Patent Review
Inhibitors of emerging epigenetic targets for cancer therapy: a patent review (2010–2014)
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 leading cause of death (574,743 people died of cancer in 2010; 23% of all deaths) [2] . Classically, anticancer drugs have been divided into mechanistic classes, such as alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., 5-fluorouracil), topoisomerase inhibitors (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 (GleevecTM, Novartis) in 1997 brought a paradigm shift in cancer drug discovery and development: the example of directly engaging an oncoprotein (the BCR-ABL tyrosine kinase) for therapeutic benefit, here in chronic myelogenous leukemia [3] . Subsequently, therapeutic agents were successfully developed for additional
10.4155/PPA.15.16 © 2015 Future Science Ltd
Minoru Tanaka1,2, Justin M Roberts1, Jun Qi & James E Bradner*,1,2 Department of Medical Oncology, DanaFarber 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: [email protected] 1
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 number of efficacious drug molecules. Regrettably, these agents have not proven curative for the vast majority of patients, owing to evasive resistance, which enforces reactivation of downstream growth and survival pathways. Further, 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 conviction that antagonists of downstream gene regulatory pathways are urgently needed. Emergent insights from cancer genetics and cancer biology have established chromatin-associated factors as validated and pressing targets for therapeutic development. Recent advances in sequencing technologies have identified unexpected, common alterations in epigenetic regulators as driver mutations [7] . Already, alterations of specialized enzymes that write or erase post-translational
Pharm. Pat. Anal. (2015) 4(4), 261–284
part of
ISSN 2046-8954
261
Patent Review Tanaka, Roberts, Qi & Bradner
Key terms Writers: Enzymes that catalyze addition of a covalent modification to chromatinassociated proteins. Erasers: Enzymes that catalyze removal of a covalent modification from chromatin-associated proteins.
modifications (PTMs) to histone and chromatinassociated proteins, and alterations of genes encoding proteins possessing specialized folds capable of reading histone PTMs, have been identified in numerous malignancies [8,9] . The reversibility of epigenomic modifications, catalyzed by specialized enzymes (socalled writers and erasers of chromatin-associated post-translational modification), suggests the feasibility of small-molecule antagonism [10,11] . The allure of targeting chromosome-associated factors is twofold. With somatic alteration of an epigenetic 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 chromatindependent oncogenic signaling to maintain determinants of the hallmark features of cancer. Our recent research directed at the development and characterization 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, displacement 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 epigenetic target classes (DNMT and HDAC) have been approved by the FDA, and their use is presently limited 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 targets or are inventions of ‘new use’ to focus this report on structure–activity relationships. Writers A nucleosome is the basic repeating subunit of chromatin, consisting of core histones and DNA. Histone pro-
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Pharm. Pat. Anal. (2015) 4(4)
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 modifications [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 arginine 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 euchromatin, 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 trimethylation [H3K4me3]) or enhancers (H3K4me1), but it is also a characteristic feature of silenced facultative 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 methyltransferases (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 methyltransferases [22] . H3K79 methylation is observed at actively transcribed genes, suggesting a role for DOT1L in positive 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, AF10
future science group
Inhibitors of emerging epigenetic targets for cancer therapy
Patent Review
DNMT inhibitors NH2 N
N N
HO
NH2 N O
O
N O
N
HO O
OH OH
OH
VidazaTM Azacitidine Celgene MDS
DacogenTM Decitabine Eisai MDS, AML
HDAC inhibitors
H O
H N
N H
O
Me O
H
NH
H
HN
H N
O
H
N H
O
Me
IstodaxTM Romidepsin Celgene CTCL
O
Me
S HN O S
O
ZolinzaTM Vorinostat Merck CTCL
HN
O
Me HN
OH
Me
O O S N H
O N H
OH
BeleodaqTM Belinostat Spectrum Pharmaceuticals PTCL
OH
Me
FarydakTM Panobinostat Novartis MM 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.
and ENL) to increase H3K79 methylation in regulatory and coding regions of MLL target genes (e.g., HOX genes), supporting deregulated transcription [25] . Validation 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 discovery 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, significant attention has been allocated to the discovery of
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SAM-competitive DOT1L inhibitors [29] . Five patents including one on EPZ004777 have been published since 2010, four of which were filed by Epizyme. Inhibitors can be classified into two categories: urea-based inhibitors such as EPZ004777 and benzimidazolebased inhibitors such as EPZ-5676. Representative structures are shown in Figure 2. EPZ004777 is a first generation DOT1L inhibitor, containing a urea moiety [30] . Epizyme disclosed adenosine-containing analogs representative of structure 1 [31] . EPZ004777 exhibits more than 100,000fold selectivity for DOT1L over the KMTs CARM1, EHMT2, EZH1, EZH2, PRMT1, PRMT8, SETD7 and WHSC1, and 1280-fold selectivity over the arginine HMT PRMT5. Conversely, compound 1 does not show much selectivity for DOT1L over PRMT5
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263
Patent Review Tanaka, Roberts, Qi & Bradner
NH2
NH2 N Me S
+
O
N
N
O
NH2 N
N
S
HO
HO
NH2
N
N
O
O
Me N
N
NH2
HO
HO
SAM
O
N
N
HN HN
OH
N
Me
HO
O
OH
OH
SAH Me Me Me
EPZ004777
NH2 N
N
Me N
O
N
NH2
Me
N
Me N
N
N
O
N N
HN HN
HO
HO
OH
O
OH
N NH
Me
Me
Me Me
1
Me
Me
EPZ-5676
NH2
Me
N
Me N
N
N
HO
N
N
O
HO
NH
N
Me O
N
N N
HN
N
Me Me
Me
N
OH HN
NH 2
N
N
OH
Me Me
N H
N
Me
N
NH
Me
NH2
Me
3 Me
HO
OH
O 4 Me Me Me
Figure 2. DOT1L inhibitors.
(IC50 37,000-fold and the compound has a much longer drug-target
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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 target engagement [34] . Additional crystal structures of DOT1L with inhibitors have been reported, including 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 catalytic site and activation loop residues, which largely contribute to high potency and selectivity. In 2012, Epizyme initiated an ongoing Phase I clinical trial
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Inhibitors of emerging epigenetic targets for cancer therapy
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 (IC50 300 μM). It also inhibits cell growth in a panel of cancer cell lines (e.g., IC50 = 1.040 μM for MDA-MB-231 cells). A Nevada Cancer Institute-led team filed a patent disclosing guanidine 50 (IC50 = 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 inhibitor (30% inhibition at 10 μM) [124] . Inhibition mechanisms 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.
FAD·– trans
trans
NH2 2-PCPA
O N N
-
NH N
O
R
R FAD
H N
O
NH N
N R
O NH
O
N . N
H2N
+
+. NH2
N
O
N N
NH N
R
O
FAD·–
Figure 7. Proposed inactivation mechanism of FAD by 2-PCPA.
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Pharm. Pat. Anal. (2015) 4(4)
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O
Patent Review
Inhibitors of emerging epigenetic targets for cancer therapy
NH2
trans
trans
trans
N H
NH2
Me
N O
30
2-PCPA
O
N H
O
31
NH2
trans
NH
NH
trans
N
N H
N H
N H
(1R,2S)-34 33
32 NH2
N H
N H
N H
NH
NH
(1R,2S)-35
(1S,2R)-35
(1S,2R)-34
Figure 8. 2-PCPA analogs substituted on the amino group.
Readers Chromatin-associated histone modifications are recognized 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 acetylation of lysine is recognized by bromodomains, and as we have postulated perhaps also the enzymatically
compromised Class IIA histone deacetylases [126] . Epigenetic reader domains are commonly found as modules in multidomain, chromatin-associated proteins notably including many writers and erasers. In this manner, histone-binding modules may facilitate spreading of epigenetic marks and contribute to epigenetic memory. Further, epigenetic reader proteins nucleate multiprotein chromatin-associated complexes with spatial precision, prompting transcriptional activation, conferring a repressed state to heterochromatin and facilitating nucleosome remodeling [127] . In this review, we
trans O
NH
O
trans NH2
N H
HN
H N
trans
NH2 O
O
N H
O
36
O
O
37
NH
H N
O 38
trans
N
NH2 S
trans
Cl O N H
NH
O 39
H N
HN
S O O CN 40
NH2 N H
N 41
Figure 9. 2-PCPA analogs substituted on the benzene ring.
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Patent Review Tanaka, Roberts, Qi & Bradner
trans N H
N H
42
trans
Me
N H
N
Me
45
Me
O Me Me
N H
N H
NH2
44
trans
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N H
O F3C
N
O
43
Cl
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O
O
Cl
N H
trans NH2
Me O
Me Me
O
N H
O O
46
47
Me
trans NH2
O N H
O O
N
48
Figure 10. 2-PCPA analogs substituted both on the amino group and benzene ring.
will focus on the recent explosion in the development of small-molecule antagonists of bromodomains, and in particular bromodomain and extra-terminal (BET) bromodomains.
cal member BRD4, facilitate transcriptional elongation via recruitment of the positive transcription elongation factor (P-TEFb) and displacement of negative regulators (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 chromosomes and reduces expression of growth-associated genes, leading to G1 cell cycle arrest and apoptosis [133] . In 2010, our laboratory reported the first directacting bromodomain inhibitor, JQ1, in a collaborative study with Prof Stefan Knapp (Figure 12) [12] . JQ1 is a highly potent and BET-selective thienodiazepine which binds into the conserved asparagine via a methyl-triazolo chemical feature. This chemical tool has facilitated the mechanistic and translational study
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 acetyllysine-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 prototypiOH
O N
Cl Me
N H
O
NH
O O S N
H2N
O
Me
O N
N
O
N
N
NH2
O
OH
S N H
NH
50
49
N H
H N
N H
H N
H N S
H N
N O
OH
NH2
52
51
Figure 11. Other LSD1 inhibitors.
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Pharm. Pat. Anal. (2015) 4(4)
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Inhibitors of emerging epigenetic targets for cancer therapy
Patent Review
Table 3. Clinical trials of LSD1 inhibitors. Compound
Sponsor
Phase
Condition
ClinicalTrials.gov identifier
ORY–1001
Oryzon
I/IIa
Relapsed/refractory acute leukemia
EudraCT2013–002447–29
GSK2879552
GSK
I
Relapsed/refractory small-cell lung cancer
NCT02034123
I
Relapsed/refractory AML
NCT02177812
of BET bromodomains broadly in developmental and disease models. Importantly, JQ1 selectively kills several cancer cells such as AML and multiple myeloma (MM) through downregulation of MYC transcription [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 genomewide 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-kB [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 inframe fusion with the nuclear protein in testis gene (e.g., BRD4-NUT) elaborates a chimeric oncoprotein 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, associated with squamous differentiation and robust apoptosis [12] . Primary human NMC xenografts exhibit longterm survival on continuous JQ1 therapy, supporting consideration of BET inhibition as targeted therapy in this disease [12] . A drug-like derivative of JQ1 has transitioned to human clinical investigation, and is presently 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
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disclosed for BRD4 inhibitors. They are categorized into triazolothieno/benzodiazepines and their analogs, including JQ1 and I-BET762; isoxazole derivatives; pyridone derivatives and their analogs; and others (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 analogs GW841819X (an I-BET762 prototype) and compound 53 [149-152] . GW841819X inhibits the binding of tetra-acetylated lysine histone 4 peptide (H4AcK4) to a tandem bromodomain-containing construct of BRD4 (BRD4(1,2)) with an IC50 value of 16 nM. The pIC50 of compound 53 is over 5.5. Bayer filed a patent for compound 54, which has an IC50 of 27 nM for BRD4(1) [153] . A dimeric inhibitor 55 was presented in an application by Coferon [154] . No binding affinity or inhibitory activity for BRD4 was described. Constellation, which has initiated three follow-on Phase I clinical trials for CPI-0610 in leukemia, lymphoma and multiple myeloma, reported a series of patents covering isoxazolothienoazepine analogs 56 and isoxazolobenzoazepine 57 (IC50 for BRD4(1) = 26 and 14 nM, respectively) [155–157] . Oral administration of compound 56 suppresses MYC expression in a mouse xenograft model of B-cell lymphoma with MYC-dependent Raji cells (ED50 = 20–50 mg/kg). They also disclosed triazolodihydrobenzodiazepine 58 (IC50 for BRD4(1)
Inhibitors of emerging epigenetic targets for cancer therapy: a patent review (2010–2014)
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 leading cause of death (574,743 people died of cancer in 2010; 23% of all deaths) [2] . Classically, anticancer drugs have been divided into mechanistic classes, such as alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., 5-fluorouracil), topoisomerase inhibitors (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 (GleevecTM, Novartis) in 1997 brought a paradigm shift in cancer drug discovery and development: the example of directly engaging an oncoprotein (the BCR-ABL tyrosine kinase) for therapeutic benefit, here in chronic myelogenous leukemia [3] . Subsequently, therapeutic agents were successfully developed for additional
10.4155/PPA.15.16 © 2015 Future Science Ltd
Minoru Tanaka1,2, Justin M Roberts1, Jun Qi & James E Bradner*,1,2 Department of Medical Oncology, DanaFarber 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: [email protected] 1
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 number of efficacious drug molecules. Regrettably, these agents have not proven curative for the vast majority of patients, owing to evasive resistance, which enforces reactivation of downstream growth and survival pathways. Further, 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 conviction that antagonists of downstream gene regulatory pathways are urgently needed. Emergent insights from cancer genetics and cancer biology have established chromatin-associated factors as validated and pressing targets for therapeutic development. Recent advances in sequencing technologies have identified unexpected, common alterations in epigenetic regulators as driver mutations [7] . Already, alterations of specialized enzymes that write or erase post-translational
Pharm. Pat. Anal. (2015) 4(4), 261–284
part of
ISSN 2046-8954
261
Patent Review Tanaka, Roberts, Qi & Bradner
Key terms Writers: Enzymes that catalyze addition of a covalent modification to chromatinassociated proteins. Erasers: Enzymes that catalyze removal of a covalent modification from chromatin-associated proteins.
modifications (PTMs) to histone and chromatinassociated proteins, and alterations of genes encoding proteins possessing specialized folds capable of reading histone PTMs, have been identified in numerous malignancies [8,9] . The reversibility of epigenomic modifications, catalyzed by specialized enzymes (socalled writers and erasers of chromatin-associated post-translational modification), suggests the feasibility of small-molecule antagonism [10,11] . The allure of targeting chromosome-associated factors is twofold. With somatic alteration of an epigenetic 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 chromatindependent oncogenic signaling to maintain determinants of the hallmark features of cancer. Our recent research directed at the development and characterization 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, displacement 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 epigenetic target classes (DNMT and HDAC) have been approved by the FDA, and their use is presently limited 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 targets or are inventions of ‘new use’ to focus this report on structure–activity relationships. Writers A nucleosome is the basic repeating subunit of chromatin, consisting of core histones and DNA. Histone pro-
262
Pharm. Pat. Anal. (2015) 4(4)
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 modifications [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 arginine 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 euchromatin, 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 trimethylation [H3K4me3]) or enhancers (H3K4me1), but it is also a characteristic feature of silenced facultative 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 methyltransferases (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 methyltransferases [22] . H3K79 methylation is observed at actively transcribed genes, suggesting a role for DOT1L in positive 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, AF10
future science group
Inhibitors of emerging epigenetic targets for cancer therapy
Patent Review
DNMT inhibitors NH2 N
N N
HO
NH2 N O
O
N O
N
HO O
OH OH
OH
VidazaTM Azacitidine Celgene MDS
DacogenTM Decitabine Eisai MDS, AML
HDAC inhibitors
H O
H N
N H
O
Me O
H
NH
H
HN
H N
O
H
N H
O
Me
IstodaxTM Romidepsin Celgene CTCL
O
Me
S HN O S
O
ZolinzaTM Vorinostat Merck CTCL
HN
O
Me HN
OH
Me
O O S N H
O N H
OH
BeleodaqTM Belinostat Spectrum Pharmaceuticals PTCL
OH
Me
FarydakTM Panobinostat Novartis MM 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.
and ENL) to increase H3K79 methylation in regulatory and coding regions of MLL target genes (e.g., HOX genes), supporting deregulated transcription [25] . Validation 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 discovery 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, significant attention has been allocated to the discovery of
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SAM-competitive DOT1L inhibitors [29] . Five patents including one on EPZ004777 have been published since 2010, four of which were filed by Epizyme. Inhibitors can be classified into two categories: urea-based inhibitors such as EPZ004777 and benzimidazolebased inhibitors such as EPZ-5676. Representative structures are shown in Figure 2. EPZ004777 is a first generation DOT1L inhibitor, containing a urea moiety [30] . Epizyme disclosed adenosine-containing analogs representative of structure 1 [31] . EPZ004777 exhibits more than 100,000fold selectivity for DOT1L over the KMTs CARM1, EHMT2, EZH1, EZH2, PRMT1, PRMT8, SETD7 and WHSC1, and 1280-fold selectivity over the arginine HMT PRMT5. Conversely, compound 1 does not show much selectivity for DOT1L over PRMT5
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NH2
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OH HN
NH 2
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3 Me
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Figure 2. DOT1L inhibitors.
(IC50 37,000-fold and the compound has a much longer drug-target
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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 target engagement [34] . Additional crystal structures of DOT1L with inhibitors have been reported, including 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 catalytic site and activation loop residues, which largely contribute to high potency and selectivity. In 2012, Epizyme initiated an ongoing Phase I clinical trial
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Inhibitors of emerging epigenetic targets for cancer therapy
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 (IC50 300 μM). It also inhibits cell growth in a panel of cancer cell lines (e.g., IC50 = 1.040 μM for MDA-MB-231 cells). A Nevada Cancer Institute-led team filed a patent disclosing guanidine 50 (IC50 = 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 inhibitor (30% inhibition at 10 μM) [124] . Inhibition mechanisms 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.
FAD·– trans
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NH2 2-PCPA
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NH N
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FAD·–
Figure 7. Proposed inactivation mechanism of FAD by 2-PCPA.
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O
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Inhibitors of emerging epigenetic targets for cancer therapy
NH2
trans
trans
trans
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30
2-PCPA
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(1R,2S)-34 33
32 NH2
N H
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N H
NH
NH
(1R,2S)-35
(1S,2R)-35
(1S,2R)-34
Figure 8. 2-PCPA analogs substituted on the amino group.
Readers Chromatin-associated histone modifications are recognized 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 acetylation of lysine is recognized by bromodomains, and as we have postulated perhaps also the enzymatically
compromised Class IIA histone deacetylases [126] . Epigenetic reader domains are commonly found as modules in multidomain, chromatin-associated proteins notably including many writers and erasers. In this manner, histone-binding modules may facilitate spreading of epigenetic marks and contribute to epigenetic memory. Further, epigenetic reader proteins nucleate multiprotein chromatin-associated complexes with spatial precision, prompting transcriptional activation, conferring a repressed state to heterochromatin and facilitating nucleosome remodeling [127] . In this review, we
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Cl O N H
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NH2 N H
N 41
Figure 9. 2-PCPA analogs substituted on the benzene ring.
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trans N H
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Figure 10. 2-PCPA analogs substituted both on the amino group and benzene ring.
will focus on the recent explosion in the development of small-molecule antagonists of bromodomains, and in particular bromodomain and extra-terminal (BET) bromodomains.
cal member BRD4, facilitate transcriptional elongation via recruitment of the positive transcription elongation factor (P-TEFb) and displacement of negative regulators (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 chromosomes and reduces expression of growth-associated genes, leading to G1 cell cycle arrest and apoptosis [133] . In 2010, our laboratory reported the first directacting bromodomain inhibitor, JQ1, in a collaborative study with Prof Stefan Knapp (Figure 12) [12] . JQ1 is a highly potent and BET-selective thienodiazepine which binds into the conserved asparagine via a methyl-triazolo chemical feature. This chemical tool has facilitated the mechanistic and translational study
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 acetyllysine-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 prototypiOH
O N
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Figure 11. Other LSD1 inhibitors.
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Table 3. Clinical trials of LSD1 inhibitors. Compound
Sponsor
Phase
Condition
ClinicalTrials.gov identifier
ORY–1001
Oryzon
I/IIa
Relapsed/refractory acute leukemia
EudraCT2013–002447–29
GSK2879552
GSK
I
Relapsed/refractory small-cell lung cancer
NCT02034123
I
Relapsed/refractory AML
NCT02177812
of BET bromodomains broadly in developmental and disease models. Importantly, JQ1 selectively kills several cancer cells such as AML and multiple myeloma (MM) through downregulation of MYC transcription [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 genomewide 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-kB [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 inframe fusion with the nuclear protein in testis gene (e.g., BRD4-NUT) elaborates a chimeric oncoprotein 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, associated with squamous differentiation and robust apoptosis [12] . Primary human NMC xenografts exhibit longterm survival on continuous JQ1 therapy, supporting consideration of BET inhibition as targeted therapy in this disease [12] . A drug-like derivative of JQ1 has transitioned to human clinical investigation, and is presently 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
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disclosed for BRD4 inhibitors. They are categorized into triazolothieno/benzodiazepines and their analogs, including JQ1 and I-BET762; isoxazole derivatives; pyridone derivatives and their analogs; and others (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 analogs GW841819X (an I-BET762 prototype) and compound 53 [149-152] . GW841819X inhibits the binding of tetra-acetylated lysine histone 4 peptide (H4AcK4) to a tandem bromodomain-containing construct of BRD4 (BRD4(1,2)) with an IC50 value of 16 nM. The pIC50 of compound 53 is over 5.5. Bayer filed a patent for compound 54, which has an IC50 of 27 nM for BRD4(1) [153] . A dimeric inhibitor 55 was presented in an application by Coferon [154] . No binding affinity or inhibitory activity for BRD4 was described. Constellation, which has initiated three follow-on Phase I clinical trials for CPI-0610 in leukemia, lymphoma and multiple myeloma, reported a series of patents covering isoxazolothienoazepine analogs 56 and isoxazolobenzoazepine 57 (IC50 for BRD4(1) = 26 and 14 nM, respectively) [155–157] . Oral administration of compound 56 suppresses MYC expression in a mouse xenograft model of B-cell lymphoma with MYC-dependent Raji cells (ED50 = 20–50 mg/kg). They also disclosed triazolodihydrobenzodiazepine 58 (IC50 for BRD4(1)
