RESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Isoquinolinas/farmacología , Linfoma de Células del Manto/patología , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Isoquinolinas/química , Isoquinolinas/uso terapéutico , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/enzimología , Masculino , Metilación , Ratones Endogámicos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Pirimidinas/química , Pirimidinas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Nucleares snRNP/metabolismoRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacocinética , Éteres Cíclicos/farmacocinética , Isoquinolinas/farmacocinética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Pirimidinas/farmacocinética , Animales , Inhibidores del Citocromo P-450 CYP2D6/farmacocinética , Inhibidores del Citocromo P-450 CYP3A/farmacocinética , Perros , Hepatocitos/metabolismo , Humanos , Cetoconazol/farmacocinética , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Quinidina/farmacocinética , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.
Asunto(s)
Apoptosis , Neoplasias/terapia , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Tumor Rabdoide/enzimología , Tumor Rabdoide/genética , Animales , Antineoplásicos/farmacología , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Proliferación Celular , Diseño de Fármacos , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Perfilación de la Expresión Génica , Células HEK293 , Histonas/metabolismo , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Piridinas/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Bencimidazoles/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Leucemia/genética , Leucemia/terapia , Metiltransferasas/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Relación Dosis-Respuesta a Droga , Femenino , Histona Metiltransferasas , Histonas/metabolismo , Humanos , Trasplante de Neoplasias , Conformación Proteica , Ratas , Ratas DesnudasRESUMEN
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.
Asunto(s)
Aminas/metabolismo , Inhibidores Enzimáticos/metabolismo , Etilenodiaminas/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Pirazoles/metabolismo , Animales , Arilamina N-Acetiltransferasa/metabolismo , Bilis/metabolismo , Biotransformación , Perros , Cromatografía de Gases y Espectrometría de Masas , Hepatocitos/metabolismo , Humanos , Isoenzimas/metabolismo , Cinética , Hígado/metabolismo , Masculino , Redes y Vías Metabólicas , Ratas , Ratas Sprague-DawleyRESUMEN
EPZ-5676 [(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], a small-molecule inhibitor of the protein methyltransferase DOT1L, is currently under clinical investigation for acute leukemias bearing MLL-rearrangements (MLL-r). In this study, we evaluated EPZ-5676 in combination with standard of care (SOC) agents for acute leukemias as well as other chromatin-modifying drugs in cellular assays with three human acute leukemia cell lines: MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), and SKM-1 (non-MLL-r). Studies were performed to evaluate the antiproliferative effects of EPZ-5676 combinations in a cotreatment model in which the second agent was added simultaneously with EPZ-5676 at the beginning of the assay, or in a pretreatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. EPZ-5676 was found to act synergistically with the acute myeloid leukemia (AML) SOC agents cytarabine or daunorubicin in MOLM-13 and MV4-11 MLL-r cell lines. EPZ-5676 is selective for MLL-r cell lines as demonstrated by its lack of effect either alone or in combination in the nonrearranged SKM-1 cell line. In MLL-r cells, the combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the chemotherapeutic agents, suggesting that EPZ-5676 sets up a durable, altered chromatin state that enhances the chemotherapeutic effects. Our evaluation of EPZ-5676 in conjunction with other chromatin-modifying drugs also revealed a consistent combination benefit, including synergy with DNA hypomethylating agents. These results indicate that EPZ-5676 is highly efficacious as a single agent and synergistically acts with other chemotherapeutics, including AML SOC drugs and DNA hypomethylating agents in MLL-r cells.
Asunto(s)
Antineoplásicos/administración & dosificación , Bencimidazoles/administración & dosificación , Proliferación Celular/efectos de los fármacos , Inhibidores de Crecimiento/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Metiltransferasas/antagonistas & inhibidores , Línea Celular Tumoral , Sinergismo Farmacológico , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia Mieloide Aguda/patología , Metilación/efectos de los fármacos , Metiltransferasas/metabolismoRESUMEN
EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Histonas/metabolismo , Indazoles/farmacología , Linfoma/tratamiento farmacológico , Linfoma/patología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Piridonas/farmacología , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/química , Histonas/química , Humanos , Indazoles/química , Linfoma/enzimología , Linfoma/genética , Lisina/metabolismo , Metilación/efectos de los fármacos , Estructura Molecular , Mutación Puntual , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Piridonas/química , Relación Estructura-ActividadRESUMEN
H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Humanos , Metilación , Metiltransferasas/química , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Complejo Represivo Polycomb 2/química , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
(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).
Asunto(s)
Antineoplásicos/farmacocinética , Bencimidazoles/farmacocinética , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Metiltransferasas/antagonistas & inhibidores , Animales , Antineoplásicos/sangre , Bencimidazoles/sangre , Proteínas Sanguíneas/metabolismo , Perros , Hepatocitos/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Permeabilidad , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/deficiencia , Empalme Alternativo , Antineoplásicos/química , Biomarcadores , Línea Celular Tumoral , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Humanos , Metilación , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Unión Proteica , Proteína-Arginina N-Metiltransferasas/química , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas Adaptadoras de Señalización CARD/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Guanilato Ciclasa/antagonistas & inhibidores , Isoxazoles/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Pirimidinas/uso terapéutico , Compuestos de Espiro/uso terapéutico , Animales , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacocinética , Humanos , Técnicas In Vitro , Isoxazoles/farmacocinética , Masculino , Ratones , Trasplante de Neoplasias , Pirimidinas/farmacocinética , Ratas Sprague-Dawley , Compuestos de Espiro/farmacocinéticaRESUMEN
Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers. CARM1 is known to methylate histone H3 and nonhistone substrates. To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors, but no ternary structures with nucleoside and peptide substrates have been reported. Here, the crystal structures of human CARM1 with the S-adenosylmethione (SAM) mimic sinefungin and three different peptide sequences from histone H3 and PABP1 are presented, with both nonmethylated and singly methylated arginine residues exemplified. This is the first example of multiple substrate sequences solved in a single PRMT enzyme and demonstrates how the CARM1 binding site is capable of accommodating a variety of peptide sequences while maintaining a core binding mode for the unmethylated and monomethylated substrates. Comparison of these with other PRMT enzyme-peptide structures shows hydrogen bonding patterns that may be thematic of these binding sites.
Asunto(s)
Proteína-Arginina N-Metiltransferasas/metabolismo , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Regulación Enzimológica de la Expresión Génica , Humanos , Modelos Moleculares , Conformación Proteica , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacocinética , Bencimidazoles/farmacocinética , Heces/química , Metiltransferasas/antagonistas & inhibidores , Adulto , Animales , Antineoplásicos/administración & dosificación , Autorradiografía/métodos , Bencimidazoles/administración & dosificación , Cromatografía Liquida/métodos , Perros , Femenino , N-Metiltransferasa de Histona-Lisina , Humanos , Infusiones Intravenosas , Masculino , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Especificidad de la Especie , Espectrometría de Masas en Tándem/métodos , Distribución TisularRESUMEN
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).
RESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Histonas/metabolismo , Metilación/efectos de los fármacos , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Descubrimiento de Drogas , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/farmacocinética , Humanos , Ratones , Complejo Represivo Polycomb 2/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacocinética , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Artefactos , Ensayos Analíticos de Alto Rendimiento/métodos , Histona Demetilasas/metabolismo , Espectrometría de Masas/métodos , Oxidación-Reducción , Descubrimiento de Drogas/métodos , Pruebas de Enzimas , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Metilación , Bibliotecas de Moléculas PequeñasRESUMEN
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.
RESUMEN
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.
RESUMEN
Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone - a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.
Asunto(s)
Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzamidas/farmacología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Linfoma no Hodgkin/tratamiento farmacológico , Piridonas/farmacología , Animales , Compuestos de Bifenilo , Línea Celular Tumoral , Ciclofosfamida/farmacología , Dexametasona/farmacología , Doxorrubicina/farmacología , Evaluación Preclínica de Medicamentos , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Linfoma no Hodgkin/metabolismo , Ratones SCID , Morfolinas , Trasplante de Neoplasias , Prednisolona/farmacología , Prednisona/farmacología , Distribución Aleatoria , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Vincristina/farmacologíaRESUMEN
EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wild-type and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.