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1.
Nat Rev Mol Cell Biol ; 9(10): 747-58, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18813292

RESUMO

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases that regulate diverse cellular processes, such as cell growth, cell motility, cell survival and cell proliferation. RSKs are downstream effectors of the Ras-extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling cascade. Significant advances in the field of RSK and ERK/MAPK signalling have occurred in the past few years, including biological insights and the discovery of novel substrates and new RSK regulatory mechanisms. Collectively, these data expand the current models of RSK signalling and highlight potential directions of research in RSK-mediated survival, growth, proliferation and migration.


Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ciclo Celular/fisiologia , Sobrevivência Celular/fisiologia , Ativação Enzimática , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Homologia de Sequência de Aminoácidos , Transcrição Gênica
2.
Bioorg Med Chem ; 28(23): 115815, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33091850

RESUMO

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Inibidores de Proteínas Quinases/metabolismo , Quinazolinas/química , Aldeído Oxidase/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Cães , Estabilidade de Medicamentos , Meia-Vida , Hepatócitos/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Ratos , Relação Estrutura-Atividade
3.
Bioorg Med Chem ; 26(4): 913-924, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29398441

RESUMO

We have developed a series of orally efficacious IRAK4 inhibitors, based on a scaffold hopping strategy and using rational structure based design. Efforts to tackle low permeability and high efflux in our previously reported pyrrolopyrimidine series (Scott et al., 2017) led to the identification of pyrrolotriazines which contained one less formal hydrogen bond donor and were intrinsically more lipophilic. Further optimisation of substituents on this pyrrolotriazine core culminated with the discovery of 30 as a promising in vivo probe to assess the potential of IRAK4 inhibition for the treatment of MyD88 mutant DLBCL in combination with a BTK inhibitor. When tested in an ABC-DLBCL model with a dual MyD88/CD79 mutation (OCI-LY10), 30 demonstrated tumour regressions in combination with ibrutinib.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Pirróis/química , Tiazinas/química , Animais , Sítios de Ligação , Células CACO-2 , Cães , Desenho de Fármacos , Meia-Vida , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Simulação de Dinâmica Molecular , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Permeabilidade/efeitos dos fármacos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Pirróis/farmacocinética , Pirróis/farmacologia , Ratos , Relação Estrutura-Atividade , Tiazinas/farmacocinética , Tiazinas/farmacologia
4.
PLoS Genet ; 9(9): e1003749, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086144

RESUMO

Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 "phospho-occupied" serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the per(01) arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ~ 30 hrs. Interestingly, the single S613A mutation extends the period by 2-3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period.


Assuntos
Ritmo Circadiano/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Circadianas Period/genética , Fosforilação/genética , Animais , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Humanos , Mutação , Proteínas Circadianas Period/fisiologia , Fenótipo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo
5.
Proc Natl Acad Sci U S A ; 106(28): 11606-11, 2009 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-19564600

RESUMO

Most kinases are capable of recognizing and phosphorylating peptides containing short, linear sequence motifs. To measure the activation state of many kinases from the same cell lysate, we created a multiplexed, mass-spectrometry-based in vitro kinase assay. Ninety chemically synthesized peptides derived from well-characterized peptide substrates and in vivo phosphorylation sites with either known or previously unidentified upstream kinases were reacted individually in a plate format with crude cell lysates and ATP. Phosphorylation rates were directly measured based on the addition of 90 same-sequence, site-specific phosphopeptides enriched in stable isotopes to act as ideal quantitative internal standards for analysis by liquid chromatography coupled to tandem mass spectrometry. This approach concurrently measured up to 90 site-specific peptide phosphorylation rates, reporting a diagnostic fingerprint for activated kinase pathways. We applied this unique kinome-activity profiling strategy in a variety of cellular settings, including mitogen stimulation, cell cycle, pharmacological inhibition of pathways, and to a panel of breast cancer cell lines. Finally, we identified the source of activity for a peptide (derived from a PI3K regulatory subunit) from our library. This peptide substrate demonstrated mitotic and tyrosine-specific phosphorylation, which was confirmed to be a novel Src family kinase site in vivo.


Assuntos
Ciclo Celular/fisiologia , Fosfopeptídeos/metabolismo , Fosfotransferases/metabolismo , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Marcação por Isótopo , Espectrometria de Massas , Fosforilação
6.
Br J Pharmacol ; 178(3): 600-613, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33125717

RESUMO

BACKGROUND AND PURPOSE: Savolitinib (AZD6094, HMPL-504, volitinib) is an oral, potent, and highly MET receptor TK inhibitor. This series of studies aimed to develop a pharmacokinetic-pharmacodynamic (PK/PD) model to link inhibition of MET phosphorylation (pMET) by savolitinib with anti-tumour activity. EXPERIMENTAL APPROACH: Cell line-derived xenograft (CDX) experiments using human lung cancer (EBC-1) and gastric cancer (MKN-45) cells were conducted in athymic nude mice using a variety of doses and schedules of savolitinib. Tumour pMET changes and growth inhibition were calculated after 28 days. Population PK/PD techniques were used to construct a PK/PD model for savolitinib. KEY RESULTS: Savolitinib showed dose- and dose frequency-dependent anti-tumour activity in the CDX models, with more frequent, lower dosing schedules (e.g., twice daily) being more effective than intermittent, higher dosing schedules (e.g., 4 days on/3 days off or 2 days on/5 days off). There was a clear exposure-response relationship, with maximal suppression of pMET of >90%. Data from additional CDX and patient-derived xenograft (PDX) models overlapped, allowing calculation of a single EC50 of 0.38 ng·ml-1 . Tumour growth modelling demonstrated that prolonged, high levels of pMET inhibition (>90%) were required for tumour stasis and regression in the models. CONCLUSION AND IMPLICATIONS: High and persistent levels of MET inhibition by savolitinib were needed for optimal monotherapy anti-tumour activity in preclinical models. The modelling framework developed here can be used to translate tumour growth inhibition from the mouse to human and thus guide choice of clinical dose and schedule.


Assuntos
Antineoplásicos , Proteínas Proto-Oncogênicas c-met , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Pirazinas , Triazinas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Sci Transl Med ; 13(609): eabb3738, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34516823

RESUMO

The clinical efficacy of epidermal growth factor receptor (EGFR)­targeted therapy in EGFR-mutant non­small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor­resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.


Assuntos
Receptores ErbB , Neoplasias Pulmonares , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
8.
PLoS One ; 15(7): e0236192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32692756

RESUMO

Breast cancer (BC) is the foremost cause of cancer related deaths in women globally. Currently there is a scarcity of reliable biomarkers for its early stage diagnosis and theranostics monitoring. Altered DNA methylation patterns leading to the silencing of tumor suppressor genes are considered as an important mechanism underlying tumor development and progression in various cancer types, including BC. Very recently, epigenetic silencing of SHISA3, an antagonist of ß-catenin, has been reported in various types of tumor. However, the role of SHISA3 in BC has not been investigated yet. Therefore, we aimed at evaluating the contribution of SHISA3 in BC causation by analyzing its expression and methylation levels in BC cell lines (MDA-MB231, MCF-7 and BT-474) and in 103 paired BC tissue samples. The SHISA3 expression and methylation status was determined by qPCR and methylation specific PCR (MSP) respectively. The role of SHISA3 in BC tumorigenesis was evaluated by proliferation and migration assays after ectopic expression of SHISA3. The association between SHISA3 hypermethylation and clinicopathological parameters of BC patients was also studied. The downregulation of SHISA3 expression was found in three BC cell lines used and in all BC tissue samples. However, SHISA3 promoter region was hypermethylated in 61% (63/103) tumorous tissues in comparison to the 18% of their matched normal tissues. The 5-aza-2'-deoxycytidine treatment restored SHISA3 expression by reversing promoter hypermethylation in both MDA-MB231 and MCF-7 cells. Furthermore, ectopic expression of SHISA3 significantly reduced the proliferation and migration ability of these cells. Taken together, our findings for the first time reveal epigenetic silencing and tumor suppressing role of SHISA3 in BC. Henceforth, this study has identified SHISA3 as potentially powerful target for the development of new therapies against BC, as well as novel diagnostic and therapy response monitoring approaches.


Assuntos
Neoplasias da Mama/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Via de Sinalização Wnt/genética , Azacitidina/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Metilação de DNA/genética , Feminino , Humanos , Proteínas de Membrana/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Clin Cancer Res ; 26(14): 3751-3759, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32220888

RESUMO

PURPOSE: The emergence of secondary mutations is a cause of resistance to current KIT inhibitors used in the treatment of patients with gastrointestinal stromal tumors (GIST). AZD3229 is a selective inhibitor of wild-type KIT and a wide spectrum of primary and secondary mutations seen in patients with GIST. The objective of this analysis is to establish the pharmacokinetic-pharmacodynamic (PKPD) relationship of AZD3229 in a range of mouse GIST tumor models harboring primary and secondary KIT mutations, and to benchmark AZD3229 against other KIT inhibitors. EXPERIMENTAL DESIGN: A PKPD model was developed for AZD3229 linking plasma concentrations to inhibition of phosphorylated KIT using data generated from several in vivo preclinical tumor models, and in vitro data generated in a panel of Ba/F3 cell lines. RESULTS: AZD3229 drives inhibition of phosphorylated KIT in an exposure-dependent manner, and optimal efficacy is observed when >90% inhibition of KIT phosphorylation is sustained over the dosing interval. Integrating the predicted human pharmacokinetics into the mouse PKPD model predicts that an oral twice daily human dose greater than 34 mg is required to ensure adequate coverage across the mutations investigated. Benchmarking shows that compared with standard-of-care KIT inhibitors, AZD3229 has the potential to deliver the required target coverage across a wider spectrum of primary or secondary mutations. CONCLUSIONS: We demonstrate that AZD3229 warrants clinical investigation as a new treatment for patients with GIST based on its ability to inhibit both ATP-binding and A-loop mutations of KIT at clinically relevant exposures.


Assuntos
Tumores do Estroma Gastrointestinal/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Quinazolinas/farmacologia , Triazóis/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Tumores do Estroma Gastrointestinal/patologia , Humanos , Camundongos , Modelos Biológicos , Mutação , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Quinazolinas/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Triazóis/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Sci Transl Med ; 12(541)2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350132

RESUMO

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.


Assuntos
Antineoplásicos , Tumores do Estroma Gastrointestinal , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Humanos , Mutação , Naftiridinas , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Pirazóis , Pirróis , Ratos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Triazinas , Ureia/análogos & derivados , Fator A de Crescimento do Endotélio Vascular
11.
J Med Chem ; 62(21): 9918-9930, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31622099

RESUMO

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Terapia de Alvo Molecular , Fator 88 de Diferenciação Mieloide/genética , Quinazolinas/química , Quinazolinas/farmacologia , Administração Oral , Animais , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Quinases Associadas a Receptores de Interleucina-1/química , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Modelos Moleculares , Mutação , Conformação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/administração & dosagem , Quinazolinas/farmacocinética , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Curr Biol ; 15(19): 1762-7, 2005 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-16213824

RESUMO

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.


Assuntos
Apoptose/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Linhagem Celular , Sobrevivência Celular/fisiologia , Primers do DNA , Proteínas Quinases Associadas com Morte Celular , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Microscopia de Fluorescência , Mutação/genética , Interferência de RNA
13.
J Med Chem ; 61(19): 8797-8810, 2018 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-30204441

RESUMO

While the treatment of gastrointestinal stromal tumors (GISTs) has been revolutionized by the application of targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation, diverse mutations to this kinase drive resistance to established therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that can be dosed without being limited by the tolerability issues seen with multitargeted agents. This effort focused on identification and optimization of an existing kinase scaffold through the use of structure-based design. Starting from a series of previously reported phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimized, with a particular focus on reducing activity against a KDR driven cell model in order to limit the potential for hypertension commonly seen in second and third line GIST therapies. AZD3229 demonstrates potent single digit nM growth inhibition across a broad cell panel, with good margin to KDR-driven effects. Selectivity over KDR can be rationalized predominantly by the interaction of water molecules with the protein and ligand in the active site, and its kinome selectivity is similar to the best of the approved GIST agents. This compound demonstrates excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.


Assuntos
Descoberta de Drogas , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Proteínas Mutantes/antagonistas & inibidores , Mutação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Quinazolinas/química , Quinazolinas/farmacologia , Triazóis/química , Triazóis/farmacologia , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Humanos , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Quinazolinas/farmacocinética , Distribuição Tecidual , Triazóis/farmacocinética , Células Tumorais Cultivadas
14.
J Med Chem ; 60(24): 10071-10091, 2017 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-29172502

RESUMO

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.


Assuntos
Antineoplásicos/farmacologia , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cristalografia por Raios X , Cães , Feminino , Humanos , Quinases Associadas a Receptores de Interleucina-1/química , Linfoma Difuso de Grandes Células B/genética , Espectroscopia de Ressonância Magnética , Masculino , Camundongos SCID , Mutação , Fator 88 de Diferenciação Mieloide/genética , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/química , Pirróis/química , Ratos Wistar , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Clin Cancer Res ; 22(22): 5527-5538, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27780853

RESUMO

PURPOSE: Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. EXPERIMENTAL DESIGN: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. RESULTS: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. CONCLUSIONS: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Compostos Organofosforados/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Crizotinibe , Células Hep G2 , Humanos , Neoplasias Pulmonares/metabolismo , Mutação/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Sulfonas/farmacologia , Células U937
16.
J Med Chem ; 59(10): 4948-64, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27144831

RESUMO

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Neoplasias Pulmonares/tratamento farmacológico , Compostos Organofosforados/farmacologia , Fosfinas/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Administração Oral , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Conformação Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/química , Fosfinas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade
17.
Nat Commun ; 6: 8163, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26348592

RESUMO

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.


Assuntos
Proteínas Arqueais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Sulfolobus acidocaldarius/genética , Ubiquitinas/genética , Proteínas Arqueais/metabolismo , Cromatografia em Gel , Cromatografia Líquida , Dicroísmo Circular , Cristalografia por Raios X , Espectrometria de Massas , Microscopia Eletrônica , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Proteólise , Sulfolobus acidocaldarius/metabolismo , Ubiquitinas/metabolismo
18.
Clin Cancer Res ; 20(22): 5745-5755, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25239608

RESUMO

PURPOSE: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST. EXPERIMENTAL DESIGN: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents. RESULTS: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST. CONCLUSION: Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/genética , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Piridazinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Éxons , Feminino , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Mesilato de Imatinib , Imidazóis/química , Imidazóis/uso terapêutico , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Mutação , Recidiva Local de Neoplasia , Piperazinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/química , Piridazinas/química , Piridazinas/uso terapêutico , Pirimidinas/farmacologia , Pirróis/farmacologia , Sunitinibe , Tomografia Computadorizada por Raios X , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
FEBS Lett ; 586(19): 3471-6, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22967899

RESUMO

N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Tosilfenilalanil Clorometil Cetona/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sequência Conservada , Cisteína/química , Primers do DNA/genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Homologia de Sequência de Aminoácidos
20.
Chem Biol Drug Des ; 78(6): 999-1005, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22034911

RESUMO

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Crizotinibe , Humanos , Neoplasias Pulmonares , Mutação , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo
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