RESUMO
Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação , Proteínas Proto-Oncogênicas A-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas A-raf/genética , Quinases raf/antagonistas & inibidores , Animais , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Melanoma/patologia , Camundongos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas A-raf/química , Quinases raf/químicaRESUMO
The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series. In short, we found that gemcitabine, as its diphosphate metabolite, represents one of the few described active site inhibitors of RRM1. We herein describe the identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors through in-cell phenotypic screening.
Assuntos
Desoxicitidina/análogos & derivados , Proteínas Supressoras de Tumor/antagonistas & inibidores , Linhagem Celular Tumoral , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ribonucleosídeo Difosfato Redutase , Relação Estrutura-Atividade , GencitabinaRESUMO
The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders. We present previously unreported degradation activity of published papain-like protease inhibitors. Follow-up exploration of structure-activity relationships and mechanism-of-action studies points to the recruitment of the ubiquitin-proteasome machinery that is solely driven by site occupancy, regardless of molecular features of the ligand. Supported by HDX data, we hypothesize that binding-induced structural changes in NSP3 trigger the recruitment of an E3 ligase and lead to proteasomal degradation.
Assuntos
COVID-19 , Proteases Semelhantes à Papaína de Coronavírus , Papaína , Humanos , Papaína/metabolismo , Proteínas não Estruturais Virais/metabolismo , SARS-CoV-2/química , Inibidores de Proteases/metabolismoRESUMO
Purpose: This study explores the potential of preclinical in vitro cell line response data and computational modeling in identifying optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. Results: In a drug combination screen of 43 melanoma cell lines, we identified unique dosage landscapes of panRAF and MEK inhibitors for NRAS vs BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma. Computational modeling and molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated in vivo translatability of in vitro dose-response maps by accurately predicting tumor growth in xenografts. Then, we analyzed pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. Conclusion: Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range.
RESUMO
PURPOSE: This study explores the potential of pre-clinical in vitro cell line response data and computational modeling in identifying the optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. RESULTS: In a drug combination screen of 43 melanoma cell lines, we identified specific dosage landscapes of panRAF and MEK inhibitors for NRAS vs. BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma (mean Bliss score of 0.27 in NRAS vs. 0.1 in BRAF mutants). Computational modeling and follow-up molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated the in vivo translatability of in vitro dose-response maps by predicting tumor growth in xenografts with high accuracy in capturing cytostatic and cytotoxic responses. We analyzed the pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. CONCLUSION: Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range. Overall, this work presents a framework to aid dose selection in drug combinations.
RESUMO
Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.
RESUMO
Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Análise de Célula Única/métodosRESUMO
KRAS, which is mutated in â¼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.
Assuntos
Carcinogênese/metabolismo , Dimerização , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas ras/genéticaRESUMO
Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.
Assuntos
Neoplasias/tratamento farmacológico , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinonas/uso terapêutico , Quinases raf/antagonistas & inibidores , Animais , Azetidinas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Cães , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos Nus , Estrutura Molecular , Mutação , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Piperidinas/uso terapêutico , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Quinazolinonas/química , Quinazolinonas/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/genética , Quinases raf/metabolismoRESUMO
Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.
RESUMO
Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.
Assuntos
Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/efeitos dos fármacos , Linhagem Celular Tumoral , Guanosina Trifosfato/metabolismo , Humanos , Mutação/efeitos dos fármacos , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas ras/efeitos dos fármacos , Proteínas ras/genéticaRESUMO
Human BRG1, a subunit of the Swi/Snf chromatin remodeling apparatus, has been implicated in regulation of cellular proliferation and is a candidate tumor suppressor. Reintroduction of BRG1 into a breast tumor cell line, ALAB, carrying a defined mutation in the BRG1 gene, induced growth arrest. Gene expression data revealed that the arrest may in part be accounted for by down-regulation of select E2F target genes such as cyclin E, but more dramatically, by up-regulation of mRNAs for the cyclin-dependent kinase inhibitors p21 and p15. Protein levels of both p15 and p21 were induced, and p21 protein was recruited to a complex with cyclin-dependent kinase, CDK2, to inhibit its activity. BRG1 can associate with the p21 promoter in a p53-independent manner, suggesting that the induction of p21 by BRG1 may be direct. Further, using microarray and real-time PCR analysis we identified several novel BRG1-regulated genes. Our work provides further evidence for a role for BRG1 in the regulation of several genes involved in key steps in tumorigenesis and has revealed a potential mechanism for BRG1-induced growth arrest.
Assuntos
Ciclo Celular/fisiologia , Genes Supressores de Tumor , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Adenoviridae , Proteínas E2 de Adenovirus/metabolismo , Quinases relacionadas a CDC2 e CDC28/metabolismo , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , DNA Helicases , Perfilação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Mutação , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição/genéticaRESUMO
PURPOSE: We determined the safety, pharmacokinetics, pharmacodynamics, and recommended phase II dose of MK-8776 (SCH 900776), a potent, selective checkpoint kinase 1 (Chk1) inhibitor, as monotherapy and in combination with gemcitabine in a first-in-human phase I clinical trial in patients with advanced solid tumor malignancies. PATIENTS AND METHODS: Forty-three patients were treated by intravenous infusion with MK-8776 at seven dose levels ranging from 10 to 150 mg/m(2) as monotherapy and then in combination with gemcitabine 800 mg/m(2) (part A, n = 26) or gemcitabine 1,000 mg/m(2) (part B, n = 17). Forty percent of patients had three or more prior treatment regimens, and one third of patients had previously received gemcitabine. RESULTS: As monotherapy, MK-8776 was well tolerated, with QTc prolongation (19%), nausea (16%), fatigue (14%), and constipation (14%) as the most frequent adverse effects. Combination therapy demonstrated a higher frequency of adverse effects, predominantly fatigue (63%), nausea (44%), decreased appetite (37%), thrombocytopenia (32%), and neutropenia (24%), as well as dose-related, transient QTc prolongation (17%). The median number of doses of MK-8776 administered was five doses, with relative dose-intensity of 0.96. Bioactivity was assessed by γ-H2AX ex vivo assay. Of 30 patients evaluable for response, two showed partial response, and 13 exhibited stable disease. CONCLUSION: MK-8776 was well tolerated as monotherapy and in combination with gemcitabine. Early evidence of clinical efficacy was observed. The recommended phase II dose is MK-8776 200 mg plus gemcitabine 1,000 mg/m(2) on days 1 and 8 of a 21-day cycle.
Assuntos
Desoxicitidina/análogos & derivados , Neoplasias/tratamento farmacológico , Proteínas Quinases/metabolismo , Pirazóis/administração & dosagem , Pirazóis/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Quinase 1 do Ponto de Checagem , Estudos de Coortes , Citarabina/administração & dosagem , Desoxicitidina/administração & dosagem , Feminino , Histonas/metabolismo , Humanos , Infusões Intravenosas , Células K562 , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Sarcoma/tratamento farmacológico , Fatores de Tempo , GencitabinaRESUMO
Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.
Assuntos
Replicação do DNA , Histonas/metabolismo , Proteínas Quinases/metabolismo , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Desoxirribonucleotídeos/metabolismo , Humanos , Fosforilação , RNA Interferente Pequeno/metabolismo , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleotídeos/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Óxidos N-Cíclicos , Quinases Ciclina-Dependentes/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Histonas/metabolismo , Humanos , Immunoblotting , Indolizinas , Camundongos , Camundongos Nus , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/administração & dosagem , Pirazóis/química , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/química , Interferência de RNA , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Inhibition of thymidine biosynthesis is a clinically-validated therapeutic approach for multiple cancers. Inhibition of thymidylate synthetase (TS) leads to a decrease in cellular TTP levels, replication stress and increased genomic incorporation of uridine (dUMP). Thus, inhibitors of this pathway (such as methotrexate) can drive a multitude of downstream cell cycle checkpoint and DNA repair processes. Genomic dUMP is recognized by the base excision repair (BER) pathway. Using a synthetic lethal siRNA-screening approach, we systematically screened for components of BER that, when ablated, enhanced methotrexate response in a high content γ-H2A.X bioassay. We observed specific ablation of the mixed function DNA glycosylase/lyase Neil1 phenotypically enhanced several inhibitors of thymidine biosynthesis, as well as cellular phenotypes downstream of gemcitabine, cytarabine and clofarabine exposure. These synthetic lethal interactions were associated with significantly enhanced accumulation of γ-H2A.X and improved growth inhibition. Significantly, following TS pathway inhibition, addition of exogenous dTTP complemented the primary Neil1 γ-H2A.X phenotypes. Similarly, co-depletion of Neil1 with Cdc45, Cdc6, Cdc7 or DNA polymerase ß (PolB) suppressed Neil1 phenotypes. Conversely, co-depletion of Neil1 with the Rad17, Rad9 ATR, ATM and DNA-PK checkpoint/sensor proteins appears primarily epistatic to Neil1. These data suggest Neil1 may be a critical mediator of BER of incorporated dUMP following TS pathway inhibition.
Assuntos
DNA Glicosilases/metabolismo , Timidilato Sintase/antagonistas & inibidores , Biomarcadores/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , DNA Glicosilases/genética , Reparo do DNA , Inibidores Enzimáticos/metabolismo , Antagonistas do Ácido Fólico/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Metotrexato/metabolismo , Inibidores da Síntese de Ácido Nucleico/metabolismo , Fenótipo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Quinazolinas/metabolismo , Tiofenos/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismoRESUMO
Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC(50) values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters. Compared with flavopiridol, SCH 727965 exhibits superior activity with an improved therapeutic index. In cell-based assays, SCH 727965 completely suppressed retinoblastoma phosphorylation, which correlated with apoptosis onset and total inhibition of bromodeoxyuridine incorporation in >100 tumor cell lines of diverse origin and background. Moreover, short exposures to SCH 727965 were sufficient for long-lasting cellular effects. SCH 727965 induced regression of established solid tumors in a range of mouse models following intermittent scheduling of doses below the maximally tolerated level. This was associated with modulation of pharmacodynamic biomarkers in skin punch biopsies and rapidly reversible, mechanism-based effects on hematologic parameters. These results suggest that SCH 727965 is a potent and selective CDK inhibitor and a novel cytotoxic agent.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Compostos de Piridínio/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular Tumoral , Óxidos N-Cíclicos , Relação Dose-Resposta a Droga , Flavonoides/química , Flavonoides/farmacologia , Humanos , Indolizinas , Fosforilação/efeitos dos fármacos , Piperidinas/efeitos adversos , Piperidinas/química , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/química , Compostos de Piridínio/efeitos adversos , Compostos de Piridínio/química , Proteína do Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.
RESUMO
Chk1 contributes to both intra-S and DNA damage checkpoint responses. Here, we show that depletion of DNA Polalpha and not Polepsilon or Poldelta by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Polalpha and Chk1 causes an accumulation of gamma-H2A.X, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Co-depletion of DNA Polalpha with ATR yields similar phenotypes, suggesting that ATR and Chk1 are epistatic and required for maintenance of genomic integrity following replication stress. Significantly, Chk1 and DNA Polalpha can be co-immunoprecipated from native cell extracts. Moreover, following replication stress, Polalpha-associated Chk1 becomes rapidly phosphorylated on Ser345 in a TopBP1 and ATR-dependent manner. Hence, the ability to efficiently phosphorylate Chk1 in the context of DNA Polalpha complexes is correlated with suppression of DNA damage following replication stress. These findings identify DNA Polalpha as an important component of the signal transduction cascade that activates the intra-S checkpoint.
Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , Isoenzimas/metabolismo , Proteínas Quinases/metabolismo , Antimetabólitos/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Dano ao DNA , DNA Polimerase I/genética , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Isoenzimas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
A series of four bicyclic cores were prepared and evaluated as cyclin-dependent kinase-2 (CDK2) inhibitors. From the in-vitro and cell-based analysis, the pyrazolo[1,5-a]pyrimidine core (represented by 9) emerged as the superior core for further elaboration in the identification of novel CDK2 inhibitors.