RESUMO
Cyclin-dependent kinase 9 (CDK9) coordinates signaling events that regulate RNA polymerase II (Pol II) pause-release states. It is an important co-factor for transcription factors, such as MYC, that drive aberrant cell proliferation when their expression is deregulated. CDK9 modulation offers an approach for attenuating dysregulation in such transcriptional programs. As a result, numerous drug development campaigns to inhibit CDK9 kinase activity have been pursued. More recently, targeted degradation has emerged as an attractive approach. However, comprehensive evaluation of degradation versus inhibition is still critically needed to assess the biological contexts in which degradation might offer superior therapeutic benefits. We validated that CDK9 inhibition triggers a compensatory mechanism that dampens its effect on MYC expression and found that this feedback mechanism was absent when the kinase is degraded. Importantly, CDK9 degradation is more effective than its inhibition for disrupting MYC transcriptional regulatory circuitry likely through the abrogation of both enzymatic and scaffolding functions of CDK9. Highlights: - KI-CDK9d-32 is a highly potent and selective CDK9 degrader. - KI-CDK9d-32 leads to rapid downregulation of MYC protein and mRNA transcripts levels. - KI-CDK9d-32 represses canonical MYC pathways and leads to a destabilization of nucleolar homeostasis. - Multidrug resistance ABCB1 gene emerged as the strongest resistance marker for the CDK9 PROTAC degrader.
RESUMO
Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptores Androgênicos/genética , Transcrição Gênica/efeitos dos fármacos , Antagonistas de Receptores de Andrógenos/uso terapêutico , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Neoplasias de Próstata Resistentes à Castração/genética , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Cancer genomes contain large numbers of somatic mutations but few of these mutations drive tumor development. Current approaches either identify driver genes on the basis of mutational recurrence or approximate the functional consequences of nonsynonymous mutations by using bioinformatic scores. Passenger mutations are enriched in characteristic nucleotide contexts, whereas driver mutations occur in functional positions, which are not necessarily surrounded by a particular nucleotide context. We observed that mutations in contexts that deviate from the characteristic contexts around passenger mutations provide a signal in favor of driver genes. We therefore developed a method that combines this feature with the signals traditionally used for driver-gene identification. We applied our method to whole-exome sequencing data from 11,873 tumor-normal pairs and identified 460 driver genes that clustered into 21 cancer-related pathways. Our study provides a resource of driver genes across 28 tumor types with additional driver genes identified according to mutations in unusual nucleotide contexts.
Assuntos
Biologia Computacional/métodos , Mutação , Neoplasias/genética , Nucleotídeos/genética , Proteínas/genética , Análise por Conglomerados , Humanos , Proteínas/química , Sequenciamento do Exoma/métodosRESUMO
Many promising therapeutic protein targets were previously considered "undruggable" due to a deficit in structural information to guide drug design and/or a lack of an obvious binding pocket. Fortunately, array-based methods for evaluating protein binding against large chemical libraries, such as small-molecule microarray screening, have provided one of several emerging inroads to ligand discovery for these elusive targets. Despite the advance in the area of ligand discovery for poorly structured and intrinsically disordered proteins provided by array-based technologies involving cell lysates, the extension of this technology for screening proteins with short half-lives in physiologically relevant conformations has been technically challenging. In this chapter we present a protocol for leveraging in vitro translation strategies to enable array-based screening of short-lived proteins against large small-molecule libraries for ligand discovery.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Análise Serial de Proteínas/métodos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Ligantes , Ligação ProteicaRESUMO
PRMT5 catalyzes the mono- and symmetric dimethylation of the arginine N-guanidine group of a wide variety of target proteins including histones, transcriptional elongation factors, kinases and tumor suppressors by utilizing the essential co-factor S-adenosylmethionine as methyl source. PRMT5 overexpression has been linked to the progression of various diseases, including cancer, and is oftentimes associated with a poor prognosis. Therefore, PRMT5 is promoted as a valuable target for drug discovery approaches and was a subject matter in recent endeavors aiming for the development of specific PRMT5 inhibitors. This review will embrace the significance of PRMT5 as therapeutic target with respect to its molecular interdependencies in disease states as well as its implication in drug development approaches.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Antineoplásicos/química , Inibidores Enzimáticos/química , Humanos , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.
Assuntos
Terapia de Alvo Molecular , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/química , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
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
Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.
Assuntos
Autofagia/efeitos dos fármacos , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Autofagossomos/efeitos dos fármacos , Descoberta de Drogas , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirimidinas/química , Sirolimo/farmacologia , Relação Estrutura-AtividadeRESUMO
Histone acetyltransferases (HATs) are epigenetic drivers that catalyze the acetyl transfer from acetyl-CoA to lysines of both histone and non-histone substrates and thereby induce transcription either by chromatin remodeling or direct transcription factor activation. Histone deacetylases (HDACs) conduct the reverse reaction to counter HAT activity. Physiological processes such as cell cycle progression or apoptosis require a thoroughly balanced equilibrium of the interplay between acetylation and deacetylation processes to maintain or, if required, alter the global acetylome status. Aberrant HAT activity has recently been demonstrated to play a crucial role in the progression of various diseases such as prostate, lung, and colon cancers as well as glioblastomas and neurodegenerative diseases. Recent investigations have aimed for the identification of HAT modulators to further decipher the complexity of acetyl transferase related signaling cascades and discover potential leads for drug design approaches. HDACs have been extensively characterized and targeted by small molecules, including four FDA-approved HDAC inhibitors; in contrast, HATs have not been active targets for therapeutic development. This review will summarize the status of HAT associated diseases and the arsenal of currently known and available HAT inhibitors with respect to their discovery, further improvements, and current applications.
RESUMO
The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Membrana/antagonistas & inibidores , Miristatos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Membrana/genética , Modelos Moleculares , Estrutura Molecular , Mutação , Miristatos/síntese química , Miristatos/química , Proteínas de Neoplasias/genética , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antineoplásicos/química , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Bases de Dados de Compostos Químicos , Desenho de Fármacos , Receptores ErbB/genética , Humanos , Cinética , Neoplasias Pulmonares , Modelos Moleculares , Conformação Molecular , Mutação , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Bibliotecas de Moléculas Pequenas , Solubilidade , Relação Estrutura-Atividade , Quinases da Família src/química , Quinases da Família src/genéticaRESUMO
KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Pontos de Checagem do Ciclo Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA , Modelos Animais de Doenças , Xenoenxertos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Células Tumorais CultivadasRESUMO
Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Ligação Competitiva , Humanos , Modelos MolecularesRESUMO
The clinical success of covalent kinase inhibitors in the treatment of EGFR-dependent non-small cell lung cancer (NSCLC) has rejuvenated the appreciation of reactive small molecules. Acquired drug resistance against first-line EGFR inhibitors remains the major bottleneck in NSCLC and is currently addressed by the application of fine-tuned covalent drugs. Here we report the design, synthesis and biochemical evaluation of a novel class of EGFR inhibitors with a covalent yet reversible warhead. A series of WZ4002 analogs, derived from anilinopyrimidine and 3-substituted-2-cyanoacrylamide scaffolds, exhibit strong and selective inhibitory activity against clinically relevant EGFR(L858R) and EGFR(L858R/T790M).
Assuntos
Acrilamidas/química , Acrilamidas/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 , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Modelos Moleculares , Mutação , Mutação PuntualRESUMO
Progressive loss and impaired restoration of neuronal activity are hallmarks of neurological diseases, and new small molecules with neurotrophic activity are in high demand. The militarinone alkaloids and structurally simplified analogues with 4-hydroxy-2-pyridone core structure induce pronounced neurite outgrowth, but their protein target has not been identified. Reported herein is the synthesis of a militarinone-inspired 4-hydroxy-2-pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The most potent 4-hydroxy-2-pyridone is a selective ATP-competitive inhibitor of MAP4K4 but not of the other stress pathway related kinases, as proven by biochemical analysis and by a crystal structure of the inhibitor in complex with MAP4K4. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases.
Assuntos
Alcaloides/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neurogênese/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridonas/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Conformação Molecular , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Piridonas/síntese química , Piridonas/química , Relação Estrutura-AtividadeRESUMO
The cytosolic Ser/Thr kinase TBK1 was discovered to be an essential element in the mediation of signals that lead to tumor migration and progression. These findings meet the need for the identification of novel tool compounds and potential therapeutics to gain deeper insights into TBK1 related signaling and its relevance in tumor progression. Herein, we undertake the activity-based screening for unique inhibitors of TBK1 and their subsequent optimization. Initial screening approaches identified a selection of TBK1 inhibitors that were optimized using methods of medicinal chemistry. Variations of the structural characteristics of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or basic contributions to drug design approaches are anticipated for our improved small molecules.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells.
Assuntos
Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Animais , Proteína Tirosina Quinase CSK , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Humanos , Masculino , Camundongos Nus , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Método de Monte Carlo , Neuroblastoma/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Pirazóis/química , Pirimidinas/química , Pirimidinas/metabolismo , Termodinâmica , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/química , Quinases da Família src/metabolismoRESUMO
Discoidin domain-containing receptors (DDRs) exhibit a unique mechanism of action among the receptor tyrosine kinases (RTKs) because their catalytic activity is induced by extracellular collagen binding. Moreover, they are essential components in the assimilation of extracellular signals. Recently, DDRs were reported to be significantly linked to tumor progression in breast cancer by facilitating the processes of invasion, migration, and metastasis. Here, we report the successful development of a fluorescence-based, direct binding assay for the detection of type II and III DFG-out binders for DDR2. Using sequence alignments and homology modeling, we designed a DDR2 construct appropriate for fluorescent labeling. Successful assay development was validated by sensitive detection of a reference DFG-out binder. Subsequent downscaling led to convenient application to high-throughput screening formats. Screening of a representative compound library identified high-affinity DDR2 ligands validated by orthogonal activity-based assays, and a subset of identified compounds was further investigated with respect to DDR1 inhibition.