RESUMEN
BACKGROUND: Immune escape is an important feature of hepatocellular carcinoma (HCC). The overall response rate of immune checkpoint inhibitors (ICIs) in HCC is still limited. Revealing the immune regulation mechanisms and finding new immune targets are expected to further improve the efficacy of immunotherapy. Our study aims to use CRISPR screening mice models to identify potential targets that play a critical role in HCC immune evasion and further explore their value in improving immunotherapy. METHODS: We performed CRISPR screening in two mice models with different immune backgrounds (C57BL/6 and NPG mice) and identified the immunosuppressive gene Gsk3a as a candidate for further investigation. Flow cytometry was used to analyze the impact of Gsk3a on immune cell infiltration and T-cell function. RNA sequencing was used to identify the changes in neutrophil gene expression induced by Gsk3a and alterations in downstream molecules. The therapeutic value of the combination of Gsk3a inhibitors and anti-programmed cell death protein-1 (PD-1) antibody was also explored. RESULTS: Gsk3a, as an immune inhibitory target, significantly promoted tumor growth in immunocompetent mice rather than immune-deficient mice. Gsk3a inhibited cytotoxic T lymphocytes (CTLs) function by inducing neutrophil chemotaxis. Gsk3a promoted self-chemotaxis of neutrophil expression profiles and neutrophil extracellular traps (NETs) formation to block T-cell activity through leucine-rich α-2-glycoprotein 1 (LRG1). A significant synergistic effect was observed when Gsk3a inhibitor was in combination with anti-PD-1 antibody. CONCLUSIONS: We identified a potential HCC immune evasion target, Gsk3a, through CRISPR screening. Gsk3a induces neutrophil recruitment and NETs formation through the intermediate molecule LRG1, leading to the inhibition of CTLs function. Targeting Gsk3a can enhance CTLs function and improve the efficacy of ICIs.
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Carcinoma Hepatocelular , Inmunoterapia , Neoplasias Hepáticas , Animales , Humanos , Ratones , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Evasión Inmune , Inmunoterapia/métodos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/terapia , Ratones Endogámicos C57BL , Escape del Tumor/efectos de los fármacosRESUMEN
The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.
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Proteínas de Ciclo Celular , Glucógeno Sintasa Quinasa 3 , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-akt , Humanos , Células HEK293 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Ciclo Celular/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/genética , Proteínas Tirosina Quinasas/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
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Proteínas de la Matriz Extracelular , Matriz Extracelular , Degeneración Macular , Epitelio Pigmentado de la Retina , Animales , Ratones , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Degeneración Macular/patología , Degeneración Macular/genética , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Humanos , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Modelos Animales de Enfermedad , Distrofias Retinianas/metabolismo , Distrofias Retinianas/patología , Distrofias Retinianas/genética , Drusas del Disco Óptico/congénitoRESUMEN
Glycogen synthase kinase-3 (GSK3), consisting of GSK3α and GSK3ß subtypes, is a complex protein kinase that regulates numerous substrates. Research has observed increased GSK3 expression in the brains of Alzheimer's disease (AD) patients and models. AD is a neurodegenerative disorder with diverse pathogenesis and notable cognitive impairments, characterized by Aß aggregation and excessive tau phosphorylation. This article provides an overview of GSK3's structure and regulation, extensively analyzing its relationship with AD factors. GSK3 overactivation disrupts neural growth, development, and function. It directly promotes tau phosphorylation, regulates amyloid precursor protein (APP) cleavage, leading to Aß formation, and directly or indirectly triggers neuroinflammation and oxidative damage. We also summarize preclinical research highlighting the inhibition of GSK3 activity as a primary therapeutic approach for AD. Finally, pending issues like the lack of highly specific and affinity-driven GSK3 inhibitors, are raised and expected to be addressed in future research. In conclusion, GSK3 represents a target in AD treatment, filled with hope, challenges, opportunities, and obstacles.
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Enfermedad de Alzheimer , Glucógeno Sintasa Quinasa 3 , Animales , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/enzimología , Precursor de Proteína beta-Amiloide/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas tau/metabolismo , Proteínas tau/antagonistas & inhibidoresRESUMEN
Alzheimer's disease (AD) is a major cause of dementia and one of the most common chronic diseases affecting the aging population. Because AD is considered a public health priority, there is a critical need to discover novel and effective agents for the treatment of this condition. In view of the known contribution of up-regulated glutaminyl cyclase (QC) and glycogen synthase kinase-3ß (GSK-3ß) to the initiation of AD, we previously evaluated a series of dual inhibitors containing maleimide and imidazole motifs as potential anti-AD agents. Here, we assessed another series of hybrids containing maleimide and imidazole motifs to gain an in-depth understanding of the structure-activity relationship (SAR). Based on the primary screening, the introduction of 5-methyl imidazole at one side of the molecule did not enhance the QC-specific inhibitory activity of these hybrids (2, IC50 = 1.22 µM), although the potency was increased by 2' substitution on the maleimide motif at the other side of the molecule. Interestingly, compounds containing 5-methyl imidazole exhibited stronger GSK-3ß-specific inhibitory activity (2, IC50 = 0.0021 µM), and the electron-withdrawing group and 2' and 3' substitution were favorable. Further investigation of substitutions on the maleimide motif in compounds 14-35 revealed that QC-specific inhibition in the presence of piperidine was improved by introduction of a methoxy group (R2). Increasing the linker length and introduction of a methoxy group (R2) also increased the GSK-3ß-specific inhibitory potency. These findings were further confirmed by molecular docking analysis of 33 and 24 with QC and GSK-3ß. Overall, these hybrids exhibited enhanced inhibitory potency against both QC and GSK-3ß, highlighting an important strategy for improving the potency of hybrids as dual-targeting anti-AD agents.
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Aminoaciltransferasas , Glucógeno Sintasa Quinasa 3 beta , Imidazoles , Maleimidas , Relación Estructura-Actividad , Maleimidas/química , Maleimidas/farmacología , Maleimidas/síntesis química , Imidazoles/química , Imidazoles/farmacología , Imidazoles/síntesis química , Humanos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Estructura Molecular , Enfermedad de Alzheimer/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Relación Dosis-Respuesta a DrogaRESUMEN
Over the last six decades, lithium has been considered the gold standard treatment for the long-term management of bipolar disorder due to its efficacy in preventing both manic and depressive episodes as well as suicidal behaviors. Nevertheless, despite numerous observed effects on various cellular pathways and biologic systems, the precise mechanism through which lithium stabilizes mood remains elusive. Furthermore, there is recent support for the therapeutic potential of lithium in other brain diseases. This review offers a comprehensive examination of contemporary understanding and predominant theories concerning the diverse mechanisms underlying lithium's effects. These findings are based on investigations utilizing cellular and animal models of neurodegenerative and psychiatric disorders. Recent studies have provided additional support for the significance of glycogen synthase kinase-3 (GSK3) inhibition as a crucial mechanism. Furthermore, research has shed more light on the interconnections between GSK3-mediated neuroprotective, antioxidant, and neuroplasticity processes. Moreover, recent advancements in animal and human models have provided valuable insights into how lithium-induced modifications at the homeostatic synaptic plasticity level may play a pivotal role in its clinical effectiveness. We focused on findings from translational studies suggesting that lithium may interface with microRNA expression. Finally, we are exploring the repurposing potential of lithium beyond bipolar disorder. These recent findings on the therapeutic mechanisms of lithium have provided important clues toward developing predictive models of response to lithium treatment and identifying new biologic targets. SIGNIFICANCE STATEMENT: Lithium is the drug of choice for the treatment of bipolar disorder, but its mechanism of action in stabilizing mood remains elusive. This review presents the latest evidence on lithium's various mechanisms of action. Recent evidence has strengthened glycogen synthase kinase-3 (GSK3) inhibition, changes at the level of homeostatic synaptic plasticity, and regulation of microRNA expression as key mechanisms, providing an intriguing perspective that may help bridge the mechanistic gap between molecular functions and its clinical efficacy as a mood stabilizer.
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Compuestos de Litio , Humanos , Animales , Compuestos de Litio/farmacología , Compuestos de Litio/uso terapéutico , Antimaníacos/farmacología , Antimaníacos/uso terapéutico , Trastorno Bipolar/tratamiento farmacológico , Plasticidad Neuronal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidoresRESUMEN
Astrocytes and ependymal cells have been reported to be able to switch from a mature cell identity towards that of a neural stem/progenitor cell. Astrocytes are widely scattered in the brain where they exert multiple functions and are routinely targeted for in vitro and in vivo reprogramming. Ependymal cells serve more specialized functions, lining the ventricles and the central canal, and are multiciliated, epithelial-like cells that, in the spinal cord, act as bi-potent progenitors in response to injury. Here, we isolate or generate ependymal cells and post-mitotic astrocytes, respectively, from the lateral ventricles of the mouse brain and we investigate their capacity to reverse towards a progenitor-like identity in culture. Inhibition of the GSK3 and TGFß pathways facilitates the switch of mature astrocytes to Sox2-expressing, mitotic cells that generate oligodendrocytes. Although this medium allows for the expansion of quiescent NSCs, isolated from live rats by "milking of the brain", it does not fully reverse astrocytes towards the bona fide NSC identity; this is a failure correlated with a concomitant lack of neurogenic activity. Ependymal cells could be induced to enter mitosis either via exposure to neuraminidase-dependent stress or by culturing them in the presence of FGF2 and EGF. Overall, our data confirm that astrocytes and ependymal cells retain a high capacity to reverse to a progenitor identity and set up a simple and highly controlled platform for the elucidation of the molecular mechanisms that regulate this reversal.
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Astrocitos , Epéndimo , Fenotipo , Animales , Astrocitos/metabolismo , Astrocitos/citología , Epéndimo/citología , Epéndimo/metabolismo , Ratones , Células Cultivadas , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Diferenciación Celular , Encéfalo/citología , Encéfalo/metabolismo , Ratas , Factores de Transcripción SOXB1/metabolismo , Ratones Endogámicos C57BL , Mitosis , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Animales Recién NacidosRESUMEN
Epithelial ovarian cancer (EOC) is one of the most common malignant gynecological tumors with rapid growth potential and poor prognosis, however, the molecular mechanism underlying its outgrowth remained elusive. Germ cell-specific gene 2 (GSG2) was previously reported to be highly expressed in ovarian cancer and was essential for the growth of EOC. In this study, GSG2-knockdown cells and GSG2-overexpress cells were established through lentivirus-mediated transfection with Human ovarian cancer cells HO8910 and SKOV3. Knockdown of GSG2 inhibited cell proliferation and induced G2/M phase arrest in EOC. Interestingly, the expression of p27, a well-known regulator of the cell cycle showed a most significant increase after GSG2 knockdown. Further phosphorylation-protein array demonstrated the phosphorylation of GSK3αSer21 decreased in GSG2-knockdown cells to the most extent. Notably, inhibiting GSK3α activity effectively rescued GSG2 knockdown's suppression on cell cycle as well as p27 expression in EOC. Our study substantiates that GSG2 is able to phosphorylate GSK3α at Ser21 and then leads to the reduction of p27 expression, resulting in cell cycle acceleration and cell proliferation promotion. Thus, GSG2 may have the potential to become a promising target in EOC.
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Carcinoma Epitelial de Ovario , Ciclo Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Glucógeno Sintasa Quinasa 3 , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Ováricas , Proteínas Serina-Treonina Quinasas , Femenino , Humanos , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Fosforilación , Transducción de Señal , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
Trypanosomatids are an important group of parasites that predominate in tropical and subtropical areas of the planet, which cause diseases that are classified as forgotten and neglected by the world health organization. In this group of parasites, we find Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma brucei rhodesiense and Leishmania spp, for which there is no vaccine available, and its control has focused mainly on pharmacological treatment. Due to the poverty situation where these diseases are found and the biological complexity of these parasites, there are multiple variables to control, including the diversity of species, the complexity of their life cycles, drug resistance, cytotoxicity, the limited use in pregnant women, the high costs of treatment and the little-known pharmacological mechanisms of action, among others. It is therefore necessary to find new strategies and approaches for the treatment of these parasitic diseases. Among these new approaches is the rational search for new targets based on the allosteric inhibition of protein kinases, which have been little studied in trypanosomatids. Among these kinases, we find Glycogen Synthase Kinase-3 (GSK-3), a kinase of great pharmacological interest, which is under intense basic and clinical research by pharmaceutical companies for the treatment of cancer. This kinase, highly studied in the PI3K/AKT/mTOR pathway signaling in humans, has an orthologous gene in these parasites (GSK-3 s), which has proven to be essential for them in response to different challenges; Therefore, it is notable to increase research in this kinase in order to achieve a broad structural and functional characterization in the different species of trypanosomatids.
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Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Animales , Trypanosomatina/enzimología , Trypanosomatina/efectos de los fármacos , Trypanosomatina/genéticaRESUMEN
All cells and intracellular components are remodeled and recycled in order to replace the old and damaged cells. Autophagy is a process by which damaged, and unwanted cells are degraded in the lysosomes. There are three different types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Autophagy has an effect on adaptive and innate immunity, suppression of any tumour, and the elimination of various microbial pathogens. The process of autophagy has both positive and negative effects, and this pertains to any specific disease or its stage of progression. Autophagy involves various processes which are controlled by various signaling pathways, such as Jun N-terminal kinase, GSK3, ERK1, Leucine-rich repeat kinase 2, and PTEN-induced putative kinase 1 and parkin RBR E3. Protein kinases are also important for the regulation of autophagy as they regulate the process of autophagy either by activation or inhibition. The present review discusses the kinase catalyzed phosphorylated reactions, the kinase inhibitors, types of protein kinase inhibitors and their binding properties to protein kinase domains, the structures of active and inactive kinases, and the hydrophobic spine structures in active and inactive protein kinase domains. The intervention of autophagy by targeting specific kinases may form the mainstay of treatment of many diseases and lead the road to future drug discovery.
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Autofagia , Inhibidores de Proteínas Quinasas , Humanos , Autofagia/efectos de los fármacos , Autofagia/inmunología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Inmunidad Innata , Transducción de Señal , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Glycogen Synthase Kinase-3 (GSK-3) was recently implicated in the dysregulated biology of acute myeloid leukemia (AML). Low concentrations of GSK-3 inhibitors, SB216763 and BIO, suppressed the proliferation of AML cells with FLT3-ITD as early as 24 h after treatment. BIO was used in subsequent assays since it exhibited higher inhibitory effects than SB216763. BIO-induced G1 cell cycle arrest by regulating the expression of cyclin D2 and p21 in MV4-11 cells, and promoted apoptosis by regulating the cleaved-caspase3 signaling pathways. In vivo assays demonstrated that BIO suppressed tumor growth, while metabolomics assay showed that BIO reduced the levels of ATP and pyruvate in MV4-11 cells suggesting that it inhibited glycolysis. BIO markedly suppressed cell growth and induced apoptosis of AML cells with FLT3-ITD by partially inhibiting glycolysis, suggesting that BIO may be a promising therapeutic candidate for AML.
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Glucógeno Sintasa Quinasa 3 , Leucemia , Humanos , Proliferación Celular , Tirosina Quinasa 3 Similar a fms/genética , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Metabolómica , Línea Celular Tumoral , Puntos de Control del Ciclo CelularRESUMEN
BACKGROUND: Malignant pleural mesothelioma (MPM) is one of the most aggressive tumors with few effective treatments worldwide. It has been suggested that alternative splicing at the transcriptome level plays an indispensable role in MPM. METHODS: We analyzed the splicing profile of 84 MPM patients from the TCGA cohort by using seven typical splicing types. We classified MPM patients based on their splicing status and conducted a comprehensive analysis of the correlation between the splicing classification and clinical characteristics, genetic variation, pathway changes, immune heterogeneity, and potential therapeutic targets. RESULTS: The expression of the alternative splicing regulator SRPK1 is significantly higher in MPM tissues than in normal tissues, and correlates with poor survival. SRPK1 deficiency promotes MPM cell apoptosis and inhibits cell migration in vitro. We divided the MPM patients into four clusters based on their splicing profile and identified two clusters associated with the shortest (cluster 3) and longest (cluster 4) survival time. We present the different gene signatures of each cluster that are related to survival and splicing. Comprehensive analysis of data from the GDSC and TCGA databases revealed that cluster 3 MPM patients could respond well to the small-molecule inhibitor CHIR-99021, a small-molecule inhibitor of GSK-3. CONCLUSION: We performed unsupervised clustering of alternative splicing data from 84 MPM patients from the TCGA database and identified a cluster associated with the worst prognosis that was sensitive to a GSK-3 inhibitor.
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Neoplasias Pulmonares , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurales , Empalme Alternativo , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Neoplasias Pulmonares/patología , Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Mesotelioma/patología , Neoplasias Pleurales/tratamiento farmacológico , Neoplasias Pleurales/genética , Neoplasias Pleurales/patología , Proteínas Serina-Treonina QuinasasRESUMEN
ABSTRACTHIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the "Shock and Kill" strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream ß-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the ß-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3'-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.
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Linfocitos T CD4-Positivos/virología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , VIH-1/crecimiento & desarrollo , Indoles/farmacología , Oximas/farmacología , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Animales , Linfocitos T CD4-Positivos/inmunología , Línea Celular Tumoral , VIH-1/efectos de los fármacos , VIH-1/genética , Células HeLa , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Humanos , Macaca mulatta , Transcripción Genética/efectos de los fármacos , Células U937 , Activación Viral/genética , Latencia del Virus/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Merkel cell carcinoma is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). Since proliferation of MCPyV-positive MCC tumor cells strictly depends on expression of the virus-encoded T antigens (TA), these proteins theoretically represent ideal targets for different kinds of therapeutic approaches. Here we developed a cell-based assay to identify compounds which specifically inhibit growth of MCC cells by repressing TA expression. Applying this technique we screened a kinase inhibitor library and identified six compounds targeting glycogen synthase kinase 3 (GSK3) such as CHIR99021 as suppressors of TA transcription in MCC cells. Involvement of GSK3α and -ß in the regulation of TA-expression was confirmed by combining GSK3A knockout with inducible GSK3B shRNA knockdown since double knockouts could not be generated. Finally, we demonstrate that CHIR99021 exhibits in vivo antitumor activity in an MCC xenograft mouse model suggesting GSK3 inhibitors as potential therapeutics for the treatment of MCC in the future.
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Antígenos Virales de Tumores/genética , Carcinoma de Células de Merkel/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3/genética , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/patología , Carcinoma de Células de Merkel/virología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Poliomavirus de Células de Merkel/efectos de los fármacos , Poliomavirus de Células de Merkel/patogenicidad , Ratones , Piridinas/farmacología , Pirimidinas/farmacología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
Herein we present the evaluation of 11C-labeled-maleimides as radiotracers for positron emission tomography imaging of GSK-3 associated with Alzheimer's disease (AD). 3-Acetyl-4-(1-[11C]-methyl-1H-indol-3-yl)[1H]pyrrole-2,5-dione ([11C]-2) was obtained by direct methylation using [11C]-CH3I and Cs2CO3 in DMF with a 31 ± 4% radiochemical yield and a radiochemical purity of 97.7 ± 0.8%. [11C]-2 was stable both in its final formulation and in human plasma for 120 min and had a plasma protein binding of 70 ± 1% and a LogD7.4 value of 1.84 ± 0.04. [11C]-2 ex vivo biodistributions in healthy animals demonstrated significant brain uptake and retention, showing its ability to penetrate the intact blood-brain barrier. In vivo PET imaging in mice bearing AD showed, with respect to normal animals, significant differences in uptake in the hypothalamus, the striatum, and the amygdala and a significant increase in amygdala uptake in later stages of the pathology. These results are very promising, and further studies are being performed for a complete validation of this compound as novel tracer for AD.
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Enfermedad de Alzheimer/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Radioisótopos de Carbono/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/química , Tomografía de Emisión de Positrones/métodos , Inhibidores de Proteínas Quinasas/farmacología , Enfermedad de Alzheimer/diagnóstico por imagen , Animales , Encéfalo/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Prueba de Estudio Conceptual , Radioquímica , Radiofármacos/farmacologíaRESUMEN
Small-molecule drugs targeting glycogen synthase kinase 3 (GSK3) as inhibitors of the protein kinase activity are able to stimulate reparative dentine formation. To develop this approach into a viable clinical treatment for exposed pulp lesions, we synthesized a novel, small-molecule noncompetitive adenosine triphosphate (ATP) drug that can be incorporated into a biodegradable hydrogel for placement by syringe into the tooth. This new drug, named NP928, belongs to the thiadiazolidinone (TDZD) family and has equivalent activity to similar drugs of this family such as tideglusib. However, NP928 is more water soluble than other TDZD drugs, making it more suitable for direct delivery into pulp lesions. We have previously reported that biodegradable marine collagen sponges can successfully deliver TDZD drugs to pulp lesions, but this involves in-theater preparation of the material, which is not ideal in a clinical context. To improve surgical handling and delivery, here we incorporated NP928 into a specifically tailored hydrogel that can be placed by syringe into a damaged tooth. This hydrogel is based on biodegradable hyaluronic acid and can be gelled in situ upon dental blue light exposure, similarly to other common dental materials. NP928 released from hyaluronic acid-based hydrogels upregulated Wnt/ß-catenin activity in pulp stem cells and fostered reparative dentine formation compared to marine collagen sponges delivering equivalent concentrations of NP928. This drug-hydrogel combination has the potential to be rapidly developed into a therapeutic procedure that is amenable to general dental practice.
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Dentina Secundaria , Dentinogénesis , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Tiadiazoles/farmacología , Pulpa Dental , Dentinogénesis/efectos de los fármacos , Humanos , HidrogelesRESUMEN
INTRODUCTION: GSK-3 is an immune regulator that plays a role in the modulation of cytokine-producing effector T cells associated with inflammation and demyelination of the CNS in EAE. OBJECTIVE: This study aimed to evaluate the treatment paradigm of a single dose of GSK-3 inhibitor administration at various time courses for the protection of the CNS from EAE. MATERIALS AND METHODS: Effects of GSK-3 inhibition on intracellular cytokine levels were evaluated from in vitro naïve CD4+ T cell cultures. Immunized C57BL/6 female mice with MOG35-55 in conjunction with CFA and Ptx were used as a chronic inflammatory EAE disease model. Tideglusib (NP12), a Thiadiazolidinone class, selective, and non-ATP competitive GSK-3 inhibitor, was injected intraperitoneally at pre-EAE, same-day of immunization or disease onset. After 30 days post-immunization, brain, and spinal cord tissues were collected for inflammation and demyelination analysis by H&E and luxol fast blue staining, respectively, whereas cytokine profiles of the serum were assessed by cytokine beads array. RESULTS: The inhibition of GSK-3 in CD4+ T cells increased IL-10 production. The administration of Tideglusib during pre-EAE and same-day, but not during disease onset, significantly reduced clinical symptoms and delayed disease onset. Histopathological analysis of spinal cord tissues showed a significant decline in the number of inflammatory cell infiltration with a concomitant reduction in demyelination through the blocking of GSK-3, especially during pre-EAE and sameday. Upregulation of IL-10 via GSK-3 inhibition coincided with the downregulation of cytokineassociated effector T cells, including IFN-γ, IL-9, IL-17A, IL-17F, IL-21, and IL-23. Increased IL-4 production, however, was only significant in the pre-EAE group. CONCLUSION: The neuroprotective effects of Tideglusib against EAE are time-dependent. Downregulation of Th1 and Th17 hallmark cytokines by Tideglusib in EAE may be associated with IL-10 production.
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Encefalomielitis Autoinmune Experimental , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Animales , Linfocitos T CD4-Positivos/patología , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Femenino , Glucógeno Sintasa Quinasa 3 beta , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores , Células Th17/patología , Tiadiazoles/farmacología , Factores de TiempoRESUMEN
The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35-0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.
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COVID-19/prevención & control , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Compuestos de Litio/uso terapéutico , Adulto , Anciano , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Células HEK293 , Humanos , Compuestos de Litio/farmacología , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Estudios RetrospectivosRESUMEN
GSK3 is a promising target for the treatment of Alzheimer's disease. Here, we describe the design and synthesize of a series of GSK3 degraders based on a click chemistry platform. A series of highly potent GSK3 degraders were obtained. Among them, PT-65 exhibited most potent degradation potency against GSK3α (DC50 = 28.3 nM) and GSK3ß (DC50 = 34.2 nM) in SH-SY5Y cells. SPR assay confirmed that PT-65 binds to GSK3ß with high affinity (KD = 12.41 nM). The proteomic study indicated that PT-65 could selectively induced GSK3 degradation. Moreover, PT-65 could effectively suppress GSK3ß and Aß mediated tau hyperphosphorylation in a dose-dependent manner and protect SH-SY5Y cells from Aß caused cell damage. We also confirmed that PT-65 could suppress OA induced tau hyperphosphorylation and ameliorate learning and memory impairments in vivo model of AD. In summary, PT-65 might be a promising candidate for the treatment of AD.
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Enfermedad de Alzheimer/tratamiento farmacológico , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Proteolisis/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.