RESUMO
Despite the clinical efficacy of epidermal growth factor receptor (EGFR) inhibitors, a subset of patients with non-small cell lung cancer displays insertion mutations in exon20 in EGFR and Her2 with limited treatment options. Here, we present the development and characterization of the novel covalent inhibitors LDC8201 and LDC0496 based on a 1H-pyrrolo[2,3-b]pyridine scaffold. They exhibited intense inhibitory potency toward EGFR and Her2 exon20 insertion mutations as well as selectivity over wild type EGFR and within the kinome. Complex crystal structures with the inhibitors and biochemical and cellular on-target activity document their favorable binding characteristics. Ultimately, we observed tumor shrinkage in mice engrafted with patient-derived EGFR-H773_V774insNPH mutant cells during treatment with LDC8201. Together, these results highlight the potential of covalent pyrrolopyridines as inhibitors to target exon20 insertion mutations.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutagênese Insercional , Mutação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
While selective inhibition is one of the key assets for a small molecule drug, many diseases can only be tackled by simultaneous inhibition of several proteins. An example where achieving selectivity is especially challenging are ligands targeting human kinases. This difficulty arises from the high structural conservation of the kinase ATP binding sites, the area targeted by most inhibitors. We investigated the possibility to identify novel small molecule ligands with pre-defined binding profiles for a series of kinase targets and anti-targets by in silico docking. The candidate ligands originating from these calculations were assayed to determine their experimental binding profiles. Compared to previous studies, the acquired hit rates were low in this specific setup, which aimed at not only selecting multi-target kinase ligands, but also designing out binding to anti-targets. Specifically, only a single profiled substance could be verified as a sub-micromolar, dual-specific EGFR/ErbB2 ligand that indeed avoided its selected anti-target BRAF. We subsequently re-analyzed our target choice and in silico strategy based on these findings, with a particular emphasis on the hit rates that can be expected from a given target combination. To that end, we supplemented the structure-based docking calculations with bioinformatic considerations of binding pocket sequence and structure similarity as well as ligand-centric comparisons of kinases. Taken together, our results provide a multi-faceted picture of how pocket space can determine the success of docking in multi-target drug discovery efforts.
Assuntos
Simulação de Acoplamento Molecular/métodos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Simulação por Computador , Descoberta de Drogas , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Relação Estrutura-AtividadeRESUMO
Mutated or amplified Her2 serves as a driver of non-small cell lung cancer or mediates resistance toward the inhibition of its family member epidermal growth factor receptor with small-molecule inhibitors. To date, small-molecule inhibitors targeting Her2 which can be used in clinical routine are lacking, and therefore, the development of novel inhibitors was undertaken. In this study, the well-established pyrrolopyrimidine scaffold was modified with structural motifs identified from a screening campaign with more than 1600 compounds, which were applied against wild-type Her2 and its mutant variant Her2-A775_G776insYVMA. The resulting inhibitors were designed to covalently target a reactive cysteine in the binding site of Her2 and were further optimized by means of structure-based drug design utilizing a set of obtained complex crystal structures. In addition, the analysis of binding kinetics and absorption, distribution, metabolism, and excretion parameters as well as mass spectrometry experiments and western blot analysis substantiated our approach.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Receptor ErbB-2/genética , Receptor ErbB-2/isolamento & purificação , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Precision medicine has revolutionized the treatment of patients in EGFR driven non-small cell lung cancer (NSCLC). Targeted drugs show high response rates in genetically defined subsets of cancer patients and markedly increase their progression-free survival as compared to conventional chemotherapy. However, recurrent acquired drug resistance limits the success of targeted drugs in long-term treatment and requires the constant development of novel efficient inhibitors of drug resistant cancer subtypes. Herein, we present covalent inhibitors of the drug resistant gatekeeper mutant EGFR-L858R/T790M based on the pyrrolopyrimidine scaffold. Biochemical and cellular characterization, as well as kinase selectivity profiling and western blot analysis, substantiate our approach. Moreover, the developed compounds possess high activity against multi drug resistant EGFR-L858R/T790M/C797S in biochemical assays due to their highly reversible binding character, that was revealed by characterization of the binding kinetics. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR which provide detailed insight into their binding mode.
RESUMO
The emergence of acquired resistance against targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. In a subgroup of these patients we identified an association between selection of EGFRT790M-negative but EGFRG724S-positive subclones and osimertinib resistance. We demonstrate that EGFRG724S limits the activity of third-generation EGFR inhibitors both in vitro and in vivo. Structural analyses and computational modeling indicate that EGFRG724S mutations may induce a conformation of the glycine-rich loop, which is incompatible with the binding of third-generation TKIs. Systematic inhibitor screening and in-depth kinetic profiling validate these findings and show that second-generation EGFR inhibitors retain kinase affinity and overcome EGFRG724S-mediated resistance. In the case of afatinib this profile translates into a robust reduction of colony formation and tumor growth of EGFRG724S-driven cells. Our data provide a mechanistic basis for the osimertinib-induced selection of EGFRG724S-mutant clones and a rationale to treat these patients with clinically approved second-generation EGFR inhibitors.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas , Compostos de Anilina , Animais , Linhagem Celular Tumoral , Progressão da Doença , Receptores ErbB/química , Receptores ErbB/metabolismo , Feminino , Humanos , Cinética , Camundongos , Camundongos Nus , Mutação/genética , Células NIH 3T3 , Piperazinas/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Inibidores de Proteínas Quinases/químicaRESUMO
The identification of compounds for dissecting biological functions and the development of novel drug molecules are central tasks that often require screening campaigns. However, the required architecture is cost- and time-intensive. Herein we describe the devices and technologies that comprise a Robotics-Assisted Screening Platform for Efficient Ligand Discovery (RASPELD), which we set up in an academic laboratory. RASPELD provides semi-automated high-end screening, and it can be maintained by graduate students. We demonstrate its successful application in biochemical and cellular screens for the identification and validation of bioactive chemical entities as candidate cancer-relevant inhibitors. Specifically, we examined the interaction between a transcription factor, Nrf2, and its key regulator, Keap1. We also examined drug-resistant mutants of the epidermal growth factor receptor (EGFR). Screening campaigns with more than 30 000 compounds were performed in a reasonable period of time. We identified the molecule RSL6586 as a starting point for hit optimization, which is currently ongoing.
Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Robótica/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Bioensaio , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/instrumentação , Educação de Pós-Graduação , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Ligantes , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Robótica/instrumentaçãoRESUMO
The treatment of non-small cell lung cancer (NSCLC) is currently experiencing a revolution. Over the last decade, the knowledge gained about the biochemical features of biomarkers and their predictive abilities has led to the development of targeted small-molecule inhibitors that present an alternative to harsh chemotherapy. The use of these new therapies has improved the quality of life and increased the survival of patients. The occurrence of inevitable drug resistance requires the constant development of precision medicine. The detailed understanding of the target biology and the search for innovative chemical approaches has encouraged investigations in this field. Herein, we review selected aspects of the molecular targets and present an overview of current topics and challenges in the rational development of small molecules to target NSCLC.
Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Quinase do Linfoma Anaplásico/metabolismo , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Estrutura Molecular , Medicina de Precisão , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
In modern cancer therapy, the use of small organic molecules against receptor tyrosine kinases (RTKs) has been shown to be a valuable strategy. The association of cancer cells with dysregulated signaling pathways linked to RTKs represents a key element in targeted cancer therapies. The tyrosine kinase mast/stem cell growth factor receptor KIT is an example of a clinically relevant RTK. KIT is targeted for cancer therapy in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). However, acquired resistance mutations within the catalytic domain decrease the efficacy of this strategy and are the most common cause of failed therapy. Here, we present the structure-based design and synthesis of novel type II kinase inhibitors to overcome these mutations in KIT. Biochemical and cellular studies revealed promising molecules for the inhibition of mutated KIT.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mutação , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Mutação Puntual , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/química , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologiaRESUMO
Inhibition of the epidermal growth factor receptor represents one of the most promising strategies in the treatment of lung cancer. Acquired resistance compromises the clinical efficacy of EGFR inhibitors during long-term treatment. The recently discovered EGFR-C797S mutation causes resistance against third-generation EGFR inhibitors. Here we present a rational approach based on extending the inhibition profile of a p38 MAP kinase inhibitor toward mutant EGFR inhibition. We used a privileged scaffold with proven cellular potency as well as in vivo efficacy and low toxicity. Guided by molecular modeling, we synthesized and studied the structure-activity relationship of 40 compounds against clinically relevant EGFR mutants. We successfully improved the cellular EGFR inhibition down to the low nanomolar range with covalently binding inhibitors against a gefitinib resistant T790M mutant cell line. We identified additional noncovalent interactions, which allowed us to develop metabolically stable inhibitors with high activities against the osimertinib resistant L858R/T790M/C797S mutant.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Imidazóis/química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Simulação de Acoplamento Molecular , Mutação Puntual , Quinazolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. We provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors, such as AD80 or ponatinib, that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells, we identify the CCDC6-RETI788N mutation and drug-induced mitogen-activated protein kinase pathway reactivation as possible mechanisms by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors.
Assuntos
Adenocarcinoma/metabolismo , Rearranjo Gênico/genética , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/genética , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Imidazóis/farmacologia , Camundongos , Mutação , Células NIH 3T3 , Piridazinas/farmacologiaRESUMO
The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Humanos , Indazóis , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Simulação de Acoplamento Molecular , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Targeting acquired drug resistance represents the major challenge in the treatment of EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we describe the structure-based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR-mutant drug-resistant cells. Protein X-ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR-T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR-C797S.
Assuntos
Receptores ErbB/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Cinética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Fosforilação , Mutação Puntual , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologiaRESUMO
The receptor tyrosine kinase EGFR is regulated by complex conformational changes, and this conformational control is disturbed in certain types of cancer. Many ligands are known to bind EGFR in its active conformation, thereby preventing ATP from binding. Only a few ligands are known to stabilize EGFR in its inactive conformation, thus providing novel strategies for perturbing EGFR activity. We report a direct binding assay that enables the identification of novel ligands that bind to and stabilize the inactive conformation of EGFR.
Assuntos
Receptores ErbB/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Sítios de Ligação , Receptores ErbB/química , Receptores ErbB/genética , Cloridrato de Erlotinib/química , Cloridrato de Erlotinib/metabolismo , Lapatinib , Ligantes , Mutagênese Sítio-Dirigida , Ligação Proteica , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Quinazolinas/química , Quinazolinas/metabolismo , Espectrometria de FluorescênciaRESUMO
Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.