RESUMO
Antitumor immunity can be hampered by immunosuppressive mechanisms in the tumor microenvironment, including recruitment of arginase (ARG) expressing myeloid cells that deplete l-arginine essential for optimal T-cell and natural killer cell function. Hence, ARG inhibition can reverse immunosuppression enhancing antitumor immunity. We describe AZD0011, a novel peptidic boronic acid prodrug to deliver an orally available, highly potent, ARG inhibitor payload (AZD0011-PL). We demonstrate that AZD0011-PL is unable to permeate cells, suggesting that this compound will only inhibit extracellular ARG. In vivo, AZD0011 monotherapy leads to arginine increases, immune cell activation, and tumor growth inhibition in various syngeneic models. Antitumor responses increase when AZD0011 is combined with anti-PD-L1 treatment, correlating with increases in multiple tumor immune cell populations. We demonstrate a novel triple combination of AZD0011, anti-PD-L1, and anti-NKG2A, and combination benefits with type I IFN inducers, including polyI:C and radiotherapy. Our preclinical data demonstrate AZD0011's ability to reverse tumor immunosuppression and enhance immune stimulation and antitumor responses with diverse combination partners providing potential strategies to increase immuno-oncology therapies clinically.
Assuntos
Arginase , Linfócitos T , Humanos , Linhagem Celular Tumoral , Terapia de Imunossupressão , Tolerância Imunológica , Microambiente TumoralRESUMO
Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Descoberta de Drogas , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinase 9 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Background: Adenosine receptor type 2 (A2AR) inhibitor, AZD4635, has been shown to reduce immunosuppressive adenosine effects within the tumor microenvironment (TME) and to enhance the efficacy of checkpoint inhibitors across various syngeneic models. This study aims at investigating anti-tumor activity of AZD4635 alone and in combination with an anti-PD-L1-specific antibody (anti-PD-L1 mAb) across various TME conditions and at identifying, via mathematical quantitative modeling, a therapeutic combination strategy to further improve treatment efficacy. Methods: The model is represented by a set of ordinary differential equations capturing: 1) antigen-dependent T cell migration into the tumor, with subsequent proliferation and differentiation into effector T cells (Teff), leading to tumor cell lysis; 2) downregulation of processes mediated by A2AR or PD-L1, as well as other immunosuppressive mechanisms; 3) A2AR and PD-L1 inhibition by, respectively, AZD4635 and anti-PD-L1 mAb. Tumor size dynamics data from CT26, MC38, and MCA205 syngeneic mice treated with vehicle, anti-PD-L1 mAb, AZD4635, or their combination were used to inform model parameters. Between-animal and between-study variabilities (BAV, BSV) in treatment efficacy were quantified using a non-linear mixed-effects methodology. Results: The model reproduced individual and cohort trends in tumor size dynamics for all considered treatment regimens and experiments. BSV and BAV were explained by variability in T cell-to-immunosuppressive cell (ISC) ratio; BSV was additionally driven by differences in intratumoral adenosine content across the syngeneic models. Model sensitivity analysis and model-based preclinical study simulations revealed therapeutic options enabling a potential increase in AZD4635-driven efficacy; e.g., adoptive cell transfer or treatments affecting adenosine-independent immunosuppressive pathways. Conclusions: The proposed integrative modeling framework quantitatively characterized the mechanistic activity of AZD4635 and its potential added efficacy in therapy combinations, across various immune conditions prevailing in the TME. Such a model may enable further investigations, via simulations, of mechanisms of tumor resistance to treatment and of AZD4635 combination optimization strategies.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Antineoplásicos/farmacologia , Modelos Biológicos , Receptor A2A de Adenosina/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Algoritmos , Animais , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linhagem Celular Tumoral , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Isoenxertos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic-resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Compostos Macrocíclicos/farmacologia , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/fisiologia , Cicloeximida/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Compostos Macrocíclicos/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Antígenos de Histocompatibilidade Menor/biossíntese , Antígenos de Histocompatibilidade Menor/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/antagonistas & inibidores , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Piridinas/farmacologia , Sulfonamidas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Piridinas/química , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/patologia , Humanos , Camundongos , Simulação de Acoplamento Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Ratos , Solubilidade , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.
Assuntos
Antígenos CD/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Células Dendríticas/imunologia , Cadeias alfa de Integrinas/metabolismo , Neoplasias/imunologia , Receptor A2A de Adenosina/metabolismo , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Transdução de SinaisRESUMO
Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.
Assuntos
Indazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinase Syk/antagonistas & inibidores , Animais , Sítios de Ligação , Células CACO-2 , Cristalografia por Raios X , Canal de Potássio ERG1/antagonistas & inibidores , Humanos , Indazóis/síntese química , Indazóis/metabolismo , Indazóis/farmacocinética , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Ratos Wistar , Relação Estrutura-Atividade , Quinase Syk/química , Quinase Syk/metabolismoRESUMO
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 VascularRESUMO
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 XenoenxertoRESUMO
PURPOSE: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. EXPERIMENTAL DESIGN: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line- and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/effect relationship. RESULTS: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose- and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. CONCLUSIONS: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1-dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637).See related commentary by Alcon et al., p. 761.
Assuntos
Antineoplásicos , Neoplasias Hematológicas , Apoptose/efeitos dos fármacos , Quinase 9 Dependente de Ciclina , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , ProteômicaRESUMO
Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.
Assuntos
Antineoplásicos/química , Descoberta de Drogas/métodos , Mutação/genética , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Antineoplásicos/farmacologia , Desenho de Fármacos , Descoberta de Drogas/tendências , Humanos , Simulação de Acoplamento Molecular/métodos , Simulação de Acoplamento Molecular/tendências , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
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 CultivadasRESUMO
Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.
Assuntos
2,2'-Dipiridil/análogos & derivados , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Quinases raf/antagonistas & inibidores , Proteínas ras/genética , 2,2'-Dipiridil/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos Nus , Mutação , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/metabolismoRESUMO
RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.
Assuntos
2,2'-Dipiridil/análogos & derivados , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Quinases raf/antagonistas & inibidores , Proteínas ras/genética , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzamidas/química , Cristalografia por Raios X , Cães , Desenho de Fármacos , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Concentração Inibidora 50 , Camundongos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.
Assuntos
Inibidores Enzimáticos/farmacologia , Zíper de Leucina , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Cristalografia por Raios X , Inibidores Enzimáticos/químicaRESUMO
MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.
Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/normas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
PURPOSE: Oncogenic mutations in the KRAS/PI3K/AKT pathway are one of the most frequent alterations in cancer. Although PI3K or AKT inhibitors show promising results in clinical trials, drug resistance frequently emerges. We previously revealed Wnt/ß-catenin signaling hyperactivation as responsible for such resistance in colorectal cancer. Here we investigate Wnt-mediated resistance in patients treated with PI3K or AKT inhibitors in clinical trials and evaluate the efficacy of a new Wnt/tankyrase inhibitor, NVP-TNKS656, to overcome such resistance. EXPERIMENTAL DESIGN: Colorectal cancer patient-derived sphere cultures and mouse tumor xenografts were treated with NVP-TNKS656, in combination with PI3K or AKT inhibitors.We analyzed progression-free survival of patients treated with different PI3K/AKT/mTOR inhibitors in correlation with Wnt/ß-catenin pathway activation, oncogenic mutations, clinicopathological traits, and gene expression patterns in 40 colorectal cancer baseline tumors. RESULTS: Combination with NVP-TNKS656 promoted apoptosis in PI3K or AKT inhibitor-resistant cells with high nuclear ß-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear ß-catenin content and progressed earlier upon PI3K/AKT/mTOR inhibition. Nuclear ß-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not. CONCLUSIONS: High nuclear ß-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear ß-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/ß-catenin inhibition and together with ß-catenin may be predictive biomarkers of drug response providing a rationale to stratify colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/ß-catenin inhibitors.
Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Tanquirases/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
A key goal of cancer therapeutics is to selectively target the genetic lesions that initiate and maintain cancer cell proliferation and survival. While most cancers harbor multiple oncogenic mutations, a wealth of preclinical and clinical data supports that many cancers are sensitive to inhibition of single oncogenes, a concept referred to as 'oncogene addiction'. Herein, we describe the clinical evidence supporting oncogene addiction and discuss common mechanistic themes emerging from the response and acquired resistance to oncogene-targeted therapies. Finally, we suggest several opportunities toward exploiting oncogene addiction to achieve curative cancer therapies.
Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/genética , Oncogenes/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Genes Essenciais/genética , Humanos , Modelos Biológicos , Oncogenes/genéticaRESUMO
Tankyrases (TNKS) play roles in Wnt signaling, telomere homeostasis, and mitosis, offering attractive targets for anticancer treatment. Using unbiased combination screening in a large panel of cancer cell lines, we have identified a strong synergy between TNKS and MEK inhibitors (MEKi) in KRAS-mutant cancer cells. Our study uncovers a novel function of TNKS in the relief of a feedback loop induced by MEK inhibition on FGFR2 signaling pathway. Moreover, dual inhibition of TNKS and MEK leads to more robust apoptosis and antitumor activity both in vitro and in vivo than effects observed by previously reported MEKi combinations. Altogether, our results show how a novel combination of TNKS and MEK inhibitors can be highly effective in targeting KRAS-mutant cancers by suppressing a newly discovered resistance mechanism.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Tanquirases/metabolismo , Proteínas ras/genética , Acetamidas/administração & dosagem , Aminopiridinas/administração & dosagem , Compostos de Anilina/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Cloridrato de Erlotinib , Retroalimentação Fisiológica , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Nus , Morfolinas/administração & dosagem , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras) , Pirimidinonas/administração & dosagem , Quinazolinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais , Sulfonamidas/administração & dosagem , Tanquirases/antagonistas & inibidores , Tiazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tankyrases 1 and 2 are members of the poly(ADP-ribose) polymerase (PARP) family of enzymes that modulate Wnt pathway signaling. While amide- and lactam-based nicotinamide mimetics that inhibit tankyrase activity, such as XAV939, are well-known, herein we report the discovery and evaluation of a novel nicotinamide isostere that demonstrates selectivity over other PARP family members. We demonstrate the utilization of lipophilic efficiency-based structure-efficiency relationships (SER) to rapidly drive the evaluation of this series. These efforts led to a series of selective, cell-active compounds with solubility, physicochemical, and in vitro properties suitable for further optimization.