RESUMO
As members of the MAPK family, c-Jun-N-terminal kinases (JNKs) regulate the biological processes of apoptosis. In particular, the isoform JNK3 is expressed explicitly in the brain at high levels and is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this study, we prepared a series of five 6-dihydroxy-1H-benzo[d]imidazoles as JNK3 inhibitors and found them have potential as neuroprotective agents. Following a previous lead scaffold, benzimidazole moiety was modified with various aryl groups and hydroxylation, and the resulting compounds exhibited JNK3 inhibitory activity with improved potency and selectivity. Out of 37 analogues synthesized, (S)-cyclopropyl(3-((4-(2-(2,3-dihydrobenzo[b][1,4]dioxin -6-yl)-5,6-dihydroxy-1H-benzo[d]imidazol-1-yl)pyrimidin-2-yl)amino) piperidin-1-yl)methanone (35b) demonstrated the highest JNK3 inhibition (IC50 = 9.7 nM), as well as neuroprotective effects against Aß-induced neuronal cell death. As a protein kinase inhibitor, it also showed excellent selectivity over other protein kinases including isoforms JNK1 (>1000 fold) and JNK2 (-10 fold).
Assuntos
Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Peptídeos beta-Amiloides/toxicidade , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteína Quinase 10 Ativada por Mitógeno/química , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Neurônios/patologia , Síndromes Neurotóxicas/prevenção & controle , Fragmentos de Peptídeos/toxicidade , RatosRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) showed promising clinical efficacy toward COVID-19 (Coronavirus disease 2019) patients as potent painkillers and anti-inflammatory agents. However, the prospective anti-COVID-19 mechanisms of NSAIDs are not evidently exposed. Therefore, we intended to decipher the most influential NSAIDs candidate(s) and its novel mechanism(s) against COVID-19 by network pharmacology. FDA (U.S. Food & Drug Administration) approved NSAIDs (19 active drugs and one prodrug) were used for this study. Target proteins related to selected NSAIDs and COVID-19 related target proteins were identified by the Similarity Ensemble Approach, Swiss Target Prediction, and PubChem databases, respectively. Venn diagram identified overlapping target proteins between NSAIDs and COVID-19 related target proteins. The interactive networking between NSAIDs and overlapping target proteins was analyzed by STRING. RStudio plotted the bubble chart of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of overlapping target proteins. Finally, the binding affinity of NSAIDs against target proteins was determined through molecular docking test (MDT). Geneset enrichment analysis exhibited 26 signaling pathways against COVID-19. Inhibition of proinflammatory stimuli of tissues and/or cells by inactivating the RAS signaling pathway was identified as the key anti-COVID-19 mechanism of NSAIDs. Besides, MAPK8, MAPK10, and BAD target proteins were explored as the associated target proteins of the RAS. Among twenty NSAIDs, 6MNA, Rofecoxib, and Indomethacin revealed promising binding affinity with the highest docking score against three identified target proteins, respectively. Overall, our proposed three NSAIDs (6MNA, Rofecoxib, and Indomethacin) might block the RAS by inactivating its associated target proteins, thus may alleviate excessive inflammation induced by SARS-CoV-2.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas/metabolismo , SARS-CoV-2/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/metabolismo , Antivirais/metabolismo , Humanos , Proteína Quinase 10 Ativada por Mitógeno/química , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/química , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Terapia de Alvo Molecular , Mapas de Interação de Proteínas/efeitos dos fármacos , SARS-CoV-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína de Morte Celular Associada a bcl/química , Proteína de Morte Celular Associada a bcl/metabolismo , Proteínas ras/metabolismoRESUMO
Alzheimer's disease (AD), a severe age-related neurodegenerative disorder, lacks effective therapeutic methods at present. Physical approaches such as gamma frequency light flicker that can effectively reduce amyloid load have been reported recently. Our previous research showed that a physical method named photobiomodulation (PBM) therapy rescues Aß-induced dendritic atrophy in vitro. However, it remains to be further investigated the mechanism by which PBM affects AD-related multiple pathological features to improve learning and memory deficits. Here, we found that PBM attenuated Aß-induced synaptic dysfunction and neuronal death through MKP7-dependent suppression of JNK3, a brain-specific JNK isoform related to neurodegeneration. The results showed PBM-attenuated amyloid load, AMPA receptor endocytosis, dendrite injury, and inflammatory responses, thereby rescuing memory deficits in APP/PS1 mice. We noted JNK3 phosphorylation was dramatically decreased after PBM treatment in vivo and in vitro. Mechanistically, PBM activated ERK, which subsequently phosphorylated and stabilized MKP7, resulting in JNK3 inactivation. Furthermore, activation of ERK/MKP7 signaling by PBM increased the level of AMPA receptor subunit GluR 1 phosphorylation and attenuated AMPA receptor endocytosis in an AD pathological model. Collectively, these data demonstrated that PBM has potential therapeutic value in reducing multiple pathological features associated with AD, which is achieved by regulating JNK3, thus providing a noninvasive, and drug-free therapeutic strategy to impede AD progression.
Assuntos
Doença de Alzheimer/genética , Terapia com Luz de Baixa Intensidade/métodos , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Receptores de AMPA/metabolismo , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Endocitose , Humanos , Masculino , CamundongosRESUMO
Obesity, which has long since reached epidemic proportions worldwide, is associated with long-term stress to a variety of organs and results in diseases including type 2 diabetes. In the brain, overnutrition induces hypothalamic stress associated with the activation of several signalling pathways, together with central insulin and leptin resistance. This central action of nutrient overload appears very rapidly, suggesting that nutrition-induced hypothalamic stress is a major upstream initiator of obesity and associated diseases. The cellular response to nutrient overload includes the activation of the stress-activated c-Jun N-terminal kinases (JNKs) JNK1, JNK2 and JNK3, which are widely expressed in the brain. Here, we review recent findings on the regulation and effects of these kinases, with particular focus on the hypothalamus, a key brain region in the control of energy and glucose homeostasis. JNK1 blocks the hypothalamic-pituitary-thyroid axis, reducing energy expenditure and promoting obesity. Recently, opposing roles have been identified for JNK1 and JNK3 in hypothalamic agouti gene-related protein (AgRP) neurons: while JNK1 activation in AgRP neurons induces feeding and weight gain and impairs insulin and leptin signalling, JNK3 (also known as MAPK10) deletion in the same neuronal population produces very similar effects. The opposing roles of these kinases, and the unknown role of hypothalamic JNK2, reflect the complexity of JNK biology. Future studies should address the specific function of each kinase, not only in different neuronal subsets, but also in non-neuronal cells in the central nervous system. Decoding the puzzle of brain stress kinases will help to define the central stimuli and mechanisms implicated in the control of energy balance. Graphical abstract.
Assuntos
Metabolismo Energético/fisiologia , Hipotálamo/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Neurônios/metabolismo , Obesidade/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Estresse do Retículo Endoplasmático , Comportamento Alimentar/fisiologia , Glucose/metabolismo , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/citologia , Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leptina/metabolismo , Doenças Metabólicas/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Neurônios/citologia , Glândula Tireoide/metabolismo , Aumento de Peso/fisiologiaRESUMO
The role for c-Jun N-terminal Kinase (JNK) in the control of feeding and energy balance is not well understood. Here, by use of novel and highly selective JNK inhibitors, we investigated the actions of JNK in the control of feeding and body weight homeostasis. In lean mice, intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of SR-3306, a brain-penetrant and selective pan-JNK (JNK1/2/3) inhibitor, reduced food intake and body weight. Moreover, i.p. and i.c.v. administrations of SR11935, a brain-penetrant and JNK2/3 isoform-selective inhibitor, exerted similar anorectic effects as SR3306, which suggests JNK2 or JNK3 mediates aspect of the anorectic effect by pan-JNK inhibition. Furthermore, daily i.p. injection of SR3306 (7 days) prevented the increases in food intake and weight gain in lean mice upon high-fat diet feeding, and this injection paradigm reduced high-fat intake and obesity in diet-induced obese (DIO) mice. In the DIO mice, JNK inhibition sensitized leptin's anorectic effect, and enhanced leptin-induced STAT3 activation in the hypothalamus. The underlying mechanisms likely involve the downregulation of SOCS3 by JNK inhibition. Collectively, our data suggest that JNK activity promotes positive energy balance, and the therapeutic intervention inhibiting JNK activities represents a promising approach to ameliorate diet-induced obesity and leptin resistance.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Leptina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Metabolismo Energético/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Camundongos , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Obesidade/genética , Obesidade/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
The article reviews the literature regarding the role of c-Jun-N-terminal kinases (JNK) and its inhibitors in brain damage in the settings of ischemia and reperfusion injury. The implication of JNK in signaling mechanisms involved in ischemia-reperfusion-induced cerebral injury are discussed. Described effects associated with JNK inhibition using synthetic and natural substances in experimental models of ischemic and reperfusion injury of the brain. Results of experimental studies demonstrated that JNK represent promising therapeutic targets for brain protection against ischemic stroke. However, multiple physiologic functions of various JNK family members do not allow for the systemic use of non-specific JNK inhibitors for therapeutic purposes. The authors conclude that the continuous search for selective inhibitors of JNK3 remains an important task.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Acetonitrilas/farmacologia , Animais , Antracenos/farmacologia , Benzotiazóis/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Regulação da Expressão Gênica , Ginsenosídeos/isolamento & purificação , Ginsenosídeos/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Oximas/farmacologia , Extratos Vegetais/química , Quinoxalinas/farmacologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de SinaisRESUMO
Cilostazol(CTL) is a phosphodiesterase inhibitor, which has been widely used as anti-platelet agent. It also has preventive effects on various central nervous system (CNS) diseases, including ischemic stroke, Parkinson's disease and Alzheimer disease. However, the molecular mechanism underlying the protective effects of CTL is still unclear, and whether CTL can prevent I/R induced cognitive deficit has not been reported. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The open field tasks and Morris water maze were used to assess the effect of CTL on anxiety-like behavioral and cognitive impairment after I/R. Western blotting were performed to examine the expression of related proteins, and HE-staining was used to detect the percentage of neuronal death in the hippocampal CA1 region. Here we found that CTL significantly improved cognitive deficits and the behavior of rats in Morris water maze and open field tasks (P<0.05). HE staining results showed that CTL could significantly protect CA1 neurons against cerebral I/R (P<0.05). Additionally, Akt1 phosphorylation levels were evidently up-regulated (P<0.05), while the activation of JNK3, which is an important contributor to I/R-induced neuron apoptosis, was reduced by CTL after I/R (P<0.05), and caspase-3 levels were also decreased by CTL treatment. Furthermore, all of CTL's protective effects were reversed by LY294002, which is a PI3K/Akt1 inhibitor. Taken together, our results suggest that CTL could protect hippocampal neurons and ameliorate the impairment of learning/memory abilities and locomotor/ exploratory activities in ischemic stroke via a PI3K-Akt1/JNK3/caspase-3 dependent mechanism.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Transtornos Cognitivos/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Tetrazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Isquemia Encefálica/complicações , Isquemia Encefálica/enzimologia , Isquemia Encefálica/patologia , Caspase 3/metabolismo , Cilostazol , Transtornos Cognitivos/enzimologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Hipocampo/enzimologia , Hipocampo/patologia , Masculino , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologiaRESUMO
Alzheimer's and Parkinson's diseases are neurodegenerative disorders characterized by progressive neuronal dysfunction. Previous studies revealed that some natural products have neuroprotective properties, including species of the Myrtaceae family. However, the neuromodulatory potential of Calyptranthes grandifolia is not clear. In the present study, we examined the ability of the ethanol and hexane leaf extracts of C. grandifolia to prevent 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. Initially, we investigated the potential of the extracts to inhibit the neurodegenerative-related enzymes c-Jun N-terminal kinase 3 (JNK3) and acetylcholinesterase (AChE). In addition, SH-SY5Y cell viability was assessed by MTT assay after 100µM 6-OHDA-induced cell damage. In order to verify the possible effects of both extracts on 6-OHDA-induced cell death, hydrogen peroxide generation, mitochondrial potential and caspases-3 activity were assessed. Our findings revealed that ethanol extract exhibited inhibitory activity against JNK3 and AChE. In addition, when co-treating SH-SY5Y cells with 6-OHDA and the extracts, oxidative stress was inhibited by both extracts through a decrease of mitochondrial depolarization and caspases-3 activity. In summary, ethanol and hexane extracts of C. grandifolia have some suppressive property against neurotoxicity induced by 6-OHDA.
Assuntos
Myrtaceae/química , Fármacos Neuroprotetores/uso terapêutico , Síndromes Neurotóxicas/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Acetilcolinesterase/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Etanol , Hexanos , Humanos , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/enzimologia , Síndromes Neurotóxicas/patologia , Estresse Oxidativo/efeitos dos fármacos , OxidopaminaRESUMO
It has been well documented that Momordica charantia polysaccharide (MCP) has multiple biological effects such as immune enhancement, anti-oxidation and anti-cancer. However, the potential protective effects of MCP on stroke damage and its relative mechanisms remain unclear. Our present study demonstrated that MCP could scavenge reactive oxygen species (ROS) in intra-cerebral hemorrhage damage, significantly attenuating the neuronal death induced by thrombin in primary hippocampal neurons. Furthermore, we found that MCP prevented the activation of the c-Jun N-terminal protein kinase (JNK3), c-Jun and caspase-3, which was caused by the intra-cerebral hemorrhage injury. Taken together, our study demonstrated that MCP had a neuroprotective effect in response to intra-cerebral hemorrhage and its mechanisms involved the inhibition of JNK3 signaling pathway.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Hemorragia Cerebral/complicações , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Momordica charantia/química , Fármacos Neuroprotetores/farmacologia , Fitoterapia , Polissacarídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Lesões Encefálicas/etiologia , Lesões Encefálicas/metabolismo , Radicais Livres/metabolismo , Immunoblotting , Técnicas Imunoenzimáticas , Imunoprecipitação , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Momordica charantia (MC) is a medicinal plant for stroke treatment in Traditional Chinese Medicine, but its active compounds and molecular targets are unknown yet. M. charantia polysaccharide (MCP) is one of the important bioactive components in MC. In the present study, we tested the hypothesis that MCP has neuroprotective effects against cerebral ischemia/reperfusion injury through scavenging superoxide (O2(-)), nitric oxide (NO) and peroxynitrite (ONOO(-)) and inhibiting c-Jun N-terminal protein kinase (JNK3) signaling cascades. We conducted experiments with in vivo global and focal cerebral ischemia/reperfusion rat models and in vitro oxygen glucose deprivation (OGD) neural cells. The effects of MCP on apoptotic cell death and infarction volume, the bioactivities of scavenging O2(-), NO and ONOO(-), inhibiting lipid peroxidation and modulating JNK3 signaling pathway were investigated. Major results are summarized as below: (1) MCP dose-dependently attenuated apoptotic cell death in neural cells under OGD condition in vitro and reduced infarction volume in ischemic brains in vivo; (2) MCP had directing scavenging effects on NO, O2(-) and ONOO(-) and inhibited lipid peroxidation; (3) MCP inhibited the activations of JNK3/c-Jun/Fas-L and JNK3/cytochrome C/caspases-3 signaling cascades in ischemic brains in vivo. Taken together, we conclude that MCP could be a promising neuroprotective ingredient of M. charantia and its mechanisms could be at least in part attributed to its antioxidant activities and inhibiting JNK3 signaling cascades during cerebral ischemia/reperfusion injury.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Momordica charantia/química , Fármacos Neuroprotetores/administração & dosagem , Fitoterapia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Morte Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/administração & dosagem , Radicais Livres/metabolismo , Masculino , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Astragalus is a widely used traditional Chinese medicine and has been proven beneficial for many aspects of human health. It is important to explore the neuroprotective effect and mechanism of astragalus injection in cerebral ischemia reperfusion injury. METHODS: The focal cerebral ischemic model with middle cerebral artery occlusion (MCAO) reperfusion was established by Longa's method in healthy adult male Wistar rats, and treated by injecting intraperitoneally astragalus injection (3 ml/kg). The neurobehavioral function of rats was evaluated by Longa's test. The cerebral blood flow (CBF) was measured by laser Doppler flowmetry and the cerebral infarct volume was calculated by tetrazolium chloride (TTC) stain. The shape and structure of neurons in parahippocampal area was observed by HE stain and the neuronal apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and flow cytometry. The expressions of c-jun N-terminal kinase 3 (JNK3) mRNA and protein were determined by RT-PCR and immunohistochemical assay and Western blotting respectively. RESULTS: After treatment with astragalus injection, the expressions of JNK3 mRNA and protein reduced significantly, the number of neuronal apoptosis minus, the cerebral infarct volume shrink, the neuronal shape-structure and animal neurobehavioral function improved significantly than those in model rats. CONCLUSIONS: It is suggested that astragalus injection could inhibit neuronal apoptosis, reduce infarct volume and improve neurobehavioral function by down-regulating the expression of JNK3 gene after cerebral ischemia reperfusion injury in rats.
Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Saponinas/uso terapêutico , Triterpenos/uso terapêutico , Animais , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Masculino , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologiaRESUMO
Identification of a viable lead is a critical step in drug discovery. The qualities of the lead set the stage for subsequent efforts to ameliorate therapeutic efficacy through potency, selectivity, pharmacokinetics, toxicity and side effects. In a retrospective view of drug research the lead identification has been realised mainly by in vivo methodologies. However, limitations of in vivo models were found to be critical factors when analysing attrition rates that prompted research groups to introduce in vitro tests and rational approaches at the frontline of discovery programs. Virtual screening (VS) methods merge in vitro high-throughput (HTS) and rational approaches. The VS methods can be classified as ligand and structure based techniques. Structure based approaches depart from the structural information of the target to identify potential interactions between the ligands and the protein. The advantages and disadvantages and the applicability of the structure based virtual screening approaches constituted the main aim of my studies. The glycogen synthase kinase 3beta (GSK-3beta), the beta-secretase and the c-jun N-terminal kinase 3 (JNK-3) were selected as primary targets for virtual screening. The performance of virtual screens can only be validated in parallel with HTS, therefore a head to head comparative analysis was my next goal.