RESUMO
KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Desenho de Fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinazolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.
Assuntos
Benzimidazóis/uso terapêutico , Imidazóis/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirazinas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Benzimidazóis/farmacologia , Modelos Animais de Doenças , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Nus , Pirazinas/farmacologia , Pirimidinas/farmacologiaRESUMO
Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Quinazolinas/uso terapêutico , Quinolonas/uso terapêutico , Regulação Alostérica , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Células CACO-2 , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Masculino , Camundongos Nus , Conformação Molecular , Mutação , Piperazinas/síntese química , Piperazinas/farmacocinética , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinazolinas/síntese química , Quinazolinas/farmacocinética , Quinolonas/síntese química , Quinolonas/farmacocinética , Ratos Wistar , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors, where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC50 = 6 nM) as well as excellent physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging antitumor activity in preclinical models.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas , Imidazóis/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/uso terapêutico , Pirimidinas/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Apoptose , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Quimioterapia Combinada , Feminino , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Pirazinas/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral , Cães , Descoberta de Drogas , Humanos , Metilação , Inibidores de Proteínas Quinases/farmacocinéticaRESUMO
A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/enzimologia , Regulação Alostérica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Isocitrato Desidrogenase/isolamento & purificação , Isocitrato Desidrogenase/metabolismo , Leucemia Mieloide Aguda/patologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Cinamatos/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Indóis/farmacologia , Mutação/genética , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cinamatos/administração & dosagem , Avaliação Pré-Clínica de Medicamentos , Moduladores de Receptor Estrogênico/administração & dosagem , Receptor alfa de Estrogênio/química , Feminino , Humanos , Indóis/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Conformação Proteica , Ratos , Células Tumorais Cultivadas , Útero/metabolismo , Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.
Assuntos
Antineoplásicos/metabolismo , Cinamatos/química , Cinamatos/metabolismo , Antagonistas de Estrogênios/síntese química , Antagonistas de Estrogênios/farmacologia , Moduladores de Receptor Estrogênico/síntese química , Moduladores de Receptor Estrogênico/farmacologia , Indóis/química , Indóis/metabolismo , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Regulação para Baixo/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Injeções Intramusculares , Difração de Raios XRESUMO
The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.
Assuntos
Desenho de Fármacos , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/química , Inibidores de Proteínas Quinases/farmacologia , Sequência de Aminoácidos , Células Cultivadas , Cristalografia por Raios X , Humanos , Immunoblotting , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Here, we describe an approach to identify novel selective estrogen receptor downregulator (SERD) compounds with improved properties such as oral bioavailability and the potential of increased efficacy compared to currently marketed drug treatments. Previously, methodologies such as Western blotting and transient cell reporter assays have been used to identify and characterize SERD compounds, but such approaches can be limited due to low throughput and sensitivity, respectively. We have used an endogenous cell-imaging strategy that has both the throughput and sensitivity to support a large-scale hit-to-lead program to identify novel compounds. A screening cascade with a suite of assays has been developed to characterize compounds that modulate estrogen receptor α (ERα)-mediated signaling or downregulate ERα levels in cells. Initially, from a focused high-throughput screening, novel ERα binders were identified that could be modified chemically into ERα downregulators. Following this, cellular assays helped determine the mechanism of action of compounds to distinguish between on-target and off-target compounds and differentiate SERDs, selective estrogen receptor modulator (SERM) compounds, and agonist ERα ligands. Data are shown to exemplify the characterization of ERα-mediated signaling inhibitors using a selection of literature compounds and illustrate how this cascade has been used to drive the chemical design of novel SERD compounds.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Regulação para Baixo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica , Receptores de Progesterona/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5' overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z' factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.