RESUMO
The colony-stimulating factor 1 receptor (CSF1R) is an attractive target for inflammation disorders and cancers. Based on a series of pyrrolo[2,3-d]pyrimidine containing two carbo-aromatic rings, we have searched for new CSF1R inhibitors having a higher fraction of sp3-atoms. The phenyl unit in the 4-amino group could efficiently be replaced by tetrahydropyran (THP) retaining inhibitor potency. Exchanging the 6-aryl group with cyclohex-2-ene units also resulted in highly potent compounds, while fully saturated ring systems at C-6 led to a loss of activity. The structure-activity relationship study evaluating THP containing pyrrolo[2,3-d]pyrimidine derivates identified several highly active inhibitors by enzymatic studies. A comparison of 11 pairs of THP and aromatic compounds showed that inhibitors containing THP had clear benefits in terms of enzymatic potency, solubility, and cell toxicity. Guided by cellular experiments in Ba/F3 cells, five CSF1R inhibitors were further profiled in ADME assays, indicating the para-aniline derivative 16t as the most attractive compound for further development.
Assuntos
Pirimidinas , Receptores Proteína Tirosina Quinases , Pirimidinas/farmacologia , Pirróis/farmacologia , Relação Estrutura-Atividade , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The aim of our proposed concept is to find new target structures for combating cancers with unmet medical needs. This, unfortunately, still applies to the majority of the clinically most relevant tumor entities such as, for example, liver cancer, pancreatic cancer, and many others. Current target structures almost all belong to the class of oncogenic proteins caused by tumor-specific genetic alterations, such as activating mutations, gene fusions, or gene amplifications, often referred to as cancer "driver alterations" or just "drivers." However, restoring the lost function of tumor suppressor genes (TSGs) could also be a valid approach to treating cancer. TSG-derived proteins are usually considered as control systems of cells against oncogenic properties; thus, they represent the brakes in the "car-of-life." Restoring these tumor-defective brakes by gene therapy has not been successful so far, with a few exceptions. It can be assumed that most TSGs are not being inactivated by genetic alteration (class 1 TSGs) but rather by epigenetic silencing (class 2 TSGs or short "C2TSGs"). Reactivation of C2TSGs in cancer therapy is being addressed by the use of DNA demethylating agents and histone deacetylase inhibitors which act on the whole cancer cell genome. These epigenetic therapies have neither been particularly successful, probably because they are "shotgun" approaches that, although acting on C2TSGs, may also reactivate epigenetically silenced oncogenic sequences in the genome. Thus, new strategies are needed to exploit the therapeutic potential of C2TSGs, which have also been named DNA methylation cancer driver genes or "DNAme drivers" recently. Here we present a concept for a new translational and therapeutic approach that focuses on the phenotypic imitation ("mimesis") of proteins encoded by highly disease-relevant C2TSGs/DNAme drivers. Molecular knowledge on C2TSGs is used in two complementary approaches having the translational concept of defining mimetic drugs in common: First, a concept is presented how truncated and/or genetically engineered C2TSG proteins, consisting solely of domains with defined tumor suppressive function can be developed as biologicals. Second, a method is described for identifying small molecules that can mimic the effect of the C2TSG protein lost in the cancer cell. Both approaches should open up a new, previously untapped discovery space for anticancer drugs.
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
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
Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias Mamárias Experimentais/patologia , Melanoma Experimental/patologia , Metástase Neoplásica/imunologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Feminino , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/deficiência , Proteínas Proto-Oncogênicas c-cbl/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Varfarina/farmacologia , Varfarina/uso terapêutico , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
Large-scale screens in mammalian cells demand for flexible high-throughput screening platforms that allow to analyze cellular traits on a genome-wide level or to identify small-molecule inhibitors (SMIs) from complex compound libraries. In this study we developed and tested high-density cell arrays made out of polydimethylsiloxane (PDMS) that support cell growth directly on standard glass microscope objective slides. We analyzed the effect of 3 reference inhibitors (MLN-8054, VX-680, and flavopiridol) and 4 exploratory, cell permeable small-molecule kinase inhibitors (two benzothiophene-based and two 4-amino-6-arylpyrimidine-based compounds) on different cell lines, using prototype 5 × 5 and 9 × 9 array carpets. We found that high-density PDMS cell arrays support growth of a broad range of cell types, are well suited for compound screens, and are compatible with high-content screening platforms. This novel array format is of particular advantage for compound screening to identify SMIs, when a combination of flexibility with respect to culture volume, well density, and high-resolution imaging is required. In addition, we demonstrated the suitability of this format for reverse transfection and siRNA experiments.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas , Animais , Células COS , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Chlorocebus aethiops , Dimetilpolisiloxanos , Humanos , Interfase/efeitos dos fármacos , Microscopia de Fluorescência , Índice Mitótico , RNA Interferente Pequeno/genética , Análise Serial de Tecidos , TransfecçãoRESUMO
The Mycobacterium tuberculosis genome encodes for eleven eukaryotic-like Ser/Thr protein kinases. At least three of these (PknA, PknB and PknG) are essential for bacterial growth and survival. PknG is secreted by pathogenic mycobacteria, in macrophages to intervene with host cell signalling pathways and to block the fusion of the lysosomes with the phagosome by a still unknown mechanism. Based on our previously published results, we have initiated a drug discovery program, aiming to improve the potency against PknG and the physiochemical properties of the initially identified hit compound, AX20017, from the class of the tetrahydrobenzothiophenes. We have established a radioactive biochemical PknG kinase assay to test the novel analogues around AX20017. We have developed lead molecules with IC50 values in nanomolar range, and demonstrated their antituberculotic effects on human macrophages. Selected leads might ultimately serve the purpose of inducing phagosomal-lysosomal fusion and therefore destroy the residence of the intracellular mycobacteria. It is unclear at this time if these "homeless" mycobacteria are getting killed by the host, but they will be at least vulnerable to the activity of antimycobacterial agents. Released mycobacteria rely on the essential function of PknB for survival, which is our second molecular kinase target. PknB is a transmembrane protein, responsible for the cell growth and morphology. We have screened our library and synthesized novel compounds for the inhibition of PknB. A pharmacophore model was built and 70,000 molecules from our synthesizable virtual library have been screened to identify novel inhibitor scaffolds for the generation of templated compound libraries. Currently, we are using a radioactive kinase assay employing GarA as the putative, physiological substrate of PknB kinase. We have identified hits and generated optimised hit compounds with IC50 values for the inhibition of PknB in the nanomolar range. Yet those promising hits are not potent enough to yield meaningful "minimum inhibitory concentrations" in mycobacterial growth assays. In the course of our future work, we will increase the potency of the next generation of PknB inhibitors in order to improve their antibacterial activity.
Assuntos
Desenho de Fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Amidas/química , Amidas/farmacologia , Animais , Proteínas de Bactérias/metabolismo , Células Cultivadas , Concentração Inibidora 50 , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia , Tuberculose/enzimologiaRESUMO
An increasing number of kinase inhibitor candidates are entering clinical development, representing an important change in the pharmaceutical industry; notably, the development of small-molecule kinase inhibitors for signal transduction therapies. Today, kinase inhibitors garner substantial attention in cancer research. Over the last few years, three distinct small-molecule kinase inhibitors reached the market for treatment of chronic myeloid leukaemia, gastrointestinal stromal tumours, and non-small cell lung cancers. These three drugs, imatinib, gefitinib and erlotinib, act on a distinct subset of dysregulated, and often cancer-relevant kinases. Imatinib, gefitinib and erlotinib are considered the front-runners of targeted kinase inhibitor drugs. The entire research field gains tremendous insights through the ongoing research and clinical trials with these three drugs and with fast following first-generation kinase inhibitors, many of which are in different phases of clinical development. In addition, novel chemogenomic and chemoproteomic technologies are emanating from the current kinase research area, focussing efforts on the generation of spectrum-selective inhibitors for anticancer therapies as opposed to the monospecific inhibitors for the remaining therapeutic areas.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Humanos , Inibidores de Proteínas Quinases/química , Especificidade por SubstratoRESUMO
Traditionally, protein kinases have been regarded as non-druggable targets, instead they play a central role in physiological and pathophysiological processes. This changed when STI571, an inhibitor of the Bcr-Abl kinase, known as Gleevec, reached the market as the first designer drug. Ever since, kinase-directed research and development (R&D) expanded rapidly, leading to more than 45 clinically relevant kinase inhibitors. At a comparable pace the kinase-based technologies matured, cumulating in the development of sophisticated chemogenetic and chemoproteomic tools, which are referred to as chemical kinomics.: