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1.
Curr Protoc ; 1(2): e51, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33587334

RESUMO

Evaluation of in vivo potencies plays an important role in drug discovery. Traditionally, the cellular activity and percent of plasma protein binding of a test agent are evaluated separately, with the plasma protein binding-adjusted cellular potency computation used to estimate in vivo potency. This process is costly, takes weeks to complete, and is increasingly unreliable for compounds that bind extensively to plasma proteins. Described in this article is a simple, high-throughput human plasma in-cell Western (ICW) assay that directly incorporates plasma protein binding into a cellular pharmacodynamic assay to provide a rapid and accurate estimate of in vivo potencies. The assay is versatile and can be readily employed for various targets that require short treatment periods for displaying maximal biological responses. © 2021 Wiley Periodicals LLC. Basic Protocol: Concentration-dependent human plasma ICW assay to determine test compound IC50 against the target of interest.


Assuntos
Bioensaio , Descoberta de Drogas , Proteínas Sanguíneas/metabolismo , Humanos , Ligação Proteica
2.
JCI Insight ; 5(8)2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32191634

RESUMO

Acute graft-versus-host disease (aGVHD) is a T cell-mediated immunological disorder and the leading cause of nonrelapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations that protein arginine methyltransferase 5 (PRMT5) and arginine methylation are upregulated in activated memory T cells, we hypothesized that PRMT5 is involved in the pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity were upregulated in activated T cells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression was also upregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared with those who did not develop aGVHD. PRMT5 inhibition using a selective small-molecule inhibitor (C220) substantially reduced mouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration of PRMT5 small-molecule inhibitors substantially improves survival, reducing disease incidence and clinical severity in mouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retained the beneficial graft-versus-leukemia effect by maintaining cytotoxic CD8+ T cell responses. Mechanistically, we show that PRMT5 inhibition potently reduced STAT1 phosphorylation as well as transcription of proinflammatory genes, including interferon-stimulated genes and IL-17. Additionally, PRMT5 inhibition deregulates the cell cycle in activated T cells and disrupts signaling by affecting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as a regulator of T cell responses and as a therapeutic target in aGVHD.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Interferons/imunologia , Ativação Linfocitária/imunologia , Proteína-Arginina N-Metiltransferases/imunologia , Linfócitos T/imunologia , Animais , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Camundongos
3.
ACS Med Chem Lett ; 10(7): 1033-1038, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31312404

RESUMO

Protein arginine methyltransferase 5 (PRMT5) is known to symmetrically dimethylate numerous cytosolic and nuclear proteins that are involved in a variety of cellular processes. Recent findings have revealed its potential as a cancer therapeutic target. PRMT5 possesses a cysteine (C449) in the active site, unique to PRMT5. Therefore, covalent PRMT5 inhibition is an attractive chemical approach. Herein, we report an exciting discovery of a series of novel hemiaminals that under physiological conditions can be converted to aldehydes and react with C449 to form covalent adducts, which presumably undergo an unprecedented elimination to form the thiol-vinyl ethers, as indicated by electron density in the co-crystal structure of the PRMT5/MEP50 complex.

4.
Cancer Cell ; 33(2): 309-321.e5, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29438699

RESUMO

Overall shared DNA methylation patterns between senescence (Sen) and cancers have led to the model that tumor-promoting epigenetic patterns arise through senescence. We show that transformation-associated methylation changes arise stochastically and independently of programmatic changes during senescence. Promoter hypermethylation events in transformation involve primarily pro-survival and developmental genes, similarly modified in primary tumors. Senescence-associated hypermethylation mainly involves metabolic regulators and appears early in proliferating "near-senescent" cells, which can be immortalized but are refractory to transformation. Importantly, a subset of transformation-associated hypermethylated developmental genes exhibits highest methylation gains at all age-associated cancer risk states across tissue types. These epigenetic changes favoring cell self-renewal and survival, arising during tissue aging, are fundamentally important for stratifying cancer risk and concepts for cancer prevention.


Assuntos
Transformação Celular Neoplásica/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética/genética , Animais , Senescência Celular/genética , Humanos , Camundongos , Camundongos SCID , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Risco
5.
ACS Chem Biol ; 13(3): 772-781, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29381856

RESUMO

DNA methyltransferase-1 (DNMT1) plays a crucial role in the maintenance of genomic methylation patterns. The crystal structure of DNMT1 was determined in two different states in which the helix that follows the catalytic loop was either kinked (designated helix-kinked) or well folded (designated helix-straight state). Here, we show that the proper structural transition between these two states is required for DNMT1 activity. The mutations of N1248A and R1279D, which did not affect interactions between DNMT1 and substrates or cofactors, allosterically reduced enzymatic activities in vitro by decreasing kcat/ Km for AdoMet. The crystallographic data combined with molecular dynamic (MD) simulations indicated that the N1248A and R1279D mutants bias the catalytic helix to either the kinked or straight conformation. In addition, genetic complementation assays for the two mutants suggested that disturbing the conformational transition reduced DNMT1 activity in cells, which could act additively with existing DNMT inhibitors to decrease DNA methylation. Collectively, our studies provide molecular insights into conformational changes of the catalytic helix, which is essential for DNMT1 catalytic activity, and thus aid in better understanding the relationship between DNMT1 dynamic switching and enzymatic activity.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/química , Simulação de Dinâmica Molecular , Animais , Domínio Catalítico , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Humanos , Mutação , Conformação Proteica
6.
Cancer Cell ; 31(5): 653-668.e7, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28486105

RESUMO

An oncogenic role for CHD4, a NuRD component, is defined for initiating and supporting tumor suppressor gene (TSG) silencing in human colorectal cancer. CHD4 recruits repressive chromatin proteins to sites of DNA damage repair, including DNA methyltransferases where it imposes de novo DNA methylation. At TSGs, CHD4 retention helps maintain DNA hypermethylation-associated transcriptional silencing. CHD4 is recruited by the excision repair protein OGG1 for oxidative damage to interact with the damage-induced base 8-hydroxydeoxyguanosine (8-OHdG), while ZMYND8 recruits it to double-strand breaks. CHD4 knockdown activates silenced TSGs, revealing their role for blunting colorectal cancer cell proliferation, invasion, and metastases. High CHD4 and 8-OHdG levels plus low expression of TSGs strongly correlates with early disease recurrence and decreased overall survival.


Assuntos
Autoantígenos/genética , Neoplasias Colorretais/genética , Metilação de DNA , Repressão Epigenética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , 8-Hidroxi-2'-Desoxiguanosina , Animais , Autoantígenos/metabolismo , Movimento Celular , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Intervalo Livre de Doença , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células HCT116 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Estimativa de Kaplan-Meier , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Estresse Oxidativo , Modelos de Riscos Proporcionais , Interferência de RNA , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteínas Supressoras de Tumor
7.
Genome Res ; 27(4): 533-544, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28232479

RESUMO

Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Genoma Humano , Células HCT116 , Humanos , Regiões Promotoras Genéticas , Ubiquitina-Proteína Ligases
8.
Mol Cell ; 65(2): 323-335, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-28107650

RESUMO

TET proteins, by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are hypothesized, but not directly shown, to protect promoter CpG islands (CGIs) against abnormal DNA methylation (DNAm) in cancer. We define such a protective role linked to DNA damage from oxidative stress (OS) known to induce this abnormality. TET2 removes aberrant DNAm during OS through interacting with DNA methyltransferases (DNMTs) in a "Yin-Yang" complex targeted to chromatin and enhanced by p300 mediated TET2 acetylation. Abnormal gains of DNAm and 5hmC occur simultaneously in OS, and knocking down TET2 dynamically alters this balance by enhancing 5mC and reducing 5hmC. TET2 reduction results in hypermethylation of promoter CGIs and enhancers in loci largely overlapping with those induced by OS. Thus, TET2 indeed may protect against abnormal, cancer DNAm in a manner linked to DNA damage.


Assuntos
Cromatina/metabolismo , Metilação de DNA , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilação , Cromatina/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Dioxigenases , Proteína p300 Associada a E1A/metabolismo , Células HCT116 , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Neoplasias/genética , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Fatores de Tempo , Transfecção , Ubiquitinação
9.
Development ; 143(23): 4368-4380, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27660325

RESUMO

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.


Assuntos
Diferenciação Celular/fisiologia , Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Tanquirases/antagonistas & inibidores , Via de Sinalização Wnt/fisiologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Reprogramação Celular/fisiologia , Camadas Germinativas/embriologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Humanos , Janus Quinases/metabolismo , Fator Inibidor de Leucemia/metabolismo , Camundongos , Fator de Transcrição STAT3/metabolismo
10.
Proc Natl Acad Sci U S A ; 113(37): 10238-44, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27573823

RESUMO

Vitamin C deficiency is found in patients with cancer and might complicate various therapy paradigms. Here we show how this deficiency may influence the use of DNA methyltransferase inhibitors (DNMTis) for treatment of hematological neoplasias. In vitro, when vitamin C is added at physiological levels to low doses of the DNMTi 5-aza-2'-deoxycytidine (5-aza-CdR), there is a synergistic inhibition of cancer-cell proliferation and increased apoptosis. These effects are associated with enhanced immune signals including increased expression of bidirectionally transcribed endogenous retrovirus (ERV) transcripts, increased cytosolic dsRNA, and activation of an IFN-inducing cellular response. This synergistic effect is likely the result of both passive DNA demethylation by DNMTi and active conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) enzymes at LTR regions of ERVs, because vitamin C acts as a cofactor for TET proteins. In addition, TET2 knockout reduces the synergy between the two compounds. Furthermore, we show that many patients with hematological neoplasia are markedly vitamin C deficient. Thus, our data suggest that correction of vitamin C deficiency in patients with hematological and other cancers may improve responses to epigenetic therapy with DNMTis.


Assuntos
Ácido Ascórbico/administração & dosagem , Azacitidina/análogos & derivados , Inibidores Enzimáticos/administração & dosagem , Neoplasias Hematológicas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Deficiência de Ácido Ascórbico/complicações , Deficiência de Ácido Ascórbico/tratamento farmacológico , Deficiência de Ácido Ascórbico/metabolismo , Deficiência de Ácido Ascórbico/patologia , Azacitidina/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Decitabina , Dioxigenases , Sinergismo Farmacológico , Retrovirus Endógenos/genética , Feminino , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/patologia , Humanos , Interferons/genética , Masculino , Metiltransferases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA de Cadeia Dupla/efeitos dos fármacos
11.
Cancer Cell ; 20(5): 606-19, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22094255

RESUMO

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.


Assuntos
DNA (Citosina-5-)-Metiltransferases/fisiologia , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , Sirtuína 1/fisiologia , Animais , Cromatina/metabolismo , Colite/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HCT116 , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Sirtuína 1/metabolismo , DNA Metiltransferase 3B
12.
Mol Cell Biol ; 31(2): 375-84, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21078873

RESUMO

Transcriptional corepressors play complex roles in developmental gene regulation. These proteins control transcription by recruiting diverse chromatin-modifying enzymes, but it is not known whether corepressor activities are finely regulated in different developmental settings or whether their basic activities are identical in most contexts. The evolutionarily conserved C-terminal binding protein (CtBP) is recruited by a variety of transcription factors that play crucial roles in development and disease. CtBP contains a central NAD(H) binding core domain that is homologous to D2 hydroxy acid dehydrogenase enzymes, as well as an unstructured C-terminal domain. NAD(H) binding is important for CtBP function, but the significance of its intrinsic dehydrogenase activity, as well as that of the unstructured C terminus, is poorly understood. To clarify the biological relevance of these features, we established genetic rescue assays to determine how different forms of CtBP function in the context of Drosophila melanogaster development. The mutant phenotypes and specific gene regulatory effects indicate that both the catalytic site of CtBP and the C-terminal extension play important, if nonessential roles in development. Our results indicate that the structural and enzymatic features of CtBP, previously thought to be dispensable for overall transcriptional control, are critical for modulating this protein's activity in diverse developmental settings.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Gênica , Oxirredutases do Álcool/química , Animais , Animais Geneticamente Modificados , Proteínas Correpressoras/química , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Fenótipo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
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