RESUMO
The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.
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 natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells. Among the identified 14-3-3 target proteins are THEMIS2, receptor interacting protein kinase 2 (RIPK2), EIF2AK2, and several members of the LDB1 complex. Biophysical and structural biology studies confirm these 14-3-3 PPIs as physical targets of FC stabilization, and transcriptome as well as pathway analyses suggest possible explanations for the observed synergistic effect of IFNα/FC treatment on cancer cells. This study elucidates the polypharmacological effects of FCs in cancer cells and identifies potential targets from the vast interactome of 14-3-3s for therapeutic intervention in oncology.
Assuntos
Interferon-alfa , Neoplasias Ovarianas , Humanos , Feminino , Interferon-alfa/farmacologia , Apoptose , Linhagem Celular Tumoral , Morte CelularRESUMO
The colony-stimulating factor 1 receptor (CSF1R) plays an important role in the regulation of many inflammatory processes, and overexpression of the kinase is implicated in several disease states. Identifying selective, small-molecule inhibitors of CSF1R may be a crucial step toward treating these disorders. Through modelling, synthesis, and a systematic structure-activity relationship study, we have identified a number of potent and highly selective purine-based inhibitors of CSF1R. The optimized 6,8-disubstituted antagonist, compound 9, has enzymatic IC50 of 0.2 nM, and displays a strong affinity toward the autoinhibited form of CSF1R, contrasting that of other previously reported inhibitors. As a result of its binding mode, the inhibitor shows excellent selectivity (Selectivity score: 0.06), evidenced by profiling towards a panel of 468 kinases. In cell-based assays, this inhibitor shows dose-dependent blockade of CSF1-mediated downstream signalling in murine bone marrow-derived macrophages (IC50 = 106 nM) as well as disruption of osteoclast differentiation at nanomolar levels. In vivo experiments, however, indicate that improve metabolic stability is needed in order to further progress this compound class.
Assuntos
Macrófagos , Osteoclastos , Animais , Camundongos , Receptores Proteína Tirosina Quinases , Diferenciação Celular , Purinas/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e MacrófagosRESUMO
Herpesviral nuclear egress is a regulated process of viral capsid nucleocytoplasmic release. Due to the large capsid size, a regular transport via the nuclear pores is unfeasible, so that a multistage-regulated export pathway through the nuclear lamina and both leaflets of the nuclear membrane has evolved. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. The transmembrane NEC protein pUL50 serves as a multi-interacting determinant that recruits regulatory proteins by direct and indirect contacts. The nucleoplasmic core NEC component pUL53 is strictly associated with pUL50 in a structurally defined hook-into-groove complex and is considered as the potential capsid-binding factor. Recently, we validated the concept of blocking the pUL50-pUL53 interaction by small molecules as well as cell-penetrating peptides or an overexpression of hook-like constructs, which can lead to a pronounced degree of antiviral activity. In this study, we extended this strategy by utilizing covalently binding warhead compounds, originally designed as binders of distinct cysteine residues in target proteins, such as regulatory kinases. Here, we addressed the possibility that warheads may likewise target viral NEC proteins, building on our previous crystallization-based structural analyses that revealed distinct cysteine residues in positions exposed from the hook-into-groove binding surface. To this end, the antiviral and NEC-binding properties of a selection of 21 warhead compounds were investigated. The combined findings are as follows: (i) warhead compounds exhibited a pronounced anti-HCMV potential in cell-culture-based infection models; (ii) computational analysis of NEC primary sequences and 3D structures revealed cysteine residues exposed to the hook-into-groove interaction surface; (iii) several of the active hit compounds exhibited NEC-blocking activity, as shown at the single-cell level by confocal imaging; (iv) the clinically approved warhead drug ibrutinib exerted a strong inhibitory impact on the pUL50-pUL53 core NEC interaction, as demonstrated by the NanoBiT assay system; and (v) the generation of recombinant HCMV ∆UL50-ΣUL53, allowing the assessment of viral replication under conditional expression of the viral core NEC proteins, was used for characterizing viral replication and a mechanistic evaluation of ibrutinib antiviral efficacy. Combined, the results point to a rate-limiting importance of the HCMV core NEC for viral replication and to the option of exploiting this determinant by the targeting of covalently NEC-binding warhead compounds.
Assuntos
Antivirais , Citomegalovirus , Humanos , Antivirais/farmacologia , Antivirais/metabolismo , Cisteína/metabolismo , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , Proteínas Virais/metabolismoRESUMO
mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.
Assuntos
Aminoácidos , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Aminoácidos/genética , Aminoácidos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Humanos , Animais , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs' antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, ß- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.
Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Farmacorresistência Viral/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Replicação Viral/genética , Aminopiridinas/farmacologia , Animais , Antivirais/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Combinação de Medicamentos , Ganciclovir/farmacologia , Humanos , Camundongos , Pirazóis/farmacologia , Ribonucleosídeos/farmacologia , Triazinas/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
In this work, we demonstrate that the indole-oxazole-pyrrole framework of the breitfussin family of natural products is a promising scaffold for kinase inhibition. Six new halogenated natural products, breitfussin C-H (3 - 8) were isolated and characterized from the Arctic, marine hydrozoan Thuiaria breitfussi. The structures of two of the new natural products were also confirmed by total synthesis. Two of the breitfussins (3 and 4) were found to selectively inhibit the survival of several cancer cell lines, with the lowest IC50 value of 340 nM measured against the drug-resistant triple negative breast cancer cell line MDA-MB-468, while leaving the majority of the tested cell lines not or significantly less affected. When tested against panels of protein kinases, 3 gave IC50 and Kd values as low as 200 and 390 nM against the PIM1 and DRAK1 kinases, respectively. The activity was confirmed to be mediated through ATP competitive binding in the ATP binding pocket of the kinases. Furthermore, evaluation of potential off-target and toxicological effects, as well as relevant in vitro ADME parameters for 3 revealed that the breitfussin scaffold holds promise for the development of selective kinase inhibitors.
Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/química , Regiões Árticas , Sítios de Ligação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Embrião não Mamífero/efeitos dos fármacos , Feminino , Humanos , Hidrocarbonetos Bromados/química , Hidrozoários/química , Indóis/química , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/toxicidade , Proteínas Proto-Oncogênicas c-pim-1/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Testes de Toxicidade , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Peixe-Zebra/embriologiaRESUMO
The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIß (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.
Assuntos
Antineoplásicos/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cílios/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor/genética , Morfolinas/farmacologia , Osteogênese/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Purinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Relação Estrutura-Atividade , Tiofenos/químicaRESUMO
The death of a cell is an inevitable part of its biology. During homeostasis, most cells die through apoptosis. If homeostasis is disturbed, cell death can switch to proinflammatory forms of death, such as necroptosis, pyroptosis, or NETosis. We demonstrate that the formation of neutrophil extracellular traps (NETs), a special form of neutrophil cell death that releases chromatin structures to the extracellular space, is dependent on gasdermin D (GSDMD). GSDMD is a pore-forming protein and an executor of pyroptosis. We screened a chemical library and found a small molecule based on the pyrazolo-oxazepine scaffold that efficiently blocks NET formation and GSDMD-mediated pyroptotic cell death in human cells. During NETosis, GSDMD is proteolytically activated by neutrophil proteases and, in turn, affects protease activation and nuclear expansion in a feed-forward loop. In addition to the central role of GSDMD in pyroptosis, we propose that GSDMD also plays an essential function in NETosis.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Morte Celular/fisiologia , Armadilhas Extracelulares/fisiologia , Proteínas de Neoplasias/fisiologia , Neutrófilos/fisiologia , Animais , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos Mutantes , Peptídeo Hidrolases/farmacologia , Proteínas de Ligação a FosfatoRESUMO
Onset of progression even during therapy with novel drugs remains an issue in chronic lymphocytic leukemia (CLL). Thus, there is ongoing demand for novel agents. Approaches targeting cyclin-dependent kinases (CDK) have reached the clinical trial stage. CDK9 mediating RNA transcriptional elongation is the evolving pivotal CLL CDK inhibitor target. However, more CDK9 selective compounds are desirable. Here, we describe the CDK9 inhibitor LDC526 displaying a low nanomolar biochemical activity against CDK9 and an at least 50-fold selectivity against other CDKs. After demonstrating in vitro MEC-1 cell line and primary human CLL cell cytotoxicity we evaluated the LDC526 in vivo effect on human CLL cells transplanted into NOD/scid/γcnull (NSG) mice. LDC526 administration (75 mg/kg) for 5 days resulted in a 77% reduction of human CLL cells in NSG spleens compared to carrier control treatment. Next, we longitudinally studied the LDC526 impact on circulating CLL cells in the TCL1 transgenic mouse model. LDC526 (50 mg/kg) administration for two days led to a 16-fold reduction of blood CLL cell numbers. Remarkably, residual CLL cells exhibited significantly increased intracellular BCL-2 levels. However, the LDC526 cytotoxic effect was not restricted to CLL cells as also declining numbers of normal B and T lymphocytes were observed in LDC526 treated TCL1 mice. Taken together, our in vivo data provide a strong rational for continued LDC526 development in CLL therapy and argue for the combination with BCL-2 inhibitors.
RESUMO
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
Selective inhibition of exclusively transcription-regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY-958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAYâ 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAYâ 1143572 exhibited the best overall profile inâ vitro and inâ vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAYâ 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/uso terapêutico , Triazinas/uso terapêutico , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Quinase 9 Dependente de Ciclina/metabolismo , Meia-Vida , Células HeLa , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Camundongos Nus , Conformação Molecular , Simulação de Acoplamento Molecular , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/toxicidade , Estrutura Terciária de Proteína , Ratos , Ratos Nus , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/toxicidade , Transplante Heterólogo , Triazinas/química , Triazinas/toxicidadeRESUMO
The RON tyrosine kinase receptor is under investigation as a novel target in pancreatic cancer. While RON mutations are uncommon, RON isoforms are produced in cancer cells via a variety of mechanisms. In this study we sought to: 1) characterize RON isoform expression in pancreatic cancer, 2) investigate mechanisms that regulate isoform expression, and 3) determine how various isoforms effect gene expression, oncogenic phenotypes and responses to RON directed therapies. We quantified RON transcripts in human pancreatic cancer and found expression levels 2500 fold that of normal pancreas with RON isoform expression comprising nearly 50% of total transcript. RNA seq studies revealed that the short form (sfRON) and P5P6 isoforms which have ligand independent activity, induce markedly different patterns of gene expression than wild type RON. We found that transcription of RON isoforms is regulated by promoter hypermethylation as the DNA demethylating agent 5-aza-2'-deoxycytidine decreased all RON transcripts in a subset of pancreatic cancer cell lines. The viability of sfRON-expressing HPDE cells was reduced by a RON specific small molecule inhibitor, while a therapeutic monoclonal antibody had no demonstrable effects. In summary, RON isoforms may comprise half of total RON transcript in human pancreatic cancer and their expression is regulated at least in part by promoter hypermethylation. RON isoforms activate distinct patterns of gene expression, have transforming activity and differential responses to RON directed therapies. These findings further our understanding of RON biology in pancreatic cancer and have implications for therapeutic strategies to target RON activity.
Assuntos
Carcinoma Ductal Pancreático/enzimologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Pancreáticas/enzimologia , Receptores Proteína Tirosina Quinases/biossíntese , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Metilação de DNA/genética , Xenoenxertos , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Receptores Proteína Tirosina Quinases/genética , Células Tumorais CultivadasRESUMO
Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.
Assuntos
Antivirais/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Pirazóis/farmacologia , Triazinas/farmacologia , Adenoviridae/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Fibroblastos/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesviridae/efeitos dos fármacos , Humanos , Camundongos , Fosforilação , Replicação Viral/efeitos dos fármacosRESUMO
Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Triazinas/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Neoplasias/genética , Fosforilação , Roscovitina , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
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
The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.
Assuntos
Miocárdio/enzimologia , Miocárdio/patologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/induzido quimicamente , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Estrutura Terciária de Proteína , Piridinas/farmacologia , Pirimidinas/farmacologia , Ratos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Knowledge about molecular drug action is critical for the development of protein kinase inhibitors for cancer therapy. Here, we establish a chemical proteomic approach to profile the anticancer drug SU6668, which was originally designed as a selective inhibitor of receptor tyrosine kinases involved in tumor vascularization. By employing immobilized SU6668 for the affinity capture of cellular drug targets in combination with mass spectrometry, we identified previously unknown targets of SU6668 including Aurora kinases and TANK-binding kinase 1. Importantly, a cell cycle block induced by SU6668 could be attributed to inhibition of Aurora kinase activity. Moreover, SU6668 potently suppressed antiviral and inflammatory responses by interfering with TANK-binding kinase 1-mediated signal transmission. These results show the potential of chemical proteomics to provide rationales for the development of potent kinase inhibitors, which combine rather unexpected biological modes of action by simultaneously targeting defined sets of both serine/threonine and tyrosine kinases involved in cancer progression.
Assuntos
Inibidores da Angiogênese/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Animais , Aurora Quinases , Células COS , Divisão Celular/efeitos dos fármacos , Chlorocebus aethiops , Células HeLa , Humanos , Oxindóis , Propionatos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , TransfecçãoRESUMO
The activation of NF-kappaB has long been considered a positive factor for human cytomegalovirus (HCMV) replication. The HCMV immediate-early promoter, the initial transcriptional element in the HCMV replication cycle, is activated by the transcription factor NF-kappaB, and several HCMV gene products have been demonstrated to activate this transcription factor. However, the role of NF-kappaB in the full replication cycle of the virus has not been carefully examined. A series of experiments that demonstrate an important inhibitory role of NF-kappaB for HCMV replication in fibroblasts is presented here. Using both genetic and pharmaceutical methods, it was shown that blocking NF-kappaB activation in cell culture does not inhibit HCMV replication, but rather leads to a modest increase in replication. Two cytokines inhibitory for HCMV, tumour necrosis factor-alpha and interferon-gamma, no longer inhibit HCMV when NF-kappaB activation is blocked. Furthermore, forced expression of the NF-kappaB activating IkappaB kinase beta (IKKbeta), but not a kinase inactive mutant, also inhibits HCMV replication. In addition, it was shown that NF-kappaB signalling is essential for the production of an anti-viral factor in the supernatant of HCMV-infected fibroblasts, and identified interferon-beta as this factor. Thus, the role of NF-kappaB in fibroblasts is to activate a host defence against HCMV.