A microglia clonal inflammatory disorder in Alzheimer's Disease.

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimer's Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.

Conformational-Design-Driven Discovery of EZM0414: A Selective, Potent SETD2 Inhibitor for Clinical Studies.

SETD2, a lysine N-methyltransferase, is a histone methyltransferase that plays an important role in various cellular processes and was identified as a target of interest in multiple myeloma that features a t(4,14) translocation. We recently reported the discovery of a novel small-molecule SETD2 inhibitor tool compound that is suitable for preclinical studies. Herein we describe the conformational-design-driven evolution of the advanced chemistry lead, which resulted in compounds appropriate for clinical evaluation. Further optimization of this chemical series led to the discovery of EZM0414, which is a potent, selective, and orally bioavailable inhibitor of SETD2 with good pharmacokinetic properties and robust pharmacodynamic activity in a mouse xenograft model.

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.

Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma.

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC50 = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.

Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850-60. ©2017 AACR.

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.

Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666.

The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591).

The Importance of Being Me: Magic Methyls, Methyltransferase Inhibitors, and the Discovery of Tazemetostat.

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor.

1. Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent. 2. Structure elucidation via high resolution, accurate mass LC-MS(n) revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction. 3. The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes. 4. Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.

Metabolism and disposition of the DOT1L inhibitor, pinometostat (EPZ-5676), in rat, dog and human.

PURPOSE: The metabolism and disposition of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was investigated in rat and dog. Metabolite profiles were compared with those from adult patients in the first-in-man phase 1 study as well as the cross-species metabolism observed in vitro. METHODS: EPZ-5676 was administered to rat and dog as a 24-h IV infusion of [(14)C]-EPZ-5676 for determination of pharmacokinetics, mass balance, metabolite profiling and biodistribution by quantitative whole-body autoradiography (QWBA). Metabolite profiling and identification was performed by radiometric and LC-MS/MS analysis. RESULTS: Fecal excretion was the major route of elimination, representing 79 and 81% of the total dose in and rat and dog, respectively. QWBA in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 168 h. Fecal recovery of dose-related material in bile duct-cannulated animals as well as higher radioactivity concentrations in the wall of the large intestine relative to liver implicated intestinal secretion as well as biliary elimination. EPZ-5676 underwent extensive oxidative metabolism with the major metabolic pathways being hydroxylation of the t-butyl group (EPZ007769) and N-dealkylation of the central nitrogen. Loss of adenine from parent EPZ-5676 (M7) was observed only in rat and dog feces, suggesting the involvement of gut microbiota. In rat and dog, steady-state plasma levels of total radioactivity and parent EPZ-5676 were attained rapidly and maintained through the infusion period before declining rapidly on cessation of dosing. Unchanged EPZ-5676 was the predominant circulating species in rat, dog and man. CONCLUSIONS: The excretory and metabolic pathways for EPZ-5676 were very similar across species. Renal excretion of both parent EPZ-5676 and EPZ-5676-related material was low, and in preclinical species fecal excretion of parent EPZ-5676 and EPZ007769 accounted for the majority of drug-related elimination.

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.

EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.

Structural and Kinetic Characterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652.

Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl}methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography - mass spectrometry (LC-MS(n)) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylene-diamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent Km values of >1000 µM for NAT1 and 165 ± 14.1 µM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 µL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.

A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.

Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers.

Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor.

(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of distribution 2-3 fold higher than total body water. EPZ-5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half-life (t1/2 ) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro-in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu -corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ-5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated product (M4) as well as an N-oxide (M6).

Potent inhibition of DOT1L as treatment of MLL-fusion leukemia.

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.