RESUMO
Signaling through the Rho family of small GTPases has been intensely investigated for its crucial roles in a wide variety of human diseases. Although RhoA and Rac1 signaling pathways are frequently exploited with the aid of effective small molecule modulators, studies of the Cdc42 subclass have lagged because of a lack of such means. We have applied high-throughput in silico screening and identified compounds that are able to fit into the surface groove of Cdc42, which is critical for guanine nucleotide exchange factor binding. Based on the interaction between Cdc42 and intersectin (ITSN), a specific Cdc42 guanine nucleotide exchange factor, we discovered compounds that rendered ITSN-like interactions in the binding pocket. By using in vitro binding and imaging as well as biochemical and cell-based assays, we demonstrated that ZCL278 has emerged as a selective Cdc42 small molecule modulator that directly binds to Cdc42 and inhibits its functions. In Swiss 3T3 fibroblast cultures, ZCL278 abolished microspike formation and disrupted GM130-docked Golgi structures, two of the most prominent Cdc42-mediated subcellular events. ZCL278 reduces the perinuclear accumulation of active Cdc42 in contrast to NSC23766, a selective Rac inhibitor. ZCL278 suppresses Cdc42-mediated neuronal branching and growth cone dynamics as well as actin-based motility and migration in a metastatic prostate cancer cell line (i.e., PC-3) without disrupting cell viability. Thus, ZCL278 is a small molecule that specifically targets Cdc42-ITSN interaction and inhibits Cdc42-mediated cellular processes, thus providing a powerful tool for research of Cdc42 subclass of Rho GTPases in human pathogenesis, such as those of cancer and neurological disorders.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/química , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/química , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/fisiologia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Células Swiss 3T3 , Interface Usuário-Computador , Cicatrização/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/fisiologiaRESUMO
Variations in circadian rhythms are evident in the incidence of cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. The suprachiasmatic nucleus is the central circadian pacemaker that regulates the daily rhythm of peripheral organs. Diurnal rhythms have more recently been shown to exist in myocardial tissue and are involved in metabolism and contractile function. Thus we sought to determine whether the functional deletion of the circadian rhythm mouse periodic gene 2 (mPer2) would protect the heart against ischemic injury. Nonreperfused myocardial infarction was induced in anesthetized, ventilated C57 (n = 17) and mPer2 mutant (mPer2-M; n = 15) mice via permanent ligation of the left anterior descending coronary artery. At 4 days post-myocardial infarction, we observed a 43% reduction of infarct area in mPer2-M mice compared with wild-type mice. This is coincident with 25% less macrophage infiltration, 43% higher capillary density, 17% increase in hypertrophy, and 15% less cardiomyocyte apoptosis in the infarct zone. Also, matrix metalloproteinase-9 was expressed in inflammatory cells in both groups, but total protein was 40% higher in wild-type mice, whereas it was not elevated in mPer2-M mice in response to injury. The functional deletion of the mPer2 gene reduces the severity of myocardial infarct injury by limiting the inflammatory response, reducing apoptosis, and inducing cardiomyocyte hypertrophy, thus preserving cardiac function. These findings collectively imply that the disruption of the circadian clock gene mPer2 is protective. Understanding the interactions between circadian rhythm genes and cardiovascular disease may provide insights into potential preventative and therapeutic strategies for susceptible populations.
Assuntos
Deleção de Genes , Infarto do Miocárdio/genética , Infarto do Miocárdio/prevenção & controle , Proteínas Circadianas Period/genética , Animais , Apoptose , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Hipertrofia , Ligadura/efeitos adversos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Infarto do Miocárdio/etiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
Cisplatin is an effective and widely used first-line chemotherapeutic drug for treating cancers. However, many patients sustain cisplatin-induced peripheral neuropathy (CIPN), often leading to a reduction in drug dosages or complete cessation of treatment altogether. Therefore, it is important to understand cisplatin mechanisms in peripheral nerve tissue mediating its toxicity and identify signaling pathways for potential intervention. Rho GTPase activation is increased following trauma in several models of neuronal injury. Thus, we investigated whether components of the Rho signaling pathway represent important neuroprotective targets with the potential to ameliorate CIPN and thereby optimize current chemotherapy treatment regimens. We have developed a novel CIPN model in the mouse. Using this model and primary neuronal culture, we determined whether LM11A-31, a small-molecule, orally bioavailable ligand of the p75 neurotrophin receptor (p75(NTR)), can modulate Rho GTPase signaling and reduce CIPN. Von Frey filament analysis of sural nerve function showed that LM11A-31 treatment prevented decreases in peripheral nerve sensation seen with cisplatin treatment. Morphometric analysis of harvested sural nerves revealed that cisplatin-induced abnormal nerve fiber morphology and the decreases in fiber area were alleviated with concurrent LM11A-31 treatment. Cisplatin treatment increased RhoA activity accompanied by the reduced tyrosine phosphorylation of SHP2, which was reversed by LM11A-31. LM11A-31 also countered the effects of calpeptin, which activated RhoA by inhibiting SHP2 tyrosine phosphatase. Therefore, suppression of RhoA signaling by LM11A-31 that modulates p75(NTR) or activates SHP2 tyrosine phosphatase downstream of the NGF receptor enhances neuroprotection in experimental CIPN in mouse model.