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1.
J Med Chem ; 64(4): 2228-2241, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33570945

RESUMO

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.


Assuntos
Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Janus Quinase 2/metabolismo , Estrutura Molecular , Nitrilas , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Pirimidinas , Pirrolidinas/química , Pirrolidinas/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/metabolismo
2.
Mol Cancer Ther ; 17(12): 2796-2810, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30242092

RESUMO

Resistance to androgen receptor (AR) antagonists is a significant problem in the treatment of castration-resistant prostate cancers (CRPC). Identification of the mechanisms by which CRPCs evade androgen deprivation therapies (ADT) is critical to develop novel therapeutics. We uncovered that CRPCs rely on BRD4-HOXB13 epigenetic reprogramming for androgen-independent cell proliferation. Mechanistically, BRD4, a member of the BET bromodomain family, epigenetically promotes HOXB13 expression. Consistently, genetic disruption of HOXB13 or pharmacological suppression of its mRNA and protein expression by the novel dual-activity BET bromodomain-kinase inhibitors directly correlates with rapid induction of apoptosis, potent inhibition of tumor cell proliferation and cell migration, and suppression of CRPC growth. Integrative analysis revealed that the BRD4-HOXB13 transcriptome comprises a proliferative gene network implicated in cell-cycle progression, nucleotide metabolism, and chromatin assembly. Notably, although the core HOXB13 target genes responsive to BET inhibitors (HOTBIN10) are overexpressed in metastatic cases, in ADT-treated CRPC cell lines and patient-derived circulating tumor cells (CTC) they are insensitive to AR depletion or blockade. Among the HOTBIN10 genes, AURKB and MELK expression correlates with HOXB13 expression in CTCs of mCRPC patients who did not respond to abiraterone (ABR), suggesting that AURKB inhibitors could be used additionally against high-risk HOXB13-positive metastatic prostate cancers. Combined, our study demonstrates that BRD4-HOXB13-HOTBIN10 regulatory circuit maintains the malignant state of CRPCs and identifies a core proproliferative network driving ADT resistance that is targetable with potent dual-activity bromodomain-kinase inhibitors.


Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Loci Gênicos , Humanos , Masculino , Camundongos SCID , Metástase Neoplásica , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Biol Chem ; 293(16): 6187-6200, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29449372

RESUMO

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.


Assuntos
Proteínas Culina/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Azepinas/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Lenalidomida/farmacologia , Ligantes , Camundongos , Sondas Moleculares , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linfócitos T/metabolismo , Talidomida/análogos & derivados , Talidomida/metabolismo , Talidomida/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Ubiquitina/metabolismo
4.
Mol Cancer Ther ; 16(6): 1054-1067, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28336808

RESUMO

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054-67. ©2017 AACR.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Inibidores de Proteínas Quinases/química , Proteínas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Sci Transl Med ; 9(372)2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-28077684

RESUMO

Graft-versus-host disease (GVHD) is a leading cause of nonrelapse mortality after allogeneic hematopoietic cell transplantation. T cell costimulation by CD28 contributes to GVHD, but prevention is incomplete when targeting CD28, downstream mammalian target of rapamycin (mTOR), or Aurora A. Likewise, interleukin-6 (IL-6)-mediated Janus kinase 2 (JAK2) signaling promotes alloreactivity, yet JAK2 inhibition does not eliminate GVHD. We provide evidence that blocking Aurora A and JAK2 in human T cells is synergistic in vitro, prevents xenogeneic GVHD, and maintains antitumor responses by cytotoxic T lymphocytes (CTLs). Aurora A/JAK2 inhibition is immunosuppressive but permits the differentiation of inducible regulatory T cells (iTregs) that are hyperfunctional and CD39 bright and efficiently scavenge adenosine triphosphate (ATP). Increased iTreg potency is primarily a function of Aurora A blockade, whereas JAK2 inhibition suppresses T helper 17 (TH17) differentiation. Inhibiting either Aurora A or JAK2 significantly suppresses TH1 T cells. However, CTL generated in vivo retains tumor-specific killing despite Aurora A/JAK2 blockade. Thus, inhibiting CD28 and IL-6 signal transduction pathways in donor T cells can increase the Treg/Tconv ratio, prevent GVHD, and preserve antitumor CTL.


Assuntos
Aurora Quinase A/antagonistas & inibidores , Doença Enxerto-Hospedeiro/prevenção & controle , Janus Quinase 2/antagonistas & inibidores , Leucemia/terapia , Linfócitos T Citotóxicos/citologia , Linfócitos T Reguladores/citologia , Animais , Antineoplásicos/farmacologia , Aurora Quinase A/metabolismo , Azepinas/farmacologia , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Feminino , Humanos , Imunossupressores/farmacologia , Interleucina-6/metabolismo , Janus Quinase 2/metabolismo , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Recidiva Local de Neoplasia/prevenção & controle , Transplante de Neoplasias , Pirimidinas/farmacologia , Pirrolidinas/farmacologia , Transdução de Sinais , Sulfonamidas/farmacologia , Células Th17/citologia
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