RESUMEN
Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.
Asunto(s)
Antineoplásicos/farmacología , Histonas/metabolismo , Lisina/metabolismo , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Sulfonas/química , Sulfonas/farmacología , Triazoles/química , Triazoles/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Antineoplásicos/química , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Histonas/química , Humanos , Lisina/química , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Relación Estructura-Actividad , Sulfonas/metabolismo , Triazoles/metabolismo , Células Tumorales CultivadasRESUMEN
The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular , Cromatina/genética , Cristalografía por Rayos X , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Humanos , Lisina/química , Metilación , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Proteína 4 de Unión a Retinoblastoma/química , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Transcripción GenéticaRESUMEN
The EED (embryonic ectoderm development) subunit of the Polycomb repressive complex 2 (PRC2) plays an important role in the feed forward regulation of the PRC2 enzymatic activity. We recently identified a new class of allosteric PRC2 inhibitors that bind to the H3K27me3 pocket of EED. Multiple assays were developed and used to identify and characterize this type of PRC2 inhibitors. One of them is a genetically encoded EED biosensor based on the EED[G255D] mutant and the split firefly luciferase. This EED biosensor can detect the compound binding in the transfected cells and in the in vitro biochemical assays. Compared to other commonly used cellular assays, the EED biosensor assay has the advantage of shorter compound incubation with cells. The in vitro EED biosensor is much more sensitive than other label-free biophysical assays (e.g. DSF, ITC). Based on the crystal structure, the DSF data as well as the biosensor assay data, it's most likely that compound-induced increase in the luciferase activity of the EED[G255D] biosensor results from the decreased non-productive interactions between the EED subdomain and other subdomains within the biosensor construct. This new insight of the mechanism might help to broaden the use of the split luciferase based biosensors.
Asunto(s)
Bioensayo/métodos , Luciferasas de Luciérnaga/metabolismo , Mediciones Luminiscentes/métodos , Mutación Missense , Complejo Represivo Polycomb 2/metabolismo , Sustitución de Aminoácidos , Línea Celular , Humanos , Luciferasas de Luciérnaga/genética , Complejo Represivo Polycomb 2/genética , Unión Proteica , Dominios ProteicosRESUMEN
Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.
Asunto(s)
Linfoma de Células B Grandes Difuso/patología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Embrión de Mamíferos/citología , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Linfoma de Células B Grandes Difuso/genética , Metilación/efectos de los fármacos , Ratones , Mutación/genética , Fenotipo , Complejo Represivo Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Ensayo de Tumor de Célula Madre , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368-375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1-282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel ß7 and ß8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/química , Neoplasias/metabolismo , Proteína p53 Supresora de Tumor/química , Calorimetría/métodos , Línea Celular Tumoral , Cristalografía por Rayos X/métodos , Genes Supresores de Tumor , Histonas/química , Humanos , Cinética , Lisina/química , Metilación , Conformación Molecular , Unión Proteica , Transcripción GenéticaRESUMEN
Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the low-molecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development.
Asunto(s)
Histonas , Neoplasias , Animales , Inhibidores Enzimáticos , Histonas/metabolismo , Humanos , Metilación , Ratones , Neoplasias/tratamiento farmacológico , Complejo Represivo Polycomb 2RESUMEN
LSD1 (Lysine Specific Demethylase1)/KDM1A (Lysine Demethylase 1A), a flavin adenine dinucleotide (FAD)-dependent histone H3K4/K9 demethylase, sustains oncogenic potential of leukemia stem cells in primary human leukemia cells. However, the pro-differentiation and anti-proliferation effects of LSD1 inhibition in acute myeloid leukemia (AML) are not yet fully understood. Here, we report that small hairpin RNA (shRNA) mediated LSD1 inhibition causes a remarkable transcriptional activation of myeloid lineage marker genes (CD11b/ITGAM and CD86), reduction of cell proliferation and decrease of clonogenic ability of human AML cells. Cell surface expression of CD11b and CD86 is significantly and dynamically increased in human AML cells upon sustained LSD1 inhibition. Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) analyses of histone marks revealed that there is a specific increase of H3K4me2 modification and an accompanied increase of H3K4me3 modification at the respective CD11b and CD86 promoter region, whereas the global H3K4me2 level remains constant. Consistently, inhibition of LSD1 in vivo significantly blocks tumor growth and induces a prominent increase of CD11b and CD86. Taken together, our results demonstrate the anti-tumor properties of LSD1 inhibition on human AML cell line and mouse xenograft model. Our findings provide mechanistic insights into the LSD1 functions in controlling both differentiation and proliferation in AML.
RESUMEN
PRC2 is a multisubunit methyltransferase involved in epigenetic regulation of early embryonic development and cell growth. The catalytic subunit EZH2 methylates primarily lysine 27 of histone H3, leading to chromatin compaction and repression of tumor suppressor genes. Inhibiting this activity by small molecules targeting EZH2 was shown to result in antitumor efficacy. Here, we describe the optimization of a chemical series representing a new class of PRC2 inhibitors which acts allosterically via the trimethyllysine pocket of the noncatalytic EED subunit. Deconstruction of a larger and complex screening hit to a simple fragment-sized molecule followed by structure-guided regrowth and careful property modulation were employed to yield compounds which achieve submicromolar inhibition in functional assays and cellular activity. The resulting molecules can serve as a simplified entry point for lead optimization and can be utilized to study this new mechanism of PRC2 inhibition and the associated biology in detail.
Asunto(s)
Inhibidores Enzimáticos/química , Epigénesis Genética , Metiltransferasas/antagonistas & inhibidores , Complejo Represivo Polycomb 2/química , Regulación Alostérica , Células CACO-2 , Cromatografía Liquida , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas , Estructura Molecular , Espectroscopía de Protones por Resonancia Magnética , Relación Estructura-ActividadRESUMEN
Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound 43 (EED226). Guided by X-ray crystallography, compound 43 was discovered by fragmentation and regrowth of compound 7, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of 43. Compound 43 induces robust and sustained tumor regression in EZH2MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Sulfonas/química , Sulfonas/farmacología , Triazoles/química , Triazoles/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Femenino , Haplorrinos , Histonas/metabolismo , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/metabolismo , Lisina/metabolismo , Masculino , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Ratas , Sulfonas/farmacocinética , Sulfonas/uso terapéutico , Triazoles/farmacocinética , Triazoles/uso terapéuticoRESUMEN
Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Sulfonas/farmacología , Triazoles/farmacología , Animales , Sitios de Unión , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Ratones , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Relación Estructura-Actividad Cuantitativa , Sulfonas/química , Triazoles/químicaRESUMEN
5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metionina/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Desoxiadenosinas/metabolismo , Eliminación de Gen , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferasas/genética , Purina-Nucleósido Fosforilasa/genética , ARN Interferente Pequeño/genética , Tionucleósidos/metabolismoRESUMEN
Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)- multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation.