RESUMO
PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types, influencing pro-tumor macrophage polarization as well as suppressing the antitumor inflammation response by modulating IFN-γ and IL-4 signaling. PARP14 is a 203â kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates. PARP14 also contains three macrodomains and a WWE domain which are binding modules for mono-ADP-ribose and poly-ADP-ribose, respectively, in addition to two RNA recognition motifs. Catalytic inhibitors of PARP14 have been shown to reverse IL-4 driven pro-tumor gene expression in macrophages, however it is not clear what roles the non-enzymatic biomolecular recognition motifs play in PARP14-driven immunology and inflammation. To further understand this, we have discovered a heterobifunctional small molecule designed based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+ -binding site and recruits cereblon to ubiquitinate it and selectively target it for degradation.
Assuntos
Poli(ADP-Ribose) Polimerases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Isoquinolinas/farmacologia , Linfoma de Célula do Manto/patologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Isoquinolinas/química , Isoquinolinas/uso terapêutico , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/enzimologia , Masculino , Metilação , Camundongos Endogâmicos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Pirimidinas/química , Pirimidinas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Centrais de snRNP/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Histonas/metabolismo , Indazóis/farmacologia , Linfoma/tratamento farmacológico , Linfoma/patologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Piridonas/farmacologia , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/química , Histonas/química , Humanos , Indazóis/química , Linfoma/enzimologia , Linfoma/genética , Lisina/metabolismo , Metilação/efeitos dos fármacos , Estrutura Molecular , Mutação Puntual , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Piridonas/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Sequência de Aminoácidos , Biocatálise , Humanos , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Dados de Sequência Molecular , Complexo Repressor Polycomb 2/química , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 µM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Inflamação/tratamento farmacológico , Interleucina-4/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-4/genética , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Poli(ADP-Ribose) Polimerases/genética , Células RAW 264.7 , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Interferon Tipo I/metabolismo , Neoplasias/tratamento farmacológico , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Imunidade Adaptativa/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Inibidores Enzimáticos/isolamento & purificação , Ensaios de Triagem em Larga Escala , Inibidores de Poli(ADP-Ribose) Polimerases/isolamento & purificação , Processamento de Proteína Pós-Traducional/genética , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP-Ribosilação/genética , Adenosina Difosfato Ribose/genética , Inibidores Enzimáticos/farmacologia , Humanos , NAD/química , Poli ADP Ribosilação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Especificidade por SubstratoRESUMO
Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.
Assuntos
Corantes Fluorescentes/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Competitiva , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , NAD/química , NAD/metabolismo , Nanopartículas/química , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
This paper has been written by the SLAS Sample Management Special Interest Group to serve as a guide to the best practices and methods in establishing and maintaining a high-quality sample management system. The topics covered are applicable to sample types ranging from small molecules to biologics to tissue samples. It has been put together using the collective experience of the authors in start-up companies, small pharma, agricultural research, IT, academia, biorepositories, and large pharma companies. Our hope is that sharing our experience will streamline the process of setting up a new sample management system and help others avoid some of the problems that we have encountered.
Assuntos
Pesquisa Biomédica/métodos , Laboratórios/organização & administração , Manejo de Espécimes/métodosRESUMO
Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.
Assuntos
Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Splicing de RNA/genética , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo/genética , Antineoplásicos , Arginina/análogos & derivados , Arginina/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Proteínas Centrais de snRNP/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Carcinogênese/genética , Histona-Lisina N-Metiltransferase/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Sistemas CRISPR-Cas , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/química , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/antagonistas & inibidores , Interferência de RNA , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
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.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histonas/metabolismo , Metilação/efeitos dos fármacos , Complexo Repressor Polycomb 2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Descoberta de Drogas , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/farmacocinética , Humanos , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacocinética , Relação Estrutura-AtividadeRESUMO
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).
RESUMO
Heterozygous point mutations at Y641 and A677 in the EZH2 SET domain are prevalent in about 10-24% of Non-Hodgkin lymphomas (NHL). Previous studies indicate that these are gain-of-function mutations leading to the hypertrimethylation of H3K27. These EZH2 mutations may drive the proliferation of lymphoma and make EZH2 a molecular target for patients harboring these mutations. Here, another EZH2 SET domain point mutation, A687V, occurring in about 1-2% of lymphoma patients, is also shown to be a gain-of-function mutation that greatly enhances its ability to perform dimethylation relative to wild-type EZH2 and is equally proficient at catalyzing trimethylation. We propose that A687V EZH2 also leads to hypertrimethylation of H3K27 and may thus be a driver mutation in NHL.
Assuntos
Linfoma não Hodgkin/enzimologia , Linfoma não Hodgkin/genética , Proteínas Mutantes/genética , Proteínas de Neoplasias/genética , Mutação Puntual , Complexo Repressor Polycomb 2/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Heterozigoto , Histonas/química , Histonas/metabolismo , Humanos , Cinética , Metilação , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
DOT1L is the human protein methyltransferase responsible for catalyzing the methylation of histone H3 on lysine 79 (H3K79). The ectopic activity of DOT1L, associated with the chromosomal translocation that is a universal hallmark of MLL-rearranged leukemia, is a required driver of leukemogenesis in this malignancy. Here, we present studies on the structure-activity relationship of aminonucleoside-based DOT1L inhibitors. Within this series, we find that improvements in target enzyme affinity and selectivity are driven entirely by diminution of the dissociation rate constant for the enzyme-inhibitor complex, leading to long residence times for the binary complex. The biochemical K(i) and residence times measured for these inhibitors correlate well with their effects on intracellular H3K79 methylation and MLL-rearranged leukemic cell killing. Crystallographic studies reveal a conformational adaptation mechanism associated with high-affinity inhibitor binding and prolonged residence time; these studies also suggest that conformational adaptation likewise plays a critical role in natural ligand interactions with the enzyme, hence, facilitating enzyme turnover. These results provide critical insights into the role of conformational adaptation in the enzymatic mechanism of catalysis and in pharmacologic intervention for DOT1L and other members of this enzyme class.
Assuntos
Inibidores Enzimáticos/química , Metiltransferases/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Cinética , Metilação , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Nucleosídeos/química , Nucleosídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
A survey of the human genome was performed to understand the constituency of protein methyltransferases (both protein arginine and lysine methyltransferases) and the relatedness of their catalytic domains. We identified 51 protein lysine methyltransferase proteins based on similarity to the canonical Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain. Disruptor of telomeric silencing-1-like, a known protein lysine methyltransferase, did not fit within the protein lysine methyltransferase family, but did group with the protein arginine methyltransferases, along with 44 other proteins, including the METTL and NOP2/Sun domain family proteins. We show that a representative METTL, METTL11A, demonstrates catalytic activity as a histone methyltransferase. We also solved the co-crystal structures of disruptor of telomeric silencing-1-like with S-adenosylmethionine and S-adenosylhomocysteine bound in its active site. The conformation of both ligands is virtually identical to that found in known protein arginine methyltransferases, METTL and NOP2/Sun domain family proteins and is distinct from that seen in the Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain protein lysine methyltransferases. We have developed biochemical assays for 11 members of the protein methyltransferase target class and have profiled the affinity of three ligands for these enzymes: the common methyl-donating substrate S-adenosylmethionine; the common reaction product S-adenosylhomocysteine; and the natural product sinefungin. The affinity of each of these ligands is mapped onto the family trees of the protein lysine methyltransferases and protein arginine methyltransferases to reveal patterns of ligand recognition by these enzymes.
Assuntos
Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Modelos Moleculares , Sítios de Ligação , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Ligantes , Estrutura Molecular , FilogeniaRESUMO
Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.
Assuntos
Inibidores Enzimáticos/farmacologia , Leucemia Aguda Bifenotípica/patologia , Metiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico/efeitos dos fármacos , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/metabolismo , Humanos , Leucemia Aguda Bifenotípica/genética , Lisina/metabolismo , Metilação/efeitos dos fármacos , Metiltransferases/metabolismo , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by "relamination" (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.
Assuntos
Gástrula/fisiologia , Mesoderma/fisiologia , Morfogênese , Xenopus laevis/embriologia , Xenopus/embriologia , Animais , Biotinilação , Movimento Celular , Indução Embrionária , Gástrula/ultraestrutura , Humanos , Mesoderma/ultraestrutura , Filogenia , Fatores de Tempo , Transplante de Tecidos , Xenopus/anatomia & histologia , Xenopus laevis/anatomia & histologiaRESUMO
Urodeles begin gastrulation with much of their presumptive mesoderm in the superficial cell layer, all of which must move into the deep layers during development. We studied the morphogenesis of superficial mesoderm in the urodeles Ambystoma maculatum, Ambystoma mexicanum, and Taricha granulosa. In all three species, somitic, lateral, and ventral mesoderm move into the deep layer during gastrulation, ingressing through a "bilateral primitive streak" just inside the blastopore. The mesodermal epithelium appears to slide under the endodermal epithelium by a mechanism we term "subduction." Subduction removes the large expanse of superficial presumptive somitic and lateral-ventral mesoderm that initially separates the sub-blastoporal endoderm from the notochord, leaving the endoderm bounding the still epithelial notochord along the gastrocoel roof. Subduction may be a common feature of urodele gastrulation, differing in this regard from anurans. Subducting cells constrict their apices and become bottle-shaped as they approach the junction of the mesodermal and endodermal epithelia. Subducting bottle cells endocytose apical membrane and withdraw the tight junctional component cingulin from the contracting circumferential tight junctions. Either in conjunction with or immediately after subducting, the mesodermal cells undergo an epithelial-to-mesenchymal transition. The mechanism by which epithelial cells release their apical junctions to become mesenchymal, without disrupting the integrity of the epithelium, remains mysterious, but this system should prove useful in understanding this process in a developmental context.