RESUMO
The molecular mechanisms underlying seizure generation remain elusive, yet they are crucial for developing effective treatments for epilepsy. The current study shows that inhibiting c-Abl tyrosine kinase prevents apoptosis, reduces dendritic spine loss, and maintains N-methyl-d-aspartate (NMDA) receptor subunit 2B (NR2B) phosphorylated in in vitro models of excitotoxicity. Pilocarpine-induced status epilepticus (SE) in mice promotes c-Abl phosphorylation, and disrupting c-Abl activity leads to fewer seizures, increases latency toward SE, and improved animal survival. Currently, clinically used c-Abl inhibitors are non-selective and have poor brain penetration. The allosteric c-Abl inhibitor, neurotinib, used here has favorable potency, selectivity, pharmacokinetics, and vastly improved brain penetration. Neurotinib-administered mice have fewer seizures and improved survival following pilocarpine-SE induction. Our findings reveal c-Abl kinase activation as a key factor in ictogenesis and highlight the impact of its inhibition in preventing the insurgence of epileptic-like seizures in rodents and humans.
Assuntos
Pilocarpina , Proteínas Proto-Oncogênicas c-abl , Convulsões , Animais , Masculino , Camundongos , Apoptose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/patologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/patologiaRESUMO
Brain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways (MAPK, PI3K, PLC-γ), leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors' signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Neurônios , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Neurônios/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Transdução de Sinais , Proteínas Proto-Oncogênicas c-ablRESUMO
Abelson kinase (c-Abl) is a non-receptor tyrosine kinase involved in several biological processes essential for cell differentiation, migration, proliferation, and survival. This enzyme's activation might be an alternative strategy for treating diseases such as neutropenia induced by chemotherapy, prostate, and breast cancer. Recently, a series of compounds that promote the activation of c-Abl has been identified, opening a promising ground for c-Abl drug development. Structure-based drug design (SBDD) and ligand-based drug design (LBDD) methodologies have significantly impacted recent drug development initiatives. Here, we combined SBDD and LBDD approaches to characterize critical chemical properties and interactions of identified c-Abl's activators. We used molecular docking simulations combined with tree-based machine learning models-decision tree, AdaBoost, and random forest to understand the c-Abl activators' structural features required for binding to myristoyl pocket, and consequently, to promote enzyme and cellular activation. We obtained predictive and robust models with Matthews correlation coefficient values higher than 0.4 for all endpoints and identified characteristics that led to constructing a structure-activity relationship model (SAR).
Assuntos
Aprendizado de Máquina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-abl/química , Sítios de Ligação , Desenho de Fármacos , Humanos , Ligantes , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Relação Quantitativa Estrutura-AtividadeRESUMO
Langerhans cell histiocytosis (LCH), a disorder of antigen-presenting cells, is the commonest disorder of the mononuclear phagocytic system. Diagnosis is always challenging due to heterogeneous clinical presentation. However, with the evolution and better understanding of its biology, many of these children are being diagnosed early and offered appropriate therapy. Despite these advances, in developing countries, an early diagnosis is still challenging due to resource constraints for specialized tests. As a result, many patients succumb to their disease. Autopsy data on LCH is notably lacking in the literature. We sought to analyze the clinical (including mutational) and morphologic features at autopsy in six proven cases of LCH. This study includes a detailed clinico-pathological and mutational analysis of 6 proven cases of LCH. Presence of BRAF V600E mutation was assessed by both Real Time PCR and Sanger sequencing. A varied spectrum of organ involvement was noted with some rare and novel morphological findings, like nodular bronchiolocentric infiltration of LCH cells, lymphovascular emboli of LCH cells, and paucity of eosinophils within the infiltrate; these features have not been described earlier. Surprisingly, all cases were negative for BRAF V600E mutation on both RQ-PCR and Sanger sequencing. The present study is perhaps the first autopsy series on LCH. This extensive autopsy analysis represents a correlation of pathological features with clinical symptoms which provides clues for a timely diagnosis and appropriate therapeutic intervention. Also, our findings hint at the low frequency of BRAF V600E mutation in our LCH patients.
Assuntos
Humanos , Masculino , Lactente , Pré-Escolar , Histiocitose de Células de Langerhans/patologia , Autopsia , Proteínas Proto-Oncogênicas c-abl , Quinases de Proteína Quinase Ativadas por Mitógeno , Diagnóstico PrecoceRESUMO
Drug resistance is a serious problem in cancer, viral, bacterial, fungal and parasitic diseases. Examination of crystal structures of protein-drug complexes is often not enough to explain why a certain mutation leads to drug resistance. As an example, the crystal structure of the kinase inhibitor dasatinib bound to the Abl1 kinase shows a hydrogen bond between the drug and residue Thr315 and very few contacts between the drug and residues Val299 and Phe317, yet mutations in those residues lead to drug resistance. In the first case, it is tempting to suggest that the loss of a hydrogen bond leads to drug resistance, whereas in the other two cases it is not known why mutations lead to drug resistance in the first place. We carried out extensive molecular dynamics (MD) simulations and free energy calculations to explain drug resistance to dasatinib from a molecular point of view and show that resistance is due to a multitude of subtle effects. Importantly, our calculations could reproduce the experimental values for the binding energies upon mutations in all three cases and shed light on their origin.
Assuntos
Dasatinibe/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Dasatinibe/química , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , TermodinâmicaRESUMO
The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.
Assuntos
Sobrevivência Celular/genética , Reparo do DNA , Proteínas de Drosophila/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA/genética , Animais , Dano ao DNA , Proteínas de Drosophila/genética , Drosophila melanogaster , Endorribonucleases/genética , Feminino , Fibroblastos , Instabilidade Genômica , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética , Proteostase/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , RNA Mensageiro/metabolismoAssuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/uso terapêutico , Linfócitos B/efeitos dos fármacos , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Fatores Etários , Antineoplásicos/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Transporte Biológico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Mesilato de Imatinib/metabolismo , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Progesterona/farmacologia , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Fatores Sexuais , Resultado do TratamentoRESUMO
Two new complexes of Ru(II) with mixed ligands were prepared: [Ru(bpy)2smp](PF6) (1) and [Ru(phen)2smp](PF6) (2), in which smp = sulfamethoxypyridazine; bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline. The complexes have been characterized by elemental and conductivity analyses; infrared, NMR, and electrospray ionization mass spectroscopies; and X-ray diffraction of single crystal. Structural analyses reveal a distorted octahedral geometry around Ru(II) that is bound to two bpy (in 1) or two phen (in 2) via their two heterocyclic nitrogens and to two nitrogen atoms from sulfamethoxypyridazine-one of the methoxypyridazine ring and the sulfonamidic nitrogen, which is deprotonated. Both complexes inhibit the growth of chronic myelogenous leukemia cells. The interaction of the complexes with bovine serum albumin and DNA is described. DNA footprinting using an oligonucleotide as substrate showed the complexes' preference for thymine base rich sites. It is worth notifying that the complexes interact with the Src homology SH3 domain of the Abl tyrosine kinase protein. Abl protein is involved in signal transduction and implicated in the development of chronic myelogenous leukemia. Nuclear magnetic resonance (NMR) studies of the interaction of complex 2 with the Abl-SH3 domain showed that the most affected residues were T79, G97, W99, and Y115.
Assuntos
Antineoplásicos/síntese química , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Compostos Organometálicos/síntese química , Rutênio/química , Sulfametoxipiridazina/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Dicroísmo Circular , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Estrutura Molecular , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Difração de Raios X , Domínios de Homologia de srcRESUMO
Alzheimer's disease (AD) is characterized by progressive memory loss and dementia. The strong correlation between cognitive decline and the loss of synapses supports the idea that synaptic damage is a relevant pathogenic mechanism underlying AD progression. It has been shown that amyloid beta oligomers (AßOs) induce synaptotoxicity ultimately leading to the reduction of dendritic spine density, which underlies cognitive damage. However, the signaling pathways connecting AßOs to synaptic dysfunction have not been completely elucidated. In this review, we have gathered evidence on AßOs receptors and the signaling pathways involved in synaptic damage. We make special emphasis on a new AßOs induced axis that involves the tyrosine kinase ephrin receptor A4 (EphA4) and c-Abl tyrosine kinase activation. EphA4 is a key player in homeostatic plasticity, mediating dendritic spine remodeling and retraction. AßOs aberrantly activate EphA4 leading to dendritic spine elimination. c-Abl is activated in AßOs exposed neurons and in AD patient's brain, and the inhibition of activated c-Abl ameliorates cognitive deficits in AD mouse model. The EphA4 receptor activates c-Abl intracellular signaling. Therefore EphA4 is an emerging AßOs receptor and the activation of the EphA4/c-Abl axis would explain the synaptic spine alterations found in AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Receptor EphA4/metabolismo , Transdução de Sinais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-abl/genética , Receptor EphA4/genética , Sinapses/genética , Sinapses/metabolismo , Sinapses/patologiaRESUMO
Philadelphia chromosome (Ph)-like B-cell acute lymphoblastic leukemia (Ph-like ALL) is associated with activated JAK/STAT, Abelson kinase (ABL), and/or phosphatidylinositol 3-kinase (PI3K) signaling and poor clinical outcomes. PI3K pathway signaling inhibitors have been minimally investigated in Ph-like ALL. We hypothesized that targeted inhibition of PI3Kα, PI3Kδ, PI3K/mTOR, or target of rapamycin complex 1/2 (TORC1/TORC2) would decrease leukemia proliferation and abrogate aberrant kinase signaling and that combined PI3K pathway and JAK inhibition or PI3K pathway and SRC/ABL inhibition would have superior efficacy compared to inhibitor monotherapy. We treated 10 childhood ALL patient-derived xenograft models harboring various Ph-like genomic alterations with 4 discrete PI3K pathway protein inhibitors and observed marked leukemia reduction and in vivo signaling inhibition in all models. Treatment with dual PI3K/mTOR inhibitor gedatolisib resulted in near eradication of ALL in cytokine receptor-like factor 2 (CRLF2)/JAK-mutant models with mean 92.2% (range, 86.0%-99.4%) reduction vs vehicle controls (P < .0001) and in prolonged animal survival. Gedatolisib also inhibited ALL proliferation in ABL/platelet-derived growth factor receptor (PDGFR)-mutant models with mean 66.9% (range, 42.0%-87.6%) reduction vs vehicle (P < .0001). Combined gedatolisib and ruxolitinib treatment of CRLF2/JAK-mutant models more effectively inhibited ALL proliferation than either inhibitor alone (P < .001) and further enhanced survival. Similarly, superior efficacy of combined gedatolisib and dasatinib was observed in ABL/PDGFR-mutant models (P < .001). Overall, PI3K/mTOR inhibition potently decreased ALL burden in vivo; antileukemia activity was further enhanced with combination inhibitor therapy. Clinical trials testing combinations of kinase inhibitors in Ph-like ALL patients are indicated.