of analogs having <10 nM potency against EZH2 [21]. Furthermore, morpholine was replaced with N-isopropyl piperazine, the larger group hypothesized to be more sol-vent exposed. Compound UNC1999 (5, Figure 3) which was subjected to extensive biological profiling is a prom-ising competitor to EPZ005678 and GSK126 [22]. Along with controlling proliferation of DLBCL cells (mutant EZH2 Y641N) and causing minimal toxicity to normal breast cells (MCF10A), pharmacokinetic studies revealed that at 50 mg/kg dose UNC1999 displays excellent oral bioavailability in male Swiss albino mice.

In 2014 (US20140107122), Epizyme published bio-

Figure 3. EZH2 inhibitors by Epizyme. DB: Diffuse B; EZH2: Enhancer of zeste homolog 2; PRC2: Polycomb repressive complex 2; WT: Wild-type.

EPZ005678 (1)

EZH2 (PRC2) IC50 = 54 nMH3K27Me2 IC50 = 2.9 µMDOHH2 (EZH2 WT) IC50 = 3.8 µMPfeiffer (EZH2 A677G) IC50 = 0.0052 µM

Proposed binding mode

NN

HN

O

HNO

N

O

EPZ-6438, tazemetostat (2)

H3K27 IC50 = 0.2 µM

WO 2015195848

SKLB-1049 (3)

EZH2 (WT) IC50 = 7.3 nMEZH2 (Y641N) IC50 = 39 nMEZH2 (A677G) IC50 = 5.9 nMSU-DHL-6 IC50 = 16 µMPfeiffer IC50 = 12.5 µM

WO 2013138361

EPZ-011989 (4)

EZH2 (WT) IC50 <3 nMEZH2 (Y641N) IC50 <3 nMH3K27Me3 (HeLa) IC50 = 94 nM

US 20140107122

SolventExposure

UNC1999 (5)

EZH2 IC50 <10 nMDB cells IC50 = 633 nM

EZH2 (WT) IC50 = 0.9 nMEZH2 (A677G) IC50 = 0.8 nMEZH2 (A687V) IC50 = 1.1 nM

8

9

WO 2015200650

EZH2 (WT/Y641N) IC50 <100 nM

7

EZH2 IC50 <0.01 µMH3K27 IC50 = 0.058–0.150 µMWSU IC50 = 0.325 µM

H3K27 IC50 = 0.077 µMWSU IC50 = 0.023 µM

6

HIE689

ASN688

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HNO

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Review

chemical and in vivo data of three benzamide-derived EZH2 inhibitors [23]. The compounds were found to be submicromolar potent against inhibiting the EZH2 PRC2 protein complex in vitro, as well as controlling cellular proliferation and global H3K27Me3 levels in a DLBCL cell line, WSU. Apart from having a moderate intrinsic clearance rate of 42 ml/min/kg, compound 7 was also found to inhibit the cytochrome P450 enzymes CYP2D6 and CYP3A4 at 10 μM. When compound 6 was tested in a Karpas-422 mouse xenograft model of cancer, tumor volumes were significantly reduced f ollowing 28 days of oral treatment.

A patent application (WO2013138361) by Epi-zyme in 2013 covers 75 very potent (<100 nM) EZH2 inhibitors exemplifying different scaffolds connected to 2-pyridones [24]. These compounds were tested against WT EZH2 as well as three of its mutant forms namely, Y641F, A677G and A687V; the mutants being char-acterized by elevated H3K27Me3 levels and mainly detected in B-cell lymphomas. Most of the compounds were found to inhibit these mutant EZH2 forms at simi-lar concentrations, although no particular trend across the mutants was observed for the inhibitors. Scaffold hopping efforts retaining the pyridone moiety resulted in approximately 5 nM phenyl and biphenyls attached to different saturated heterocycles. The most potent com-pound is a heterobiaryl (8, Figure 3) with a potency of 0.9 nM against WT EZH2.

In a recent patent application (WO2015200650), Epizyme studied the importance of the critical 2-pyri-done, a substructure common to almost all the potent EZH2 inhibitors reported as potential anticancer thera-peutics [25]. Compounds were assessed for their EZH2 inhibitory activities in PRC2 enzyme assays using two peptide substrates: EZH2 WT and one of its mutant forms (Y641F). The significance of the 2-pyridone moi-ety connected to a benzamide was established by sub-jecting it to different types of modifications. Reduction of the pyridone ring or its replacement with 2-pyrazo-lones completely abolishes the activity, regardless of the substitutions on the benzene ring. The 2-position of carbonyl group is essential to keep the activity intact, although changing the 6-position methyl to methoxy enhances the potency. The most potent compound of the series 9 (<0.1 μM) (Figure 3) possessed an indole-3-carboxamide linked to the 2-pyridone, and interest-ingly the racemic mixture of this compound as well as its resolved stereoisomers was found to be equally active.

In 2013, Constellation Pharmaceuticals (MA, USA) published 206 compounds in a patent application (WO2013075083) claiming variations around aryl ether linkages [26]. Synthesis and chemical structures were revealed only for half of the library. There was lit-tle correlation between the potency of the compounds

against EZH2 WT and the Y641N mutant forms, for example, compound 116 in the patent) displaying sub-micromolar potency against EZH2 WT had no inhibi-tion against EZH2 Y641N at the highest concentration tested. Compounds having p-aryl/p-alicyclic substitu-tions on the benzamide, for example, 10 (Figure 4) were found to display potencies between 1 and 10 μM. The nature of connection between the benzamide and the aromatic rings was found to be an important fac-tor; methyl ethers (11)/methyl amines were found to be significantly better than only oxygen/nitrogen link-ers. Furthermore, in the case of the most potent com-pounds, replacing benzene with p yridine, retained the EZH2 inhibitory activity.

A library of 170 indole-3-carboxamides that were evaluated against EZH2 WT, its mutant Y641N as well as for their ability to cause inhibition of the characteris-tic methylation marker H2K27Me3 in HeLa cells was recently reported in WO2015023915 by Constellation Pharmaceuticals [28]. The indole nitrogen is connected to 4-methyl piperidine; with most of the variations in the molecules centered on different aryl substitu-tions like p-fluorobenzyl 12 (Figure 4) and aliphatic substitutions attached to the N-1 piperidine. Most of the compounds were <400 nM potent against EZH2 WT/Y641N, although the doses for cellular biomarker inhibition varied and were found to be >2 μM in many cases, indicating the interplay of multiple factors in con-tributing to a biological response inside the cell. Data for some compounds are not reported and there is no general trend with respect to the nature of substituents, although longer and bulkier groups on piperidine were not tolerated. Two close analogs of 12, differing only in the N-piperidine substituent, were tested in the Kar-pass-422 mouse xenograft model, described in another patent application WO2014124418 [29]. The two com-pounds (e.g., 362 and 365 from the patent) gave >90% tumor growth inhibition when tested at 160 mg/kg. These compounds also had IC

50 values of <100 nM for

EZH2 and <400 nM for H3K27 methylation in cells.In 2015, Gehling et al. reported a series of submi-

cromolar potent indole derivatives as EZH2 inhibi-tors [27]. The inhibitors were assessed for their activities on EZH2 WT and Y641N mutant, and their ability to inhibit biomarker H3K27Me3 in HeLa cells. A diverse range of heterocycles attached to 2-pyridones was tested in vitro against EZH2, indicating 2-methyl indoles as an optimal scaffold for further development. Within a limited series, indole nitrogen substitutions with satu-rated heterocycles, similar to the previous example from Constellation, were found to be at least tenfold more potent than substituents with aromatic rings. Of par-ticular interest was the N-ethylsulfonamide piperidine derivative 13 (Figure 4), which, owing to its optimal

Figure 4. EZH2 inhibitors by Constellation Pharmaceuticals and Gehling et al. EZH2: Enhancer of zeste homolog 2; WT: Wild-type; TGI: Tumor Growth Inhibition.Data taken from [27].

WO 2013075083

WO 20150023915

1011

EZH2 (WT) IC50 = 1–10 µMEZH2 (Y641N) IC50 = 10–50 µM

EZH2 (WT) IC50 = 0.002 µMEZH2 (Y641N) IC50 = 0.003 µMH3K27 IC50 (HeLa) = 0.080 µM

EZH2 (WT and Y641N)IC50 <1 µM

12

EZH2 IC50 (WT/Y641N) <400 nMH3K27Me3 EC50 <400 nM

WO 2014124418

Example 362: R = CF2 (TGI = 92.74%)Example 365: R = CF3 (TGI = 97.25%)

13

OHN

O

HNO

HN

O

HNO

O

HNO

ONH

O

N

N

F

HNO

ONH

O

N

NR

HNO

ONH

O

N

NSO

O



balance of potency and other pharmacokinetic param-eters, was advanced to preclinical testing in mice. In a Karpass-422 mouse xenograft model, compound 13 reduced H3K27Me3 levels significantly, while also causing a dose-dependent reduction in tumor volume.

GlaxoSmithKline (GSK) previously reported their clinical candidate, GSK126 (Figure 5), in a seminal paper in 2012 [22] and prior to that in a published pat-ent application (WO2011140324) (e.g., 270) [30]. In a more recent patent application, WO2015143424 [31], GSK disclosed the discovery that tumors bearing a t(4;14) translocation in the methyl transferase multiple myeloma SET domain (MMSET), which methylates

lysine 36 on histone H3, H3K36, to give H3K36Me2 and/or a decrease in the levels of UTX, a histone demethylase protein, are particularly sensitive to EZH2 inhibitors. An increased level of MMSET also leads to an increase in H3K27Me3, the product of EZH2. Cells overexpressing MMSET display an increased sen-sitivity to EZH2 inhibitors. Around 20% of multiple myeloma cases overexpress the MMSET gene due to the t(4;14) translocation. In this patent, GSK research-ers have impressively elucidated the mechanisms by which MMSET alters gene expression and the interplay between H3K36 and H3K27 methylation in t(4;14) myeloma. A wide range of EZH2 inhibitors are claimed

Figure 5. EZH2 inhibitors reported by GlaxoSmithKline.

clinicalcandidate

WO 2015143424

Compound ‘C’Compound ‘B’(GSK-126 – clinical candidate)

15

GSK343 (17)GSK926 (16)

H3K27Me3 IC50 (nM) = 174 nMH3K27Me3 IC50 (nM) = 324 nM

14

NR2

R1

O NH

NH

OHN

O

HNO

N

N

N

HN

HN

O

HNO

N

O

N

O

NN

O NH

HNO

NN

N N

O NH

HNO

NN

NN


Review

for this application, most prominently GSK126 (14, Figure 5), which has now entered the clinic (see Table 2).

In 2012, GSK reported two indazole derivatives – GSK343 and GSK926 (Figure 5) – connecting pyr-idine-containing biaryl systems to 2-pyridones [32]. Both these SAM-competitive drugs, GSK926 (16) and GSK343 (17), exhibited <10 nM (K

iapp) potency against

EZH2 and were found to have over 100-fold selectivity when tested against a panel of MTs. Although the bio-marker H3K27Me3 IC

50s in breast cancer HCC1806

is <500 nM, the doses for producing 50% cell growth inhibition are at least 25-fold higher (∼10 μM).

In 2013 (WO2013173441), GSK claimed a series of low-molecular-weight 2-pyridones targeted at inhibit-ing cellular proliferation in EZH2-related cancers [33]. A limited set of compounds were exemplified bearing n-propyl or benzyl and the 4-position of the pyridine (18, Figure 6). Replacing the propyl group with a ben-zyl at the 4-position of the pyridone led to a tenfold increase in the potency. They examined the 5-position of the benzamide phenyl ring with different substitu-ents which was found to be a crucial factor: a change from cyano group to bromo enhanced the potency sig-nificantly. Replacing bromo with a much larger pipera-zine substituted pyridine led to 19 (Figure 6), one of the more potent molecules in this series.

In 2013, GSK filed a patent (WO2013039988) on

a library of 125 azaindazoles as EZH2 inhibitors with potential utility in anticancer therapy [34]. The com-pounds showed a broad range of EZH2 inhibitory potencies (IC

50 = 18–5747 nM), except 20 (25 μM)

(Figure 6) that bore a sulfonamide at the N-1 posi-tion of the azaindazole. In the 2-pyridone moiety, C-5 substitution (4647 nM) was unfavorable leading to a 15-fold drop in potency. Although C-6 could not with-stand bulky groups, aromatic and saturated cycles such as the cyclopropyl group at C-4 rendered more potent molecules.

Similar to the aforementioned compounds from GSK, GSK926 (16) and GSK343 (17) (Figure 5), position-6 of the azaindazole scaffold was found to be amenable to a wide variety of substitutions. While an unsubstituted pyridyl group at this position resulted in the most potent compound 21 (18 nM) (Figure 6), most of the pyridine-substituted compounds also exhibited potencies of <100 nM. Barring a phenyl ring, most of the described aryl ethers, amines, heteroaromatic and fused substitutions were well tolerated at C-6 of the azaindazole scaffold.

Because of the unavailability of the inhibitor bound crystal structure of EZH2, KaliniĆ et al. developed an EZH2 model using available crystal structures and examined enzyme–inhibitor interactions contributing to EZH2 binding and thus inhibition [35]. From the

Figure 6. EZH2 inhibitors by GlaxoSmithKline. EZH2: Enhancer of zeste homolog 2.

WO 2013173441

HNO

R

HN

O

X

HN

R = n-propyl, BnX = CN, Br

18

20

EZH2 IC50 = 25 uM EZH2 IC50 = 18 nM

EZH2 IC50 = 40 nM

21

WO 2013039988

19

N

N

HN

N

O

HN

HNO

NH

HNO

O

N

NN

HNS

OO

NH

HNO

O

N

NN

N



published structure–activity relationship (SAR) and the binding pattern of SAM, they deduced the pos-sible hydrogen bonding interactions crucial for EZH2 inhibition (Figure 7). The two closely situated amide NHs in the core structure are hypothesized to interact with H689/N688, the significance of which is exempli-fied by the catastrophic loss in activity on methylating the two NHs. Substitutions at N-1 and position-6 that project toward the lysine binding channel and solvent, respectively, also play a significant role.

In 2014 (WO2014195919), GSK disclosed an exten-sive series of 85 macrocycles featuring an olefin linker connecting a substituted phenyl with a 2-pyridone [36]. The compounds were synthesized using Grubb’s cata-lyst via a ring-closing metathesis reaction and were evaluated for their EZH2 inhibitory efficacies within the PRC2 complex. The presence of the double bond in the five-carbon chain provided an opportunity to study the effect of geometric isomerism and substitutions. Although saturation of the alkenyl bond had minimal effect on the activity, the spatial arrangement of substit-uents appears to play a key role. For example, E-isomers were found to be 80–100-times more potent than their respective Z-isomers (22, 23, Figure 8). To optimize the nature of the alkenyl carbon chain, a six-atom linker was compared with the effect of replacing one of the carbon atoms with heteroatoms like oxygen and nitrogen. While

longer carbon chains maintained the potency (IC50

= 40 nM), introduction of ether/amine in the linker resulted in a remarkable drop in the EZH2 inhibitory activity. Adding methyl or hydroxymethyl substituents to the chain reduces the potency by four to eightfold, which could be due to unfavorable interactions. On the benzene ring, replacing the cyclohexyl with a methoxy group led to about tenfold reduction in EZH2 inhibi-tion. Presence of piperidine substitutions at this position provided a good boost to the activity, while the most potent compound 24 (Figure 8) was d istinguished by a geminal difluorocyclohexane ring (Z).

GSK further reported (WO2015132765) six mol-ecules analogous to the previous, by encompassing an indole ring into the macrocyclic structure [37]. Keeping the pyridone moiety unaltered, C-6 chloro indole with an E-double bond 25 (Figure 8), as well as bulky groups like piperazine substituted pyridine (IC

50 = 40 nM)

exhibited good potencies. Other substituents such as cyano and alkyne were slightly less potent. Similar to the phenyl series, the E-isomer was found to be eight-times more potent than the Z-isomer. Saturation of the double bond was accompanied by methylation of the pyridone and hence the tenfold drop in potency could be due to one or either of these changes. Indole macro-cycles are an interesting novel scaffold; however, with only a small set of compounds studied, it is challenging

Figure 8. EZH2 inhibitors reported by GlaxoSmithKline. EZH2: Enhancer of zeste homolog 2.

Figure 7. Proposed binding pattern of indazole-based EZH2 inhibitors.

Lysine bindingchannel

Solventexposed

H-bonding to N688side chain

H-bonding to H689side chain2

13

45

6

7

Y

XN

R3

R2

HN

O

HNO

R1

WO 2014195919

WO 2015132765

WO 2014177982

Z-isomer E-isomer E-isomer

EZH2 IC50 = 32 nMEZH2 IC50 = 2512 nM

EZH2 IC50 <10 nM

EZH2 IC50 = 16 nM EZH2 IC50 = 40 nM

22 23 24 25

26

EZH2 IC50 = 200 nM

NHO

Cl O

NH2

HNO

NHO

Cl O

NH2

HNO

NHO

N

HNO

F F

NHO

HNO

Cl N

HNO

NH

O

S

NO

NO

O

HNO

HN

O

S

N

27


Review

to assign the importance of the different functionalities.A series of thiophenamides as potential EZH2

inhibitors claimed by GSK (WO2014177982) were evaluated for their potencies by scintillation proxim-ity assay (SPA) using a biotinylated H3 substrate [38]. Replacing the thiophene ring with N-substituted pyr-azoles proved catastrophic to the EZH2 inhibitory activities; hence, the structural variations were focused on position-5 of the thiophene-3-amides. C-5 does not tolerate the introduction of bulky groups consist-ing of multiple rings, although thiophene is agreeable to accommodating substituents at all four positions (26, Figure 8). Chiral centers present in piperidine or cyclohexane side chains appeared to be important: ste-reoisomers having 30-fold difference in the activities. Furthermore, presence of a double bond at the carbon linked to both the thiophene and the saturated rings led to compounds with potency below 10 nM (27).

A patent application (WO2014049488) published by Pfizer (NY, USA) features 150 derivatives of ben-zamides and heterobenzamides as EZH2 inhibitors for the treatment of abnormal cellular proliferation disor-ders like cancer [39]. The compounds were synthesized



by HATU (amide-coupling agent) mediated amide couplings between aminomethyl-2-pyridones and ben-zene/pyridine/pyrimidine carboxylic acids. The com-pounds were assessed for their potency mainly against WT EZH2 but also against EZH2 mutant Y641N for a selected set of compounds. The inhibitors exhibited a wide range of activities (IC

50 = 15 nM to 200 μM);

IC50

s being 10–100-times higher against mutant EZH2 Y641N. Most of the phenyl derivatives with substituted 3-imidazoles displayed submicromolar potencies. Presence of bulky groups like pyrimidines substituted like in 28 (Figure 9) with saturated hetero-cycles were favorable to promoting EZH2 inhibition. In heterobenzamide derivatives such as pyridines and aminopyrimidines, the presence of a basic nitrogen remarkably lowered the potencies, compared with their nonbasic phenyl counterparts. Further, fusing of the 2-pyridone head group with cyclohexyl group was found to be unfavorable.

Pfizer claimed a series of dihydroisoquinolinone-substituted pyridines in a recently published patent application US20150361067 [40]. Over 200 compounds were exemplified with a focus on extensive exploration of the 7-position of the dihydroisoquinolinone ring, but also important SAR was seen with changes to the 4-position of the pyridone ring. In general, the inhibi-tors were 2–200-times more potent against EZH2 WT compared with its Y641N mutant form, exhibiting some of the highest EZH2 potencies yet recorded.

Replacement of the pyridone 4-methyl substituent with methoxy and fluorinated derivatives gives com-pounds with contrasting metabolic profiles (OCHF

2

and OCH3); the labile OCH

3 analogs were remarkably

more potent than methyl or its fluorinated analogs.Turning to the dihydroisoquinolinone, 7-position

hydroxymethyl analogs yielded some very potent com-pounds including a range of chiral tetrahydrofuran-derived side chains; some with low nanomolar potency against both WT and the Y641N mutant forms of EZH2. Further substitution of the C-7 methenyl inves-tigated the effect of heterocycles (O, N and S). Although most substituents (including N-formyl and ketones) were well tolerated, carboxylic acid (7.81 μM) proved detrimental, resulting in an 8000-fold drop in potency. Multiple isomers resulting from two chiral centers in the molecules exhibited striking differences in their inhibi-tory potencies – the effects being more prominent than changes in ring size, heteroatom or the point of linkage. A 200–500-fold difference of potencies in some isomer pairs demonstrates the significance of spatial orienta-tion of the molecule critical to EZH2 binding. Dihy-droisoquinolone flanked by C-7 methylazetidine and 4-methoxypyridone on either side resulted in the most potent molecule 29 (EZH2 IC

50 = 10 pM, Figure 9).

In a patent application (WO2014155301) published in 2014, Piramal Enterprises (Mumbai, India) intro-duced a series of biaryls consisting of N-substituted indolines linked to 3-pyridines, targeting specifically EZH2-regulated cancers [41]. Biological data against EZH2 were reported in three potency ranges only for selected examples. The most critical part of the mol-ecule, the 2-pyridone bore various alkyl groups at the 4-position, similar to indazole from UNC (5) and GSK (17). Indolines withstood a wide variety of sub-stituents; varying the length of N-alkyl and cycloalkyl substituents as well as spirocyclopentyl at the 3-posi-tion yielded drugs with submicromolar potencies, for example, 30 (Figure 9). On the other hand, substitu-tion of the pyridine of the biaryl system favored only heteroalicyclic rings. Replacing these heterocycles with smaller groups like CF

3 proved fatal to the biological

activity. Disrupting the core of the biaryl system, such as replacing pyridine with benzene or indoline with cyclopentenylpyrimidine, for example, 31 (Figure 9) destroys the EZH2 inhibitory activity.

In two patents applications (WO2015110999 and WO2015104677), Piramal Enterprises revealed a set of around 300 compounds that were evaluated for EZH2 inhibitory activities in vitro by an Alpha-LISA assay that measures the rate of formation of H3K27Me3 [42,43]. Biological data are only provided for a subset of compounds categorizing them as + (51–100 nM) and ++ (<50 nM). This work again demonstrates that potent EZH2 inhibitors can be developed by linking the critical 2-pyridone moiety to a system of planar biaryls via a carboxamide bond. Five- and six-membered alicyclic rings such as piper-idine fused to the 2-pyridone, as in compound 32 (Figure 9) did not significantly change the potency but did provide a novel core structure with a basic center which may increase solubility. The biaryls consist of a sterically congested phenyl ring linked to different aromatic rings such as pyridine, pyrazole or phenyl. This series was further expanded into N-alkyl indoles substituted by pyridine connected to various saturated heterocycles like piperidine, piperazine or morpholine (33) led to compounds with IC

50 <50 nM.

Bristol-Myers Squibb (BMS [NJ, USA]) published a patent application (WO2015077193) describing EZH2 inhibitors with 2-pyridones fused with substituted phe-nyls giving dihydroisoquinolones, while also modifying the biaryl system [44]. Modification of the dihydroisoqui-nolone was found to be ring specific; phenyl was found to tolerate being reduced to cyclohexane, although pyri-done reduction deteriorated EZH2 inhibition capaci-ties as expected. This dataset also highlights that posi-tions-6 and -8 of the dihydroisoquinolone are agreeable

Figure 9. EZH2 inhibitors by Pfizer and Piramal. EZH2: Enhancer of zeste homolog 2; WT: Wild-type.

WO 2015193765 or US 20150361067

2829

30 31

32 33

WO 2014049488 (Pfizer)

WO 2014155301

EZH2 (WT) IC50 = 35 nMEZH2 (A641N) IC50 = 300 nM

EZH2 IC50 = 0.001–1 µM EZH2 IC50 = 10–50 µM

EZH2 IC50 = 0.01–50 nMEZH2 IC50 = 0.1–50 nM

EZH2 (WT) IC50 = 10 pMEZH2 (Y641N) IC50 = 2 nM

HNO

NH

ON

N

HN

NN

HN

O

O

N

OCl

Cl

N

HNO

NHO

NO

N N

N

HNO

NHO

N

N

N N

N

O

O

HNO

HN

O N

N

O

N

O

HNO

HN

O

N

N

N

O


Review

to hosting both electron-donating and -withdrawing substituents like F (34), OCH

3, CF

3 and CH

3.

BMS also developed another series of 2-pyridones by exploring various heteroaromatics in the biaryl system such as pyrimidine and pyridine (WO2015077194) [45]. These compounds were assessed for their EZH2 inhib-itory activities by an SPA which detects the incorpora-

tion of radioactive methyl groups. Dihydrobenzofuran linked to pyridine represented the core structure in most of the potent compounds (IC

50 <0.5 μM). Expan-

sion of furan to 6- or 7-membered rings or replacement of the gem dimethyls was found to impair effective inhibition of EZH2. The second part of the biaryl sys-tem is comprised of 3-pyridine attached to a saturated



heterocycle like piperazine, 35 (Figure 10) or morpho-line; replacing these heterocycles with smaller groups like methoxy was found to be unfavorable. Most of the other less potent compounds (>1 μM) were either biphenyls or lacked the essential substitutions on the dihydrobenzofuran.

In a Chinese patent (WO2015141616), Daiichi San-kyo (Tokyo, Japan) revealed 76 examples of compounds linking substituted 1,3-benzodioxoles to pyridones via an amide bond [46]. The drugs were evaluated for their ability to inhibit the two isomers of EZH (1/2) and H3K27Me3 – an end point marker of their catalytic activity. The modifications were primarily focused on the chiral carbon of the 1,3-dioxole, with some compounds having minor changes on the benzene ring. All the com-pounds were submicromolar potent in the three assays; most of the compounds were either equipotent against the EZH isoforms or were more potent against EZH2. Compound 36 (Figure 10) was found to be equally active against EZH1 and EZH2 (∼8–9 nM) and impressively potent in inhibiting H3K27Me3 (IC

50 = 0.48 nM).

4-Pyridone-based EZH2 inhibitorsIn a patent application (WO2014107277) published in 2014, GSK revealed a small set of 20 compounds as EZH2 inhibitors aimed at controlling abnormal proliferation of cancer cells [47]. The molecular skel-eton consists of pyridones linked to benzamides; one of the distinguishing features of the pharmacophore is the presence of 4-pyridones, as opposed to most of the other reported inhibitors that bear the carbonyl group at the 2-position. The primary focus was 3-posi-tion substitutions on the benzamide phenyl ring with different saturated rings through oxygen or nitrogen; however, aryl ethers were significantly less potent than aryl amine linkages. Chloro substitution of the phenyl was found to be tenfold more potent than the bromo analog. The most potent compound (IC

50 = 3 nM) bore

a stereospecific dimethylaminocyclohexane attached to the benzamide through a tertiary amine linkage (37).

In another patent application (WO2015004618), GSK introduced novel 4-pyridone thiophenecarbox-amide derivatives aimed at different types of cancer that are mediated by EZH2 [48]. In this case, these compounds distinguish themselves with a 3-amino pyridine substituent. The 3-aminomethyl-4-pyridones were coupled with substituted thiophene-3-carboxylic acids. EZH2 inhibitory assays were reported using two different types of peptide substrates: mononucelosome-containing histone H3 (Assay 1) and biotinylated pep-tide substrate H3 (Assay 2). Compound 38 (Figure 11, 16 nM), the best performer in both the assays, has an amino group attached at the 3-position of the pyridone ring, the absence of which led to a compound which

was found to be sevenfold less potent in the second assay (IC

50 = 100 nM).

Nonpyridone-based EZH2 inhibitorsConstellation Pharmaceuticals have elaborately dis-cussed the hit-to-lead optimization of 39 (Figure 12) as a SAM competitive inhibitor of EZH2, one of the first inhibitors devoid of the pyridone pharmacophore [49,50]. After identifying a suitable aryl ether linkage, they further modified the phenyl at the meta-position to form a biaryl system that was best complemented by a pyridazine ring. In vitro assays revealed a potency of 21 nM against EZH2, although its effects on H3K27 dimethylation and trimethylation were apparent only at approximately 30 μM on HeLa cells indicating potentially poor cell penetration; no growth inhibi-tion effects on prostate cancer cells were apparent. The 2-position cyano and immediate substituents around it were found to enhance EZH2 inhibition significantly; hence, they were hypothesized to form important interactions with the enzyme cavity.

A chemically distinct series comprising of 5-methoxy-quinolines, inspired by BIX01294 (G9a/GLP inhibi-tor), was reported as weak inhibitors of EZH2 by Xiang et al. [51]. These compounds were tested against the EZH2 enzyme, as well as against human cancer cells (HCT15 and MDA-MB-231). The compounds dis-played EZH2 inhibitory activities in the range of 5.6–39.7 μM and were similar against human cancer cells. There was little correlation between the biochemical and phenotypic assay results. The most potent EZH2 inhibi-tor 40 (Figure 12, IC

50 = 1.2 μM) inhibited H3K27Me3

inhibition at around 16 μM, with 20–70-fold selectivity against a panel of six other MTs.

Six Tanshinones obtained from the roots of Salvia miltiorrhiza were reported as EZH2 inhibitors by Woo et al. [52]. Two of the stereoisomeric diterpenoids, Tan-shindiol B and C, characterized by vicinal hydroxyl groups on ring A were found to be 10–50-times more potent than rest of the series. The activity of Tanshin-diol C 41 (Figure 12) widely varied depending on the type of cancer and cell line tested against. Increased levels of cleaved caspases, further to H3K27Me3 s uppression at 3 μM, were reported.

Docking studies were performed on the set of com-pounds against EZH2 SAM binding pocket and the interactions were compared with that of GSK126 (Figure 5), a known SAM competitive inhibitor. As established earlier, the NH of the 2-pyridones and piperazine in GSK126 form hydrogen bonding inter-actions with the C=O of the amino acid residues sur-rounding them. Though structurally diverse, Tanshin-diol C mimics the piperazine NH retaining the two interactions, and is postulated to form an additional

Figure 10. EZH2 inhibitors by Bristol–Myers Squibb. EZH2: Enhancer of zeste homolog 2.

Figure 11. 4-Pyridone EZH2 inhibitors reported by GlaxoSmithKline. EZH2: Enhancer of zeste homolog 2.

34

EZH2 IC50 <50 nM EZH2 IC50 <500 nM

EZH1 IC50 = 8.9 nMEZH2 IC50 = 8.0 nMH3K27Me3 EC50 = 0.48 nM

35 36

WO 2015141616

HNO

NH

O

F

N

O

N

O

HNO

NH

O

N

O

N

HN

HNO

HN

O

OO

N

WO 2014107277 WO 2015004618

37 38

EZH2 IC50 = 3 nM EZH2 IC50 Assay 1: 32 nM Assay 2: 16 nM

HN

ONH

O

Cl

N

N HN

ONH

O

NH2S

N

N


Review

hydrogen bond owing to the presence of hydrogen bond donors (-OH) in ring A. Although docking stud-ies support their binding mode being similar to that of known inhibitor GSK126, enzyme kinetics studies are necessary to validate their mechanism of inhibition.

Kung et al. reported a novel series of EZH2 inhibi-tors derived from the end product of the MT catalytic process, SAH [53]. They linked different amine sub-stitutions to the amino acid side chain, thus leading to amide bond bearing analogs of SAH. Most of the amine substituents having aromatic biaryl or fused rings yielded compounds with IC

50s higher than SAH itself

(11 μM). The only submicromolar potent derivative 42 (Figure 12) reported in the series comprised of a planar biaryl pyrazole methylamine system, which displayed excellent selectivity over SAH against other MTs.

EHMT2 (G9a) inhibitorsHigh-throughput screening of a large library of com-pounds against G9a (EHMT2) MT led to the dis-

covery of a small-molecule inhibitor BIX01294 (43, Figure 13) that served as a template for the development of more specific and potent G9a inhibitors [54]. Having a facile synthetic route, the BIX01294 scaffold offered primarily two positions (C-2 and C-4) for variations that were extensively explored to generate a series of 2,4-diaminoalkoxyquinazolines. The favorable substi-tutions at C-4 were limited to N-alkyl amino piperi-dines, other analogs were >10 μM against the target as determined by an AlphaScreen assay that detects the methylated histone mark H3K9Me2. Amine substitu-tions at the 2-position were better tolerated, although these compounds were less potent than BIX01294.

This necessitated variations in other parts of the quinazoline scaffold which was pursued initially by analyzing the available crystal structure of BIX01294 with GLP, a G9a-like protein that aids G9a in its func-tions by forming a complex with it. The BIX–GLP crystal structure revealed a tunnel space occupied by the substrate lysine present on histone (H3K9), which

Figure 12. Nonpyridone EZH2 inhibitors. EZH2: Enhancer of zeste homolog 2; WT: Wild type.



provided an opportunity to grow the molecule into the lysine channel to achieve specific inhibition and higher potency. To this end, they extended the methoxy ether linkage at position-7 to a three-carbon chain terminat-ing with a dimethyl amine. The prototype of this 7-sub-stituted series, UNC0224 (44, Figure 13), was fivefold more potent than BIX0129 in biochemical assays. The first ever reported G9a crystal structure, with UNC0224 bound in the peptide binding site, validated the lysine-binding channel occupancy hypothesis.

Liu et al. further continued to investigate position-7 of the quinazolines varying the linker size, terminating with different aliphatic and alicyclic groups [55]. This led to the discovery of UNC0321 (45, Figure 13) that displayed single-digit nanomolar potency in enzyme-coupled SAH detection and chemiluminescence-based oxygen tunneling assays that detect SAH and H3K9Me2 production, respectively, to quantify G9a inhibition. Although these reported inhibitors are found to affect a common end point biomarker (H3K9Me2), BIX was concluded to be a GLP-specific inhibitor, while UNC0224 and UNC0321 were found to be more spe-cific toward G9a. This finding affirms that the 7-posi-tion alkoxy linker with a dimethylamine terminus c onfers specificity and affinity to G9a inhibitors.

Although these molecules were potent G9a inhibi-tors, they appeared to lack desirable drug-like proper-

ties, such as low metabolism. To address these issues, Liu et al. developed efficient synthetic strategies to modify C-2, C-4 and C-7 positions with a wide variety of substituents [56]. Different substituted homopipera-zines were tested at position-2 of the quinazoline scaf-fold, although cyclohexane, the substituent that offered best potency and toxicity/function ratio, was devoid of the additional basic center. Iso-propyl was found to be best suited as a piperidine substituent due to optimal logP, a surrogate indicator of cellular permeability.

Finally, establishing position-7 with propoxy pyr-rolidine side chain led to the development of the most widely studied G9a inhibitor: UNC0638 (46, Figure 13). Apart from being equipotent against G9a/GLP and 600-fold selective over other epigenetic and nonepigenetic targets, UNC0638 also has lower cytotoxicity, thus offering a better therapeutic window. Kinetic studies in the presence of histone and SAM concluded that the drug is a histone peptide competitive inhibitor, which was further confirmed from the crystal structure of UNC0638 bound to G9a protein [57].

The cyclohexyl group at position-2 of UNC0638 pres-ents a classical metabolic hotspot, thus making it an inap-propriate candidate for in vivo studies. Liu et al. further modified position-2 with more robust substituents [58]. Most of the nitrogen-containing saturated heterocycles exhibited excellent potencies (<2.5 nM) against G9a.

EZH2 (WT) IC50 = 32 nMEZH2 (Y641N) IC50 = 197 nM

EZH2 IC50 = 1.2 µMHCT15 IC50 = 7.60 µMMDA-MB-231 IC50 = 2.45 µM

EZH2 IC50 = 0.55 µMPfeiffer IC50 = 1.5 µMDaudi IC50 = 10.6 µM

EZH2 (WT) IC50 = 0.27 µMEZH2 (Y641N) IC50 = 0.07 µM

39 40 41

42

O

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Figure 13. Reported G9a inhibitors. CLOT: Chemiluminescence-based oxygen tunneling; SPA: Scintillation proximity assay.

GLP (CLOT) IC50

G9a (CLOT) IC50

G9a (SPA) IC50

UNC0224 (44)BIX01294 (43)

UNC0642 (47)

250 nM

27 nM

57 nM

58 nM

UNC0321 (45)

6.0 nM

23 nM

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<2.5 nM <2.5 nM

A-366 (48)G9a IC50 = 3.3 nMH3K2Me2 EC50 ~3 µM

N

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F

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Review

UNC0642 (47, Figure 13) possessing a 4,4-difluoropi-peridine substitution at C-2 succeeded UNC0638. With a plasma half-life (T

½) of >90 min, intraperitoneal injec-

tion of UNC0642 at 5 mg/kg resulted in tenfold higher plasma concentrations than UNC0638 (46). Although these pharmacokinetic data support UNC0642’s util-ity as an in vivo probe for G9a/GLP, there are insuf-ficient pharmacodynamic data reported to derive any c onclusion about its in vivo efficacy.

The first potent nonquinazoline G9a inhibitor scaf-fold, reported by Abbvie (IL, USA), comprises of a novel amino spirocyclobutyl indole moiety [59]. Interestingly, the more potent molecules in this series, such as A-366 (48, Figure 13), preserve the right-hand side of the UNC G9a inhibitors; x-ray crystallography confirmed the interactions of the propoxy pyrrolidine side chain were in the lysine binding channel. The pyrrolidine nitrogen – an important hydrogen bond donor – when replaced with a carbon atom leads to a 13,000-fold drop in G9a inhibition, as measured by AlphaLISA® assay.

Like UNC0638, A-366 causes 50% reduction in H3K9Me2 levels in prostate cancer cells (PC-3) at 3 μM, although it exhibits no effects on cellular pro-liferation even at 10 μM when tested against an exten-sive panel of 38 cancer cell lines. This indicates that UNC0638 might be acting via additional mechanisms to affect cell viability, and thus highlights the draw-

back of highly selective monotargeted therapies (like A-366) that can fail to produce a desired phenotypic response. A-366 when tested in an acute myeloid leu-kemia flank xenograft model at 30 mg/kg/day for 14 days showed 45% inhibition of tumor volume, accompanied by a reduction in methylated histone marker levels [60]. The consensus of different biological assay results opines that a desirable clinical response in cancer can be realized by combining chemothera-peutic agents with A-366 as opposed to its utility as a single-agent regimen.

With much emphasis on the lysine-binding channel side chains, Vedadi et al. hypothesized that G9a inhibi-tion capacities will be retained by replacing the right-hand side of the molecule with equivalent chemical groups [61]. Pyrimidines with similar substituents as BIX01294 were fused with different heterocylic rings like furan, thio-phene and imidazole, and their G9a inhibitory potencies were assessed at three doses using SPA. The assay results refuted their hypothesis – even at 50 μM doses, most of the compounds could not cause significant G9a inhi-bition. The 6,7-dimethoxy groups forming hydropho-bic interactions were found to be indispensable to G9a inhibition; both desmethoxy and dioxolane analogs of BIX01294 did not contribute to G9a inhibition.

SAM competitive G9a inhibitors

Figure 14. EZH2–G9a dual inhibitors.

Compound 005 (49)

Lymphoma (WSU-FSCLL) IC50 = 3.405 µMBreast cancer (MDA-MB-231) IC50 = 4.3 µM

Compounds which upregulatedboth KRT17 and FBXO32:

011 005 022 BIX01294N

NR

OMe

OMe

HN

N Ph N

N

N

NN N

N

R =



There has been limited focus on developing SAM com-petitive inhibitors of G9a; the reported compounds have been found to have micromolar potencies [62,63], depending on the assay format. The most potent SAM competitive inhibitor Chaetocin that owes its potency to the presence of the disulfide linkage, is at least t enfold less potent than BIX01294.

Combinations of EZH2 with EHMT2 (G9a) inhibitionCurry et al. reported the synergistic effect of hitting two MT targets, namely EZH2 and EHMT2 (G9a), by a single molecule as a potential therapy for triple nega-tive breast cancer, as opposed to single target inhibi-tion [64]. The drug-treated cancer cells (MDA-MB-231) were evaluated for their growth inhibitory potencies as well as analyzed in a microarray to study the changes in gene expression pattern. Selected analogs of BIX01294 that qualified as dual (EZH2/EHMT2) inhibitors were found to upregulate three characteristic genes that are repressed by EZH2: KRT17, FBX032 and JMJD3 (demethylase) at 10 μM doses. These dual effects were neither observed with G9a inhibitor prototypes (BIX01294, UNC0638) nor with EZH2 inhibitors (GSK343). Drug 49 (Figure 14) at 2.5 μM was found to inhibit the levels of H3K9Me2 as well as H3K27Me3 – the characteristic methyl biomarkers of both enzymes. Interestingly, this drug was also found to be peptide substrate competitive with respect to EZH2, unlike all

the other reported inhibitors. These compounds were also protected in a patent application (WO2013140148) where they were disclosed to inhibit growth of BT474 and SKBR3 cell lines in the 1–2 μM range [65]. Further-more, the compounds were selective against the normal breast cell MCF10a (when its growth was stimulated with EGF) with inhibition in the concentration range 14–45 μM. This work is significant since it is not only an example of a nonpyridone EZH2 inhibitor but also the first example of a dual EZH2–EHMT2 inhibitor.

Apart from cancer, concerted actions by G9a and EZH2 MTs are also implicated in disorders like idiopathic pul-monary fibrosis [66]. The hypermethylation of histone tar-gets caused by these enzymes result in a cascade of events including DNA methylation and histone acetylation, eventually resulting in silencing of the antifibrotic gene COX-2. Treatment with G9a/GLP inhibitor BIX01294 and EZH2 inhibitor DZNep significantly reduces their respective characteristic methylation markers (H3K9Me3 and H3K27Me3), and they thus offer a promising thera-peutic avenue for the treatment of id iopathic pulmonary fibrosis by reactivating silenced genes.

EZH2 inhibitors in the clinicCurrently, there are three targeted EZH2 inhibi-tors registered in the clinicaltrials.gov database: tazemetostat (EPZ-6438, E7438), CPI-1205 (Con-stellation P harmaceuticals) and GSK2816126 (GlaxoSmithKline) (Table 2).

Table 1. Phase I clinical trial of tazemetostat in heavily pretreated patients non-Hodgkin’s lymphoma patients.

Lymphoma subtype Evaluable patients (n = 15)

DLBCL (n = 9) FL (n = 5) MZL (n =1)

CR + PR 5 3 1

SD 0 1 0

CR: Complete response; DLBCL: Diffuse large B-cell lymphoma; FL: Follicular lymphoma; MZL: Marginal zone nodule lymphoma; PR: Partial response; SD: Stable disease.Data taken with permission from [19].


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Specific clinical data are disclosed in a patent WO2015195848 by Epizyme (probably from the NCT01897571 Trial) where a population of NHL patients were treated with tazemetostat in some cases for over 1 year with complete response, partial response or stable disease in two-thirds of these heavily pretreated patients [19]. Of the 15 evaluable patients, 9 had an objec-tive response with 1 patient of the 5 DLBCL patients remaining on the study at 18 months and another with an EZH2 mutation at 6 months (Table 1). Two patients from the three follicular lymphoma responders remained on study at 12 months. A patient with marginal zone nodule lymphoma was on the study for near 12 months with a gradually improving partial response. Regarding adverse events, fewer than a third of patients suffered a grade 3 or higher adverse event with only five confirmed as treatment related. Thrombocytopenia was observed as the only dose-limiting toxicity and this occurred at the highest does of 1600 mg b.i.d. Regarding pharma-cokinetics exposures were linear across all dose ranges (100–1600 mg b.i.d.). Evidence of H3K27Me3 reduc-tions in the skin was observed at week 4 in all dose groups. This study provides very encouraging responses in patients with tazemetostat and the results from larger Phase II studies with this drug, and others, will be eagerly awaited.

Future perspectiveWith challenging pharmacokinetics and limited in vivo efficacy hampering the progress of potent com-pounds like A-366 and UNC0642 toward the clinic, it is likely that other chemotypes will populate the chemical space of future G9a inhibitors. EZH2 inhib-itor development has been progressing by leaps and bounds, despite the lack of a protein–inhibitor crystal

structure. Encouraging efficacy and safety data are now just beginning to be reported from clinical trials of lymphoma patients being treated with tazemeto-stat, the most advanced of the three EZH2 inhibi-tors. Further data with tazemetostat and other EZH2 inhibitors are eagerly awaited as the burgeoning field of MT inhibitors matures into c linical t esting.

The moderate in vivo efficacy reported for the most potent G9a inhibitor, A-366, elucidates the downside of selective inhibition of a single target, and thus empha-sizes the need for a paradigm shift in the approach to combating multifactorial diseases. The recent publica-tion describing EZH2–EHMT2 methyltransferase dual inhibition with a strong rationale holds promise for more efficacious therapies in oncology in the coming decade. Additionally, designing inhibitors against two targets affecting similar pathways, biomarkers and mechanisms of cell death can potentially lead to a therapeutic path of least ‘resistance’. With the success of selective multi-targeted kinase inhibitors and their combinations with other therapies in cancer, inhibitors of the epigenetic enzymes EZH2 and G9a are expected to venture into similar areas of multiple inhibition and combination therapy forging further into the clinic with a drug on the market in perhaps 5 years.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involve-

ment with any organization or entity with a financial inter-

est in or financial conflict with the subject matter or mate-

rials discussed in the manuscript. This includes employment,

consultancies, honoraria, stock ownership or options, expert

testimony, grants or patents received or pending, or royalties.


manuscript.

Executive summary

• Despite originating from a similar structural and functional class of enzymes, EZH2 and G9a methyltransferases present different challenges and stories of progress in the development of their inhibitors.

Development of EZH2 inhibitors• EZH2 inhibitors have made commendable progress reaching the clinic within a short span of time, and could

be a promising strategy for cancer therapy, particularly lymphoma.• Multiple patent applications and publications by pharmaceutical companies, primarily Epizyme,

GlaxoSmithKline and Constellation, have investigated and protected many potent small molecules that have performed well in preclinical testing.

• Most of the chemistry of the published inhibitors is based around 2-pyridones connected to aryl carboxamides, with limited efforts in the ‘nonpyridone’ inhibitor category.

Development of G9a inhibitors• Crystallized G9a–inhibitor complexes have supported the development of specific and potent molecules but

none have yet reached clinical testing.• The discovery of the propoxypyrrolidine side chain binding in the lysine channel of G9a and its application

in two distinct scaffolds (UNC/Abbvie) has been a significant breakthrough, although utilization of chemical space in this area remains at an early stage. One of the major challenges G9a inhibitors have faced that has hampered their entry into clinical trials is their poor efficacy in cells and in vivo performance. Further data on improved compounds, such as the more metabolically stable UNC0642, are eagerly awaited.


Review

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epigenetics - future science · keywords: bromodomains • chemical probes • chromatin •...

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