Inhibitors of emerging epigenetic targets for cancer therapy a patent review (2010–2014)

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part of 10.4155/PPA.15.16 ? 2015 Future Science Ltd Gene regulatory pathways comprise an emerging and active area of chemical probe discovery and investigational drug development. Emerging insights from cancer genome sequencing and chromatin biology have identified leveraged opportunities for development of chromatin-directed small molecules as cancer therapies. At present, only six agents in two epigenetic target classes have been approved by the US FDA, limited to treatment of hematological malignancies. Recently, new classes of epigenetic inhibitors have appeared in literatures. First-in-class compounds have successfully transitioned to clinical investigation, importantly also in solid tumors and pediatric malignancies. This review considers patent applications for small-molecule inhibitors of selected epigenetic targets from 2010 to 2014. Included are exemplary classes of chromatin-associated epigenomic writers (DOT1L and EZH2), erasers (LSD1) and readers (BRD4).

Although more than 170 anticancer drugs have been approved by the US FDA, cancer remains a profound unmet medical need. Indeed in the USA, cancer is the second lead-ing cause of death (574,743 people died of cancer in 2010; 23% of all deaths) [2]. Classi-cally, anticancer drugs have been divided into mechanistic classes, such as alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., 5-fluorouracil), topoisomerase inhibi-tors (e.g., irinotecan), antimicrotubule agents (e.g., vinblastine and paclitaxel) and cytotoxic antibiotics (e.g., doxorubicin). While highly efficacious in a small number of malignancies, chemotherapeutic agents are widely utilized for marginal clinical benefit, complicated by significant cytotoxicity (myelosuppression, anorexia and alopecia). A pressing need exists for new classes of targeted cancer therapies.The development of imatinib (Gleevec TM

, Novartis) in 1997 brought a paradigm shift in cancer drug discovery and development: the example of directly engaging an oncopro-tein (the BCR-ABL tyrosine kinase) for ther-apeutic benefit, here in chronic myelo g enous leukemia [3]. Subsequently, therapeutic agents were successfully developed for additional oncogenic kinases including ABL , BRAF , EGFR and ALK . The feasibility of developing ATP-competitive small-molecule antagonists to kinase proteins led to a proliferation of research on signaling pathways and a num-ber of efficacious drug molecules. Regretta-bly, these agents have not proven curative for the vast majority of patients, owing to eva-sive resistance, which enforces reactivation of downstream growth and survival pathways. F urther, the most common human cancers lack actionable alterations, featuring instead a pathophysiology defined by ‘undruggable’ oncogenic drivers and tumor suppressors (e.g., KRAS, MYC, TP53 and RB1) [4–6]. This experience has redoubled our convic-tion that antagonists of downstream gene r egulatory pathways are urgently needed.Emergent insights from cancer genetics

and cancer biology have established chro-matin-associated factors as validated and pressing targets for therapeutic development. Recent advances in sequencing technologies have identified unexpected, common altera-tions in epigenetic regulators as driver muta-tions [7]. Already, alterations of specialized enzymes that write or erase post-translational

Inhibitors of emerging epigenetic targets for cancer therapy: a patent review (2010–2014)

Minoru Tanaka 1,2,

Justin M Roberts 1, Jun Qi & James E Bradner*,1,2

Department of Medical Oncology, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, MA 02215, USA 2

Department of Medicine, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA

*Author for correspondence: james_bradner@https://www.360docs.net/doc/236697777.html,

Patent Review Tanaka, Roberts, Qi & Bradner

modifications (PTMs) to histone and chromatin-associated proteins, and alterations of genes encoding proteins possessing specialized folds capable of read-ing histone PTMs, have been identified in numerous malignancies [8,9]. The reversibility of epigenomic modifications, catalyzed by specialized enzymes (so-called writers and erasers of chromatin-associated post-translational modification), suggests the f e asibility of s mall-molecule antagonism [10,11].

The allure of targeting chromosome-associated factors is twofold. With somatic alteration of an epi-genetic factor, direct antagonism may afford targeted therapeutic development. In the absence of somatic alteration, modulation of chromatin structure may undermine the ability of upstream or chromatin-dependent oncogenic signaling to maintain determi-nants of the hallmark features of cancer. Our recent research directed at the development and character-ization of the first direct antagonists of epigenetic reader proteins (BRD4), demonstrates this principle. In BRD4-rearranged lung cancer, direct inhibition with the prototypical BRD4 inhibitor JQ1 functions as targeted therapy in predictive preclinical models [12]. More broadly, in solid and hematologic tumors, dis-placement of BRD4 by JQ1 suppresses a MYC-specific coactivator function leading to significant antitumor effects [13-16]. Together, these insights and exemplary studies position epigenetic proteins as a ttractive targets for developing cancer therapeutics.

Over the past 15 years, only six agents in two epi-genetic target classes (DNMT and HDAC) have been approved by the FDA, and their use is presently lim-ited to the treatment of hematological malignancies (Figure 1).

This review covers 112 patent applications for small molecules that target the second wave of epigenomic factors approached with discovery chemistry: DOT1L, EZH2, LSD1 and BRD4. Analysis has been performed on documents published internationally after 2010 as ‘composition-of-matter’ patents. We have omitted the patent applications which seem to cover multiple tar-gets or are inventions of ‘new use’ to focus this report on structure–activity relationships.

Writers

A nucleosome is the basic repeating subunit of chroma-tin, consisting of core histones and DNA. Histone pro-teins contain many basic amino acids (especially lysine and arginine), which render them positively charged. This property allows histones to serve as a structural scaffold for the packaging of negatively charged DNA. Further, PTMs facilitate chromatin-dependent signal transduction to RNA polymerase via recruitment of protein complexes with avidity for specific modifica-tions [17]. Histone tails are modified in various ways including lysine and arginine methylation, lysine acetylation, serine and threonine phosphorylation, ubiquitination, citrullination, ADP-ribosylation and SUMOylation. Lysine acetylation and lysine or argi-nine methylation are the most abundant PTMs; they are catalyzed by histone acetyltransferases or histone methyltransferases (HMTs), respectively [18,19]. In general, lysine acetylation is a feature of open euchro-matin, and accumulation of lysine acetylation occurs at enhancer and promoter regions nearby actively transcribed genes. Histone lysine methylation may be observed at active promoters (histone 3 lysine 4 tri-methylation [H3K4me3]) or enhancers (H3K4me1), but it is also a characteristic feature of silenced facul-tative heterochromatin (H3K27me3) and constitutive heterochromatin (H3K9me3) [20].

Among epigenetic writers, early efforts to develop therapeutics have been allocated to two members of the expanded family of histone lysine methyltransfer-ases (KMTs), namely DOT1L and EZH2. Inhibitors for these enzymes first discovered by Epizyme and GlaxoSmithKline (GSK), respectively, feature high target potency and selectivity, and drug-like derivatives have entered clinical trials. We will first summarize the trends in patent applications for DOT1L and EZH2 inhibitors.

DOT1L

DOT1L is a H3K79-specific lysine methyltransferase which catalyzes mono-, di- and tri-methylation in an S-adenosyl-L-methionine (SAM)-dependent manner. DOT1L is well conserved from yeast to mammals [21]. DOT1L lacks the canonical KMT SET (Su(var)3–9, Enhancer-of-zeste, trithorax) domain, rather featuring structural analogy to protein arginine methyltransfer-ases [22]. H3K79 methylation is observed at actively transcribed genes, suggesting a role for DOT1L in pos-itive epigenetic memory. Indeed, DOT1L-mediated aberrant H3K79 methylation plays an important role in the maintenance of constitutive expression of the developmental HoxA cluster of genes that contribute to the pathogenesis of mixed-lineage leukemia (MLL)-rearranged leukemia [23,24].

Oncogenic MLL fusion proteins recruit DOT1L through a macromolecular complex assembled around the proto-oncogenic fusion partner (e.g., AF4, AF9, AF

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Figure 1. US FDA-approved epigenetic drugs.

AML: Acute myeloid leukemia; CTCL: Cutaneous T-cell lymphoma; MDS: Myelodysplastic syndrome; MM: Multilple myeloma; PTCL: Peripheral T-cell lymphoma.

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and ENL) to increase H3K79 methylation in regulatory and coding regions of MLL target genes (e.g., HOX genes), supporting deregulated transcription [25]. Vali-dation of DOT1L in MLL was established with genetic deletion, which effectively attenuated growth in faithful murine models of this disease [26]. Based on this genetic target validation, a coordinated effort in ligand discov-ery was undertaken at Epizyme, which elaborated the SAM-competitive chemical tool EPZ004777, which demonstrated profound antileukemia activity in models of MLL in vitro [27,28]. EPZ004777 is highly potent (K i = 0.3 nM) and selective for DOT1L compared with other HMTs [28]. These results render DOT1L an attractive target for therapeutic intervention.

Based on these compelling preliminary data, signifi-cant attention has been allocated to the discovery of SAM-competitive DOT1L inhibitors [29]. Five patents including one on EPZ004777 have been published since 2010, four of which were filed by Epizyme. Inhib-itors can be classified into two categories: urea-based inhibitors such as EPZ004777 and benzimidazole-based inhibitors such as EPZ-5676. R epresentative structures are shown in Figure 2.

EPZ004777 is a first generation DOT1L inhibi-tor, containing a urea moiety [30]. Epizyme disclosed adenosine-containing analogs representative of struc-ture 1 [31]. EPZ004777 exhibits more than 100,000-fold selectivity for DOT1L over the KMTs CARM1, EHMT2, EZH1, EZH2, PRMT1, PRMT8, SETD7 and WHSC1, and 1280-fold selectivity over the argi-nine HMT PRMT5. Conversely, compound 1 does not show much selectivity for DOT1L over PRMT5

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Figure 2. DOT1L inhibitors.

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(IC 50 <100 nM). No in vivo activity was reported in the patents for this compound.

The published experience, to date, with EPZ004777 suggests imperfect pharmacokinetic properties, requir-ing administration in murine models via an implanted mini-osmotic pump. The second-generation benz-imidazole-based inhibitor EPZ-5676 (K i = 0.08 nM) also reported by Epizyme, has enhanced biochemical properties and pharmacologic performance [32]. In a rat MV4–11 xenograft model, 70 mg/kg of EPZ-5676 (continuous intravenous infusion) achieves com-plete regression of established tumors. The selectivity over PRMT5 was further improved to >37,000-fold and the compound has a much longer drug-target residence time (over 24 h for EPZ-5676 and 1 h for EPZ004777) [33]. Recently, our group and others attributed the remarkable potency and residence time of the near chemical derivative of SAM, EPZ004777, to unexpected catalytic site remodeling upon tar-get engagement [34]. Additional crystal structures of DOT1L with inhibitors have been reported, includ-ing EPZ004777 (PDB 4EKI) and EPZ-5676 (PDB 4HRA) [34,35]. These structures further confirmed that these small molecules occupy the SAM binding pocket and induce conformational rearrangements in the cat-alytic site and activation loop residues, which largely contribute to high potency and selectivity. In 2012, Epizyme initiated an ongoing Phase I clinical trial

Inhibitors of emerging epigenetic targets for cancer therapy Patent Review of single-agent EPZ-5676 in patients with advanced

hematologic malignancies [36]. A fourth patent from

Epizyme includes carbocycle-containing analogs that

are represented by compound 2 (IC

50 <100 nM) [37].

A patent from Kainos Medicine disclosed aminoimid-azolotriazine analogs, including compound 3, which

has an IC

50 value of 5.9 nM [38]. No in vivo efficacy was

reported in the patents for compounds 2 and 3. Recent, ongoing structure-function research performed by our laboratory has identified the N6 position of the adenine as a permissive site for functionalization (compound 4,

50 = 46.3 nM), allowing the preparation of tools for

assay development and further optimization of cellular and pharmacologic properties [39].

We summarized the current status of clinical trials of DOT1L inhibitors in Table 1.

EZH2

The catalytic subunit of the polycomb repressive pro-tein complex 2 (PRC2) is one of two SAM-dependent methyltransferases: EZH1 and EZH2. PRC2 is a multimeric protein complex responsible for main-taining facultative heterochromatin, contributing to the transcriptionally repressive state of genes [20]. Although both EZH2 and EZH1 are SAM-dependent KMTs and share high homology (96% identity within the SET domain), they possess divergent patterns of expression and biological function [40,41]. Generally, EZH2 is expressed in actively dividing cells, whereas EZH1 is widely present in both dividing and differ-entiated cells [20]. PRC2 complexes containing EZH2 (PRC2–EZH2) efficiently catalyze the methyl transfer reaction and repress transcription through H3K27 tri-methylation. In contrast, PRC2–EZH1 has low KMT activity and likely represses transcription by alternate mechanisms [41].

EZH2 is overexpressed in several types of cancer associated with poor prognosis, including breast, kid-ney and lung cancer [42-44]. Cancer genome sequencing efforts further identified somatic alteration of EZH2 in solid and liquid tumors. Mutation of Tyrosine-641 (Y641) and Alanine-677 (A677) have been described and characterized. Both mutations arise in the cata-lytic SET domain, and influence substrate specificity. Whereas wild-type (WT) EZH2 enzyme catalyzes monomethylation of H3K27 (H3K27me1) more efficiently than di- or trimethylation, mutant EZH2 favors a H3K27me2 substrate, thus facilitating effi-cient trimethylation of chromatin [45,46]. This gain-of-function mutation is detected in follicular lymphoma (F L), the germinal center B-cell-like subtype of dif-fuse large B-cell lymphoma (DLBCL), and malignant m elanoma [47,48].

In 2011, two patents from GSK were published that described two or more chemical series of pyridone inhibitors of EZH2 [49,50]. Subsequently in 2012, GSK, Epizyme and Novartis each reported potent, selective EZH2 inhibitors in the research literature (GSK126, EPZ005687 and EI1, respectively), that inhibit both WT and mutant forms of the enzyme [51-53]. GSK126 is a SAM-competitive inhibitor with a K

app of approxi-mately 0.5–3 nM and more than 1000-fold selectivity over 20 other HMTs (150-fold selectivity over EZH1). GSK126 suppresses tumor growth in EZH2-mutant DLBCL xenograft models. A clinical trial for GSK126 in patients with relapsed or refractory DLBCL and transformed follicular lymphoma is currently planned, to the best of our knowledge [54]. EPZ005687 is a structurally analogous SAM-competitive inhibitor with a K

value of 24 nM and more than 500-fold selec-tivity over 15 other KMTs (～50-fold selectivity over EZH1). Epizyme reported a follow-up patent for their second-generation compound, EPZ-6438, which has superior potency and pharmacology. EPZ-6438 has a K

value of 2.5 nM, is orally bioavailable and leads to complete and sustained regression of tumors in EZH2-mutant non-Hodgkin lymphoma xenograft models in mice [55]. Epizyme and Eisai have jointly initiated a Phase I/II clinical trial of EPZ-6438 (E7438) for patients with advanced solid tumors or B-cell lympho-mas [56]. The structurally similar, SAM-competitive inhibitor EI1 from Novartis shows impaired prolifera-tion and induces apoptosis in DLBCL cells possessing the Y641 mutation [53]. Together, these data identify a compelling rationale for the development of EZH2 inhibitors as cancer therapy.

Structurally, EZH2 inhibitors can be classified into three classes: 2-pyridone-containing inhibitors, 4-pyridone-containing inhibitors and others. Twenty-six patents have been published on these compounds; two-third of them have been filed by GSK and Epi-zyme, including GSK126, EPZ005687 and EPZ-

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Figure 3. 2-Pyridone-containing EZH2 inhibitors (see facing page).

Figure 4. 4-Pyridone-containingEZH2 inhibitors.

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6438 [49,57,58]. Representative structures are shown below (Figures 3–5).

Most of the EZH2 inhibitors, such as GSK126 or EPZ-6438, have a common structural feature, includ-ing the 2-pyridone moiety. GSK also reported an inda-zole analog of GSK126 (compound 5, IC 50 = 2 nM for WT EZH2) and azaindazole analogs 6 and 7 (IC 50 = 18 and 24 nM, respectively) [50,59,60]. Indole derivatives with divergent substitution patterns (compound 8 and 9) were disclosed by GSK and Constellation Pharma-ceuticals, respectively [61,62]. No biological data con-cerning compound 8 were described. Compound 9 has IC 50 values of less than 100 nM for both the WT and the Y641N mutant form of EZH2. The indoline deriv-ative 10 and pyrrolopyridazine 11 were disclosed by Piramal and Constellation, respectively [63,64]. Com-pound 10 and 11 have IC 50 values of less than 1 μM and 100 nM for WT EZH2, respectively. No in vivo activity was reported in the patents for c ompounds 5–11.

Several analogs lacking a fused aromatic core were disclosed; EPZ-6438 is a typical example. Jointly with Eisai, Epizyme filed patents covering several substi-tuted benzene derivatives exemplified by compounds 12 and 13 [65,66]. They reported that compound 12 (IC 50 = 0.78 and 2.25 nM for WT and Y641F, respec-tively) inhibits tumor growth in a mouse DLBCL xenograft model at 100 mg/kg, administered twice daily (administration route was not identified). No biological data for 13 were reported. GSK and Pfizer published similar chemical scaffolds – 14, 15 and 16, respectively [67-69]. The IC 50 values for 14, 15 and 16 for WT EZH2 are 13, <10 and 22 nM, respectively. Meanwhile, the recyclized tetrahydroisoquinolone analog 17 disclosed by Pfizer has an IC 50 value of less than 3.83 nM for WT EZH2 and 5.07 nM for the

Y641N mutant [70]. The patent covers 5–7 membered fused ring analogs with various substituents. Constel-lation disclosed simple disubstituted benzene deriva-tives exemplified by compounds 18 and 19 [71,72]. They reported that both compounds have IC 50 values of <1 μM for both WT and Y641N EZH2. GSK filed a patent including conformationally restricted analogs exemplified by compound 20 [73]. Compound 20 has an IC 50 of 13 nM for WT EZH2. No in vivo efficacy was reported in the patents for compounds 13–20.F rom the end of June to July 2014, Epizyme and GSK successively disclosed patents that describe 4-pyr-idone-containing inhibitors. Compound 21 is a 4-pyri-done analog of EPZ-6438 and has an IC 50 value range of 134–149 nM [74]. In the patent, the more potent compound 22 (IC 50 = 1 and 5 nM for WT and Y641F, respectively) is also detailed. Epizyme reported a 4-pyridone analog of GSK126 (compound 23), whose IC 50 value for WT EZH2 is 28 nM [75]. GSK filed a patent disclosing compound 24 (IC 50 = 3 nM for WT) as the most potent inhibitor in the patent [76]. This compound was also described in Epizyme’s preced-ing patent [74]. No in vivo activity was reported in the

p atents for compounds 21–24.As with DOT1L, SAM mimetics can also func-tion as EZH2 inhibitors. Epizyme submitted a patent describing SAM mimetic 25 (IC 50 = 8.95 μM for WT and 2.50–7.56 μM for various Y641 mutants) [77]. The novel scaffold 26 was reported by Constellation [78]. Although compound 26 has neither the pyridone pharmacophore nor an adenosine equivalent moiety, it has an IC 50 value of less than 1 μM for WT EZH2 and an IC 50 between 1 and 10 μM for the Y641N mutant. In their patent, the GSK126 analog 27, which has the 2,2,6,6-tetramethylpiperidine moiety instead of the pyridone warhead, is also included (no biological

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Figure 5. Other EZH2 inhibitors.

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data). Another scaffold lacking the pyridone warhead was reported by Epizyme; representative compound 28 has an IC 50 value of 0.32 μM for WT EZH2 [79].

Beyond direct antagonists of EZH2 are compounds discovered from phenotypic screening. Imperial Inno-vations and Emory University jointly applied for a pat-ent that describes the quinazoline derivative 29 [80]. This compound was discovered in a phenotypic screen for compounds that upregulate mRNA levels of the genes KRT17 and FBXO32 in the breast cancer cell line MDA-MB-231; the compound inhibits H3K27 tri-methylation in the promoter regions of silenced genes. Compound 29 was shown to downregulate EZH2 and upregulate KRT17 and FBXO32 in MDA-MB-231 cells. The compound also upregulates JMJD3, which encodes for an H3K27 demethylase. In contrast, they reported that another quinazoline-based inhibitor of G9a HMT (BIX-01294) does not inhibit EZH2 expression [81]. Molecular targets of the compound have not been identified to date. No in vivo efficacy was disclosed in the patents for compounds 25–29.We summarized the current status of clinical trials of EZH2 inhibitors in Table 2.

Erasers PTMs on histone proteins are catalytically pruned by epigenomic eraser proteins so as to dynamically modu-late transcription in opposition to epigenomic writers. There are multiple categories of epigenetic erasers that target histones including HDACs, lysine demethyl-ases, protein phosphatases, deubiquitinases and argi-nine deiminases. Histone deacetylases are the most widely studied erasers, and numerous chemical probes have been reported and patented targeting either the zinc-dependent hydrolases and the NAD-dependent sirtuins. At present, four HDAC inhibitors have been approved by the FDA for cutaneous T-cell lymphoma and peripheral T-cell lymphoma (Figure 1). As HDAC inhibitors have been reviewed in depth previously (for reviews see ref [82-84]), the present analysis will focus on the lysine-specific histone demethylase (LSD1) which has very recently been the subject of successful ligand discovery research.

LSD1

Histone methylation had been considered a perma-nent histone mark until the lysine-specific demethyl-ase LSD1 was isolated in 2004 [85]. LSD1 is a flavin adenine dinucleotide (F AD)-dependent amine oxi-dase that catalyzes demethylation of H3K4me and H3K4me2.

H3K4me2 is a marker of active gene transcrip-tion [86,87]. Since formation of the iminium cation intermediate is included in the catalytic mechanism,

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the enzyme is incapable of H3K4me3 demethyl-ation (Figure 6). This behavior stands in contrast to the recently characterized Jumonji family of lysine demethylases, individual enzymes of which may effi-ciently catalyze trimethyl lysine demethylation as Fe 2+ and alpha-ketoglutarate-depedent oxygenases.

LSD1 is overexpressed in many types of cancer, such as neuroblastoma, breast, bladder, lung and colorectal cancers [88-90]. It is believed that LSD1 overexpression contributes to carcinogenesis through aberrant silencing of tumor suppressor genes. Additionally, LSD1 plays an important role in the relapse of certain malignancies. Even after complete clinical remission by chemotherapy, most patients with AML will relapse due to the survival of self-renewable leukemia initiating cells (LICs) [91]. LSD1 has proven essential to the self-renewal capabil-ity of LICs, and inhibition with tool compound LSD1 inhibitors leads to differentiation of LICs both in vitro and in vivo [92]. Thus, LSD1 inhibition appears to offer an alternative antineoplastic strategy by forcing termi-nal differentiation and growth arrest of cancer cells, in particular, acute leukemia.

LSD1 inhibition has been intently pursued for nearly a decade [93,94]. Early research identified that monoamine oxidase (MAO) inhibitors, such as trans -2-phenylcyclopropylamine (2-PCPA), possess unin-tended, weak inhibitory activity toward LSD1. The apparent activity likely relates to similarity between the catalytic domains of MAO and LSD1 [95]. 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a K i value of 242 μM and a k inact of 0.0106 s -1, that forms a covalent adduct with FAD (Figure 7) [96]. Many inhibitors with improved selectiv-ity over MAOs have been reported, some characterized by co-crystallography studies [96,97].

ORY-1001 (structure not disclosed), created by Ory-zon Genomics (Oryzon, Spain), has an IC 50 value of 20 nM for LSD1, has high selectivity over other FAD-dependent amine oxidases and reduces tumor growth in rodent AML xenograft models of MV4–11 cells with oral administrations of <0.020 mg/kg [98]. Oryzon ini-tiated a Phase I/IIa trial for ORY-1001 for AML in the EU [99]. In April 2014, Oryzon and Roche announced a collaboration to develop ORY-1001 and its backup

compounds. Additionally, GSK2879552 (structure not disclosed) is an orally available, irreversible inhibitor of LSD1 with potential antineoplastic activity from GSK. Currently two Phase I clinical t rials are enrolling for study in patients with relapsed or refractory small-cell lung cancer or AML [100,101].

Twenty-three composition-of-matter patents have been disclosed for LSD1 inhibitors since 2010, ten of them published by Oryzon. The patents can be divided into two types: 2-PCPA analogs and others.

(Figure 8–11)

Oryzon discovered that substitutions on the amino group of 2-PCPA drastically improves activity (IC 50 <100 nM) and selectivity for LSD1 over the two MAO enzymes in humans, MAO-A and MAO-B (>400-fold; compound 30–33) [102-105]. Among the 2-PCPA analogs, compound 33 has an IC 50 value of 56 nM for LSD1 and >100 μM for MAO-A/B. Com-pound 33 also shows potent activity in inducing dif-ferentiation of THP-1 leukemia cells (EC 50 = 2.1 nM). Compound 30–33 are trans-racemate, whereas chi-ral 34 and 35 were published by Oryzon and GSK, respectively [106,107]. Interestingly, although compound (1R ,2S )-34 has 20-fold stronger activity than (1S ,2R )-34 (IC 50 = 15 and 292 nM, respectively), there is no significant difference in activity between (1R ,2S )-35 and (1S ,2R )-35 (pIC 50 = 8.2 and 8.3, respectively). All four enantiomers show reasonable selectivity over MAO-B (IC 50s >10–100 μM). Compound (1R ,2S )-34 is highly potent in the THP-1 differentiation assay (EC 50 = 0.8 nM). No in vivo data were reported in the patents for compounds 30–35.

Substitution on the benzene ring of 2-PCPA also contributes to improving activity and selectivity. Ital-ian and Japanese groups independently published compounds 36 and 37, respectively [108,109]. Both com-pounds have IC 50s in the low micromolar range for LSD1 (1.3 μM for 36 and 1.92 μM for 37). The IC 50 value of compound 37 for MAO-B is >1000 μM. The Japanese group also disclosed a patent including com-pound 38 (IC 50 = 0.16 μM), which has the same sub-stituent that 37 has off of the benzene, but off of the amino group [110]. The chiral analog 39 (IC 50 <1 μM) was reported by Imago Biosciences [111]. Heteroaryl

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Figure 6. Enzymatic reaction mechanism of LSD1.

Figure 7. Proposed inactivation mechanism of FAD by 2-PCPA.

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analogs (compound 40 and 41) were disclosed by Ory-zon [112,113]. Their LSD1 inhibitory activities (IC 50s) are less than 0.1 and 1 μM, respectively. No in vivo activity was discussed in the patents for compounds 36–41.

Oryzon and Takeda Pharmaceutical filed patents including compounds 42–46 having substituents both on the amino group and benzene ring [114-118]. Every compound has an IC

50 value of less than 0.1 μM. Compound 43, 45 and 46 show good selectivity over MAO-A/B (IC 50s >10 μM). Compound 45 suppresses tumor growth in a mouse xenograft model of human erythroleukemia (HEL92.1.7 cell) via oral administra-tion (30 mg/kg, once daily). The University of Nevada disclosed a patent that covers compound 47 (IC 50 = 21.25 μM for LSD1) [119]. An Italian group published a patent concerning trisubstituted cyclopropylamines exemplified by compound 48 (IC <0.1 μM for LSD1,

>100-fold selectivity over MAO-A) [120]. No in vivo data were reported in the patents for compounds 42–48 except for 45.

The University of Utah reported benzohydrazide 49 as a novel, potent LSD1 inhibitor which does not have a cyclopropylamine structure [121]. Compound 49 has an IC 50 of 13 nM for LSD1 and exhibited no activity for MAO-A and B (IC 50s >300 μM). It also inhibits cell growth in a panel of cancer cell lines (e.g., IC 50 = 1.040 μM for MDA-MB-231 cells). A Nevada Cancer Institute-led team filed a patent disclosing guanidine 50 (IC 50 = 5.27 μM for LSD1, no data for selectivity over MAO-A and B) [122]. Polyamine-based inhibitors were disclosed by Johns Hopkins University, including compound 51 (83% inhibition at 10 μM) [123]. They also reported hydroxyamidine 52 as an LSD1 inhibi-tor (30% inhibition at 10 μM) [124]. Inhibition mecha-nisms of above-mentioned noncyclopropylamine-based inhibitors are not described in each patent. No in vivo efficacy was reported in the patents for compounds 49–52.

We summarized the current status of clinical trials of LSD1 inhibitors in Table 3.

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Figure 8. 2-PCPA analogs substituted on the amino group.

Figure 9. 2-PCPA analogs substituted on the benzene ring.

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Readers

Chromatin-associated histone modifications are recog-nized by a selective group of proteins, or readers , that contain specialized protein domains that bind directly to PTMs on histones. Lysine side-chain methylation, considered above, is recognized in a sequence-specific manner by chromo-like domains of the Royal family (chromo, tudor and malignant brain tumor domains) and plant homeodomain fingers [125]. Side-chain acet-ylation of lysine is recognized by bromodomains, and as we have postulated perhaps also the enzymatically compromised Class IIA histone deacetylases [126]. Epi-genetic reader domains are commonly found as mod-ules in multidomain, chromatin-associated proteins notably including many writers and erasers. In this manner, histone-binding modules may facilitate spread-ing of epigenetic marks and contribute to epigenetic memory. F urther, epigenetic reader proteins nucleate multiprotein chromatin-associated complexes with spa-tial precision, prompting transcriptional activation, con-ferring a repressed state to heterochromatin and facili-tating nucleosome remodeling [127]. In this review, we

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Figure 10. 2-PCPA analogs substituted both on the amino group and benzene ring.

Figure 11. Other LSD1 inhibitors.

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will focus on the recent explosion in the development of small-molecule antagonists of bromodomains, and in particular b romodomain and extra-terminal (BET) bromodomains.

BRD4

BRD4 is a member of the BET family of proteins (BRD2, BRD3, BRD4 and BRDT), all of which have tandem bromodomains [128]. A bromodomain is an antiparallel bundle of alpha helices which binds acetyl-lysine-containing peptides via molecular recognition of the acetyl cap by a conserved asparagine. There are 42 bromodomain-containing proteins, which are the subject of emerging biological and pharmacologic study [129]. BET bromodomains, such as the prototypi-cal member BRD4, facilitate transcriptional elongation via recruitment of the positive transcription elongation factor (P-TEFb) and displacement of negative regula-tors (HEXIM1 and 7SK snRNA) [130–132]. In support of this assertion, siRNA knockdown of BRD4 in HeLa cells inhibited recruitment of P-TEFb to mitotic chro-mosomes and reduces expression of growth-associated genes, leading to G1 cell cycle arrest and apoptosis [133].In 2010, our laboratory reported the first direct-acting bromodomain inhibitor, JQ1, in a collab-orative study with Prof Stefan Knapp (Figure 12) [12]. JQ1 is a highly potent and BET-selective thienodiaz-epine which binds into the conserved asparagine via a methyl-triazolo chemical feature. This chemical tool has facilitated the mechanistic and translational study

Inhibitors of emerging epigenetic targets for cancer therapy Patent Review

of BET bromodomains broadly in developmental and disease models. Importantly, JQ1 selectively kills sev-eral cancer cells such as AML and multiple myeloma (MM) through downregulation of MYC transcrip-tion [13,14], consistent with our postulated role in chromatin-dependent signal transduction from master regulatory transcription factors to RNA Polymerase II. The selectivity for MYC transcription in cancer cells is explained by asymmetric loading of BRD4 genome-wide to large so-called ‘super enhancers’, as described with Prof Richard Young [134–136]. BET inhibition as a strategy to target MYC expression and function was promptly validated in industry by Constellation, notably using JQ1 [137]. The selective downregulation of super-enhancer-associated genes was validated also in inflammatory models, where BET localization is driven by NF-k B [138].

BRD3 and BRD4 are proto-oncogenes in a highly aggressive form of poorly differentiated squamous cell carcinoma of the lung, head and neck. An in-frame fusion with the nuclear protein in testis gene (e.g., BRD4-NUT) elaborates a chimeric oncopro-tein characteristic of BET-rearranged carcinoma (also called NUT midline carcinoma [NMC]). NMC is poorly responsive to chemotherapy and radiation therapy, and to date there are few known long-term survivors [139–141]. In translational models of NMC, JQ1 exhibits a potent antiproliferative effect, associ-ated with squamous differentiation and robust apopto-sis [12]. Primary human NMC xenografts exhibit long-term survival on continuous JQ1 therapy, supporting consideration of BET inhibition as targeted therapy in this disease [12]. A drug-like derivative of JQ1 has tran-sitioned to human clinical investigation, and is pres-ently the focus of ongoing Phase I/II studies in solid and liquid tumors (TEN-010; Tensha Therapeutics). As of the end of December 2014, three additional compounds (I-BET762, OTX015 and CPI-0610) have been prepared for cancer clinical trials, including NMC [142–145].

Patent filings on BRD4 binding may be found from Mitsubishi Tanabe Pharma, where a focused set of triazolothienodiazepines was suggested as having BRD4 binding capacity [146]. No in vivo data were disclosed in the patent. Since the publication of JQ1 in 2010, 57 composition-of-matter patents have been disclosed for BRD4 inhibitors. They are categorized into triazolothieno/benzodiazepines and their ana-logs, including JQ1 and I-BET762; isoxazole deriva-tives; pyridone derivatives and their analogs; and o thers(Figures 12–15).

The patents for JQ1 and I-BET762 were published in 2011 by Dana-Farber Cancer Institute and GSK, respectively [147,148]. Several crystal structures of BRD4 with inhibitors have been reported, including JQ1 (PDB 3MXF) and I-BET762 (PDB 3P5O), and illustrate that these molecules bind to a conserved asparagine in BET bromodomains; the triazole ring acts as a mimic of acetylated lysine. GSK filed a series of patents which include triazolobenzodiazepine ana-logs GW841819X (an I-BET762 prototype) and com-pound 53[149-152]. GW841819X inhibits the binding of tetra-acetylated lysine histone 4 peptide (H4AcK

) to a tandem bromodomain-containing construct of BRD4 (BRD4(1,2)) with an IC

value of 16 nM. The pIC

of compound 53 is over 5.5. Bayer filed a patent for compound 54, which has an IC

of 27 nM for BRD4(1) [153]. A dimeric inhibitor 55 was presented in an application by Coferon [154]. No binding affin-ity or inhibitory activity for BRD4 was described. Constellation, which has initiated three follow-on Phase I clinical trials for CPI-0610 in leukemia, lym-phoma and multiple myeloma, reported a series of patents covering isoxazolothienoazepine analogs 56 and isoxazolobenzoazepine 57 (IC

for BRD4(1) = 26 and 14 nM, respectively) [155–157]. Oral adminis-tration of compound 56 suppresses MYC expression in a mouse xenograft model of B-cell lymphoma with MYC-dependent Raji cells (ED

= 20–50 mg/kg). They also disclosed triazolodihydrobenzodiazepine 58 (IC

for BRD4(1) <0.1 μM) and isoxazolodi-hydrobenzoazepine 59 (IC

for BRD4(1) = 0.03 μM) [158,159]. Recently, additional BRD4 inhibitors with alternative scaffolds, compounds 60–64, were reported by Bayer [160–164]. Compound 60 (IC

for BRD4(1) = 20 nM) has a triazolobenzazepine scaf-fold. Meanwhile, compounds 61 (IC

for BRD4(1) = 0.02 μM) and 62 (IC

for BRD4(1) = 0.14 μM) have similar triazolopyrrolodiazepine and triazolopyr-azolodiazepine scaffolds, respectively. Interestingly, isomeric benzodiazepine analogs 63 and 64, lack-ing the azole ring, have IC

s of 0.02 and 0.01 μM

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Figure 12. Triazolothieno/benzodiazepine analogs.

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Figure 13. Isoxazole derivatives.

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for BRD4(1), respectively. No in vivo efficacy was reported in the patents for compounds 53–64 except for 56.

GSK reported the isoxazole-containing compound, I-BET151 (IC 50 for BRD4(1,2) = 0.79 μM), which has an improved pharmacokinetic properties profile and shows efficacy in mouse xenograft models of human

leukemia [165–168]. Besides the antitumor activity, I-BET151 was demonstrated to suppress LPS-induced proinflammatory genes including IL-6 in peripheral blood mononuclear cells. Prophylactic injection of I-BET151 (10 mg/kg) in mice prevents death of LPS-induced endotoxic shock [166,167]. GSK also reported compound 65 (pIC 50 = 6.5–7.5) which reduces LPS

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Figure 14. Pyridone derivatives and their analogs.

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Patent Review Tanaka, Roberts, Qi & Bradner

induced IL-6 expression in mice with a comparable effect to the steroid, dexamethasone [169].

Aurigene applied for a patent including compound 66 (IC 50 <200 nM) [170]. RVX Therapeutics disclosed a simple isoxazole derivative 67 (IC 50 <30 μM for BRD4(1)), which shows efficacy in a mouse xenograft model of MM via oral administration (25–90 mg/kg, twice daily) [171]. Bristol-Myers Squibb reported compound 68 & 69 (IC 50s for both compounds <0.05 μM) [172,173]. The University of Michigan filed a pat-ent covering compound 70 [174]. This compound was assessed in fluorescent ligand-binding assays and dem-onstrated inhibition of binding for each bromodo-main in nanomolar quantities (K i for BRD4(1) and

BRD4(2) = 35.3 and 7.8 nM, respectively). It sup-presses tumor growth in mouse xenograft models of several cancers such as breast (MDA-MB-231 cell) and AML (MV4–11 cell) at various doses and administra-tion routes. Incyte, Plexxikon and Epigenetix inde-pendently reported isoxazole-containing inhibitors 71 (IC 50 <100 nM for BRD4(1)), 72 (IC 50 <1 μM for BRD4(1)) and 73 (IC 50 <1 μM for BRD4(1)), respec-tively [175-177]. Boehringer Ingelheim (Boehringer) dis-closed two patents which include compound 74 & 75 (IC 50 = 14 and 27 nM, respectively) [178,179]. Gilead Sci-ences reported compound 76 & 77 (IC 50 = 6.0 and 87.1 nM for BRD4(1), respectively) [180,181]. Compound 77 was shown to suppress tumor growth in mouse xeno-

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Figure 15. Other BRD4 inhibitors.

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graft models of MM (MM.1S cell) and large cell lym-phoma (SU-DHL-10 cell) via oral administration (10 mg/kg, once and twice daily, respectively). Beigene filed a patent covering compound 78 which has an IC 50

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value of 8 nM for BRD4(1,2) [182]. No in vivo activity was reported in the patents for compounds 66, 68, 69, 71–76 & 78.

In 2013, AbbVie disclosed a series of patents that cover pyridone derivatives and their analogs (79–84) [183-185]. They measured inhibition constants for both bromodomains of BRD4 in fluorescent ligand binding assays. Interestingly, some compounds show selectivity for the first over the second bromodomain (79, K i for BRD4(1) and BRD4(2) = 11.8 and 105 nM; 81, 151 nM and 2.07 μM and 83, 143 nM and 12.50 μM, respectively). Although the significance of tandem BET bromodomains is still not clear, each bromodomain is reported to be engaged in different molecular functions. While the first bromodomain recognizes acetylated histone proteins, there are sev-eral reports that the second bromodomain is involved in coactivation of P-TEF b [186,187]. No advantage for improved selectivity was described in the patents. Non-selective inhibitors (80, K i for BRD4(1) and BRD4(2) = 2.84 and 1.43 nM; 82, 15 and 43 nM and 84, 11 and 3.3 nM, respectively) in the patents inhibit tumor growth in several xenograft mouse models. Additional patents for similar scaffolds were disclosed in 2014 [188-192]. Representative compounds 85–89 (85, K i for BRD4(1) and BRD4(2) = 34.9 and 148 nM; 86, 1.09 and 2.08 nM; 87, 0.92 and 2.09 nM; 88, 27.3 and 318 nM and 89, 34 and 38 nM, respectively) from each patent inhibit tumor growth in a mouse xenograft model of MM (OPM-2 cell) via oral administration at various doses. GSK also filed patents, each of which

covers compound 90 and 91, respectively [193,194]. The thienopyridone analog 90 (pIC 50 for BRD4(1) >7.0) has at least tenfold selectivity over BRD4(2); meanwhile, the furanopyridone analog 91 (pIC 50 for BRD4(1) >8.0) has at least 100-fold selectivity. Novar-tis reported compound 92 which has an IC 50 value of 14 nM for BRD4(1,2) [195]. No in vivo efficacy was disclosed in the patents for compounds 90–92.

Pfizer and GSK independently discovered novel scaffolds with fragment-based drug discovery [196-199]. PF I-1 from Pfizer has an IC 50 value of 0.22 μM for BRD4(1) [200]. No in vivo efficacy was reported. GSK’s tetrahydroquinoline derivative, I-BET726, and its ana-log 93 have pIC 50s of over 6.0 for BRD4(1,2) [201,202]. In an LPS-induced mouse endotoxic shock model, 10 mg/kg of I-BET726 via intravenous administration was demonstrated to improve animal survival. They also filed a patent covering a similar scaffold [203]. Inter-estingly, most compounds in the patent show at least 100-fold selectivity for the second over the first bro-modomain; representative compound 94 has a pIC 50 value of over 7.0 for BRD4(2). Convergene filed a pat-ent which covers quinolone derivatives exemplified by compound 95 (IC 50 = 49 nM for BRD4(1,2)) [204]. No in vivo activity was reported in the patents for c ompounds 93–95.

Resverlogix discovered RVX-208 by phenotypic screening, a resveratrol derivative that increases the pro-duction of ApoA-I protein in reporter-gene assays [205]. RVX-208 was subsequently found to weakly inhibit BRD4, which may influence transcription of the APOA1 gene (although BET inhibition is mechanis-tically associated with target gene downregulation). RVX-208 exhibits some selectivity for the second bro-modomain of BET family members, such as for BRD4

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for which it has an IC 50 value of 1.8 μM for BRD4(1) and 0.04 μM for BRD4(2) [206]. BRD4(1)-dependent gene transcription, such as MYC transcription, is not downregulated by RVX-208, as it is with JQ1 [207]. We speculate that higher doses of RVX-208 may exhibit BET inhibitory behavior, if clinically achievable. Prior to its recognition as a BET inhibitor, Resverlogix devel-oped RVX-208 for the treatment of cardiovascular dis-eases associated with atherosclerosis [208,209]. RVX Ther-apeutics (the spin-out company of Resverlogix) recently filed a series of patents covering RVX-208 analogs 96 & 97 (both IC 50s for BRD4(1) are less than 30 μM, no data for BRD4(2)) [210,211]. Both compounds inhibited LPS-induced IL-6 and IL-17 p roduction in mice with oral administrations of 75 mg/kg.

Triazolopyridazine derivative 98 was reported by Constellation; triazolopyrazine 99 & 100 were dis-closed by Boehringer [212–214]. IC 50s of compounds 98, 99 & 100 for BRD4(1) are <500, 3 and 1 nM, respectively. Novartis filed three patents covering tri-azolopyridine 101 & 102 and triazolopyridazine 103. Each compound shows an IC 50 value of less than 11 nM for BRD4(1,2) [215–217]. No in vivo efficacy was discussed in the patents for compounds 98–103.

An Icahn School of Medicine at Mount Sinai-led team reported the diazobenzene derivative MS436 as tenfold selective for the first bromodomain [218,219]. The K i value of MS436 for BRD4(1) is <85 nM. No in vivo activity was reported in the patent.

Recently, some kinase inhibitors have been reported to inhibit BRD4 through binding interactions between a kinase hinge-binding motif and the conserved aspara-gine in BET bromodomains [220–222]. The PLK1 inhib-itor BI2536 was developed as a treatment for AML and non-small-cell lung cancer and has completed Phase II clinical trials [223,224]. BI2536 inhibits both PLK1 and BRD4 in the nanomolar range (IC 50s are 0.83 nM for PLK1, 25 nM for BRD4(1)) [222,225]. These findings open new possibilities for dual kinase-BRD4 inhibitors for several cancer treatments. Bayer disclosed a patent including BI2536 analog 104 which has the kinase hinge-binding motif (aminopyridine) [226]. The IC 50 value of compound 104 for BRD4(1) is 63 nM. No in vivo activity was discussed in the patent.

We summarized the current status of clinical trials of BRD4 inhibitors in Table 4.

Future perspective

At present, epigenetics -related drug discovery has been limited to DNMT and HDAC inhibitors for hematological malignancies. Novel potential drug targets have emerged since 2010 to include additional epigenetic targets that can write, erase and read histone modifications to alter gene expression. Compounds designed to effectively inhibit epigenetic proteins, such as DOT1L, EZH2, LSD1 and BRD4, have been devel-oped at a rapid rate. As a chemical biology laboratory, we found this exercise illuminating and encouraging, and hope these descriptive data and insights provide some context to a literature that is often under utilized in academic science. We expect high-quality publica-tions to arise from many of these filings, perhaps when definitive evidence of pharmacologic and biological

activity is in-hand, as could satisfy peer review. With

Patent Review Tanaka, Roberts, Qi & Bradner

advanced compounds now progressing through clini-cal investigation, an opportunity may exist for func-tionally differentiated, second-generation compounds that respond to observed toxicities, pharmacologic lia-bilities or new mechanistic opportunities. As such, this document seeks to provide a perspective on the state of ongoing innovation in epigenetic discovery chemistry. Acknowledgements

We thank JA Perry, DL Buckley, CJ Ott and CY Lin for critical reading and discussions.Financial & competing interests disclosure

M Tanaka is a visiting scientist from Mitsubishi Tanabe Pharma Corporation (MTPC). JE Bradner is a founder of Tensha Thera-peutics. The authors gratefully acknowledge nonresearch sup-port of M Tanaka by MTPC. The authors have no other rele-vant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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