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Aberrant expression of MEG3 has been shown in various cancers. The purpose of this study is to evaluate the effect of MEG3 on glioma cells and the use of potential chemotherapeutics in glioma by modulating MEG3 expression. Cell viability, migration and chemosensitivity were assayed. Cell death was evaluated in MEG3 overexpressing and MEG3 suppressed cells. MEG3 expression was compared in patient-derived glioma cells concerning IDH1 mutation and WHO grades. Silencing of MEG3 inhibited cell proliferation and reduced cell migration while overexpression of MEG3 promoted proliferation in glioma cells. MEG3 inhibition improved the chemosensitivity of glioma cells to 5-fluorouracil (5FU) but not to navitoclax. On the other hand, there is no significant effect of MEG3 expression on temozolamide (TMZ) treatment which is a standard chemotherapeutic agent in glioma. Suppression of the MEG3 gene in patient-derived oligodendroglioma cells also showed the same effect whereas glioblastoma cell proliferation and chemosensitivity were not affected by MEG3 inhibition. Further, as a possible cell death mechanism of action apoptosis was investigated. Although MEG3 is a widely known tumour suppressor gene and its loss is associated with several cancer types, here we reported that MEG3 inhibition can be used for improving the efficiency of known chemotherapeutic drug sensitivity. We propose that the level of MEG3 should be evaluated in the treatment of different glioma subtypes that are resistant to effective drugs to increase the potential effective drug applications.
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Glioma , RNA Longo não Codificante , Humanos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , RNA Longo não Codificante/genéticaRESUMO
Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, and virus neutralization assay using xCELLigence MP real-time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of montelukast both on the main protease enzyme inhibition and virus entry into the host cell (spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with montelukast for 20 h on the infected cells. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and, if its effect is proved in clinical phase studies, it should be used against coronavirus disease 2019 (COVID-19).
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Acetatos/farmacologia , Enzima de Conversão de Angiotensina 2/metabolismo , Ciclopropanos/farmacologia , Quinolinas/farmacologia , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Sulfetos/farmacologia , Células A549 , Acetatos/química , Enzima de Conversão de Angiotensina 2/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Ciclopropanos/química , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Neutralização , Conformação Proteica , Quinolinas/química , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/química , Sulfetos/química , Células Vero , Internalização do Vírus/efeitos dos fármacosRESUMO
Glycopolymers are synthetic macromolecules having pendant sugar moieties and widely utilized to target cancer cells. They are usually considered as a hydrophilic segment of amphiphilic block copolymers to fabricate micelles as drug carriers. A novel amphiphilic block copolymer, namely, poly(2-deoxy-2-methacrylamido-d-glucose-co-2-hydroxyethyl methacrylate)-b-poly(ß-amino ester) [P(MAG-co-HEMA)-b-PBAE], with active cancer cell targeting potential and pH responsivity was prepared. Tetrazine end functional P(MAG-co-HEMA) and norbornene end functional PBAE blocks were separately synthesized through reversible addition fragmentation chain transfer polymerization and Michael addition-based poly-condensation, respectively, and followed by end-group transformation. Then, inverse electron demand Diels Alder reaction between the tetrazine and the norbornene groups was performed by simply mixing to obtain the amphiphilic block copolymer. After characterization of the block copolymer in terms of chemical structure, pH responsivity, and drug loading/releasing, pH-responsive micelles were obtained with or without doxorubicin (DOX), a model anticancer drug. The micelles exhibited a sharp protonated/deprotonated transition on tertiary amine groups around pH 6.75 and the pH-specific release of DOX below this value. Eventually, the drug delivery potential was evaluated by cytotoxicity assays on both the noncancerous human umbilical vein endothelial cell (HUVEC) cell line and glioblastoma cell line, U87-MG. While the DOX-loaded polymeric micelles were not toxic in noncancerous HUVEC cells, being toxic only to the cancer cells indicates that it is a potential specific cell targeting strategy in the treatment of cancer.
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Portadores de Fármacos , Micelas , Humanos , Portadores de Fármacos/química , Ésteres , Polietilenoglicóis/química , Concentração de Íons de Hidrogênio , Doxorrubicina/química , NorbornanosRESUMO
BACKGROUND: Glioma is the most common type of brain tumors and isocitrate dehydrogenase (IDH1) gene is the most prominent molecular marker about the disease prognosis, response to therapy and patient survival. There are conflicting data about the effect of IDH1 mutation on glial cell proliferation, invasion and migration characteristics. The effect of IDH1 mutation on mTOR signaling pathway, which has key roles in tumorigenesis process, is limited and previous data is controversial. We aimed to explore the effect of wild type and mutant IDH1 overexpression on glioma cells and investigated the correlation with mTOR signaling pathway associated genes. METHODS AND RESULTS: U87-MG and A172 cells were transfected with different IDH1 mutant gene overexpressing (R132H, R132L, R132S, R132C) viral vectors. Cell proliferation, cell invasion and migration analysis as well as quantitative PCR analysis with the mutant glioma cell lines were performed. Forty-two patient derived glioma cells were obtained from patients with different glioma subtypes and cancer cells were enriched by culturing cells. Overexpression of both mutant and wild type IDH1 gene promoted the cell proliferation, but only IDH1 mutation increased cell invasion and migration. The expression of IDH1 mutation activated mTOR signaling via upregulation of WNTA, PRKAA2, GSK3B and MTOR genes as well as phosphorylated mTOR protein level. CONCLUSIONS: Our results highlighted IDH1 mutation upregulate mTOR signaling pathway and promote cell proliferation, invasion and migration.
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Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioma/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mutação/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
INTRODUCTION: Numerous efforts in natural product drug development are reported for the treatment of Coronavirus. Based on the literature, among these natural plants Artemisia annua L. shows some promise for the treatment of SARS-CoV-2. OBJECTIVE: The main objective of our study was to determine artemisinin content by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS), to investigate the in vitro biological activity of artemisinin from the A. annua plants grown in Turkey with various extracted methods, to elaborate in silico activity against SARS-CoV-2 using molecular modelling. METHODOLOGY: Twenty-one different extractions were applied. Direct and sequential extractions studies were compared with ultrasonic assisted maceration, Soxhlet, and ultra-rapid determined artemisinin active molecules by LC-ESI-MS/MS methods. The inhibition of spike protein and main protease (3CL) enzyme activity of SARS-CoV-2 virus was assessed by time resolved fluorescence energy transfer (TR-FRET) assay. RESULTS: Artemisinin content in the range 0.062-0.066%. Artemisinin showed significant inhibition of 3CL protease activity but not Spike/ACE-2 binding. The 50% effective concentration (EC50 ) of artemisinin against SARS-CoV-2 Spike pseudovirus was found greater than 50 µM (EC45 ) in HEK293T cell line whereas the cell viability was 94% of the control (P < 0.01). The immunosuppressive effects of artemisinin on TNF-α production on both pseudovirus and lipopolysaccharide (LPS)-induced THP-1 cells were found significant in a dose dependent manner. CONCLUSION: Further studies of these extracts for COVID-19 treatment will shed light to seek alternative treatment options. Moreover, these natural extracts can be used as an additional treatment option with medicines, as well as prophylactic use can be very beneficial for patients.
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Artemisia annua , Artemisininas , Tratamento Farmacológico da COVID-19 , Antivirais/farmacologia , Artemisia annua/química , Artemisininas/farmacologia , Cromatografia Líquida , Células HEK293 , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , SARS-CoV-2 , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: To identify a biomarker distinguishing patients who, despite a primary progressive multiple sclerosis (PPMS) clinical course, may nonetheless benefit from immune therapy. METHODS: The presence or absence of both immunoglobulin (Ig) G and IgM oligoclonal bands (OCB) was blindly examined in paired cerebrospinal fluid (CSF) and serum samples from a large PPMS patient cohort, and related to clinical and imaging evidence of focal inflammatory disease activity. RESULTS: Using both cross-sectional samples and serial sampling in a subgroup of patients followed prospectively as part of the placebo-controlled OLYMPUS study of rituximab in PPMS, we found that the presence of CSF-restricted IgM OCB (but not of IgG OCB) is associated with an active inflammatory disease phenotype in PPMS patients. This finding was confirmed in an independent, multicenter validation cohort. INTERPRETATION: The presence of CSF IgM OCB may be a biomarker for a subset of PPMS patients with more active inflammatory disease, who may benefit from immune-directed treatments.
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Imunoglobulina M/líquido cefalorraquidiano , Esclerose Múltipla Crônica Progressiva/imunologia , Bandas Oligoclonais/líquido cefalorraquidiano , Adulto , Biomarcadores/líquido cefalorraquidiano , Estudos Transversais , Feminino , Humanos , Inflamação/líquido cefalorraquidiano , Inflamação/imunologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/líquido cefalorraquidiano , FenótipoRESUMO
INTRODUCTION: Peritumoral brain edema (PTBE) has been widely reported with many brain tumors, especially with glioma. Since the blood-brain barrier (BBB) is essential for maintaining minimal permeability, any alteration in the interaction of BBB components, specifically in astrocytes and tight junctions (TJ), can result in disrupting the homeostasis of the BBB and making it severely leaky, which subsequently generates edema. OBJECTIVE: This study aimed to evaluate the functional gliovascular unit of the BBB by examining changes in the expression of claudin (CLDN) genes and the expression of transient receptor potential (TRP) membrane channels, additionally to define the correlation between their expressions. The evaluation was conducted using in vitro spheroid swelling models and tumor samples from glioma patients with PTBE. RESULTS: The results of the spheroid model showed that the genes TRPC3, TRPC4, TRPC5, and TRPV1 were upregulated in glioma cells either wild-type isocitrate dehydrogenase 1 (IDH1) or the IDH1 R132H mutant, with or without NaCl treatment. Furthermore, TRP genes appeared to adversely correlate with the up regulation of CLDN1, CLDN3, and CLDN5 genes. Besides, the upregulation of TRPC1 and TRPC4 in IDH1mt-R132H glioma cells. On the other hand, the correlation analysis revealed different correlations between different proteins in PTBE. CLDN1 exhibits a slight positive correlation with CLDN3. Similarly, TRPV1 displays a slight positive correlation with TRPC1. In contrast, TRPC4 shows a slight negative correlation with TRPC5. On the other hand, TRPC3 demonstrates a slight positive correlation with TRPC5, while the non-PTBE analysis highlights a moderate positive correlation between CLDN1 and TRPM4 while CLDN3 exhibits a moderate negative correlation with TRPC4. Additionally, CLDN5 demonstrates a slight negative correlation with TRPC4 but a moderate positive correlation with TRPC3. Furthermore, TRPC1 have a slight negative correlation with TRPV1, TRPC3 exhibiting a slight positive correlation with TRPC4, and TRPV1 showing a slight negative correlation with TRPC5. CONCLUSION: As a conclusion, the current study provided evidence of a slight negative correlation between TRPs and CLDN gene expression in PTBE patients and confirmatory results with some of the genes in cell model of edema.
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Edema Encefálico , Neoplasias Encefálicas , Claudina-5 , Glioma , Humanos , Edema Encefálico/genética , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Claudina-5/genética , Claudina-5/metabolismo , Regulação Neoplásica da Expressão Gênica , Claudina-3/genética , Claudina-3/metabolismo , Barreira Hematoencefálica/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Linhagem Celular Tumoral , Claudina-1/genética , Claudina-1/metabolismo , Claudinas/genética , Claudinas/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , MasculinoRESUMO
Poly(lactic-co-glycolic acid) microspheres loaded with imatinib mesylate has been developed as a new therapeutic strategy to prevent craniopharyngioma recurrence. Microspheres composed of different lactic/glycolic acid ratios, molecular weights and drug compositions were synthesized and loaded with imatinib mesylate by modified double-emulsion/solvent evaporation technique and subsequently characterized by particle-size distribution, scanning electron microscopy, encapsulation efficiency and in vitro drug release. Inhibitory potential of imatinib containing microspheres on tumor neovascularization was investigated on craniopharyngioma tumor samples by rat cornea angiogenesis assay. Results showed that microspheres in different LA:GA ratios [LA:GA 50:50 (G50), 75:25 (G25), 85:15 (G15)] considerably reduced neovascularization induced by recurrent tumor samples in an in vivo angiogenesis assay (P < 0.01). Our data indicate that local delivery of imatinib mesylate to the post-surgical tumoral cavity using biodegradable microspheres may be a promising biologically selective approach to prevent the recurrence of craniopharyngiomas, via inhibition of neovascularization.
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Antineoplásicos/administração & dosagem , Benzamidas/administração & dosagem , Craniofaringioma/irrigação sanguínea , Ácido Láctico , Microesferas , Neovascularização Patológica/prevenção & controle , Piperazinas/administração & dosagem , Ácido Poliglicólico , Pirimidinas/administração & dosagem , Cromatografia Líquida de Alta Pressão , Mesilato de Imatinib , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Espectrofotometria UltravioletaRESUMO
BACKGROUND: Predicting the long-term disability outcomes of multiple sclerosis (MS) cases is challenging. OBJECTIVE: We prospectively analysed our previous MS cohort with initial cerebrospinal fluid (CSF) proteomics data to reveal disability markers after 8.2±2.2 years of follow-up. METHODS: Patients with regular follow-up visits were assigned into two groups: those with an age-related MS severity (ARMSS) score ≥5 (unfavourable course group, N = 27) and ARMSS score <5 (favourable course group, N = 67). A machine learning-based algorithm was applied to reveal candidate poor prognosis-associated initial CSF proteins, which were measured in an independent MS cohort (verification group, N = 40) by ELISA. Additionally, the correlation of initial clinical and radiological parameters with long-term disability was analysed. RESULTS: CSF alpha-2-macroglobulin (P = 0.0015), apo-A1 (P = 0.0016), and haptoglobin (P = 0.0003) protein levels, as well as cerebral lesion load (>9 lesions) on magnetic resonance imaging, gait disturbance (P = 0.04), and bladder/bowel symptoms (P = 0.01) were significantly higher in the unfavourable course group than in the favourable course group. Optic nerve involvement evident on initial magnetic resonance imaging (P = 0.002) and optic neuritis (P = 0.01) were more frequent in the favourable course group. CONCLUSION: The herein identified initial CSF protein levels, in addition to the clinical and radiological parameters at disease onset, have predictive value for long-term disability in MS cases.
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Esclerose Múltipla , Neurite Óptica , Humanos , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/líquido cefalorraquidiano , Prognóstico , Estudos Prospectivos , Neurite Óptica/patologia , Nervo Óptico/patologia , Imageamento por Ressonância Magnética/métodos , Progressão da DoençaRESUMO
BACKGROUND: The importance of COVID-19 vaccination for patients on immunosuppressive (IS) medication has increased due to the high risk of severe disease or mortality. Different vaccines have varying efficacy rates against symptomatic COVID-19, ranging from 46.8% to 95%. The objective of this study was to examine the differences in anti-Spike IgG, anti-Spike IgA, and neutralizing antibody (NAb) activity between the inactive CoronaVac vaccine and the mRNA-based BNT162b2 vaccine in IS patients. METHOD: A total of 441 volunteers, including 104 IS patients, 263 healthy controls (HC), who received two doses of CoronaVac or BNT162b2, and 74 unvaccinated patients with a history of SARS-CoV-2 infection, were included in the study. Anti-spike IgG, IgA, and NAb activity were investigated. RESULTS: Immunogenicity with BNT162b2 was higher than with CoronaVac, but in IS groups, it was lower than HC (CoronaVac-IS: 79.3%, CoronaVac-HC: 96.5%, p < 0.001; BNT162b2-IS: 91.3%, BNT162b2-HC: 100%, p = 0.005). With CoronaVac, anti-Spike IgG levels were significantly lower than BNT162b2 (CoronaVac-IS: 234.5AU/mL, CoronaVac-HC: 457.85AU/mL; BNT162b2-IS: 5311.2AU/mL, BNT162b2-HC: 8842.8AU/mL). NAb activity in the BNT162b2 group was significantly higher. NAb and anti-Spike IgG levels were found to be correlated. Among the IS group, a significantly lower response to the vaccines was observed when using rituximab. IgA levels were found to be lower with CoronaVac. CONCLUSIONS: Although immunogenicity was lower in IS patients, an acceptable response was obtained with both vaccines, and significantly higher anti-Spike IgG, anti-Spike IgA, and NAb activity levels were obtained with BNT162b2.
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Hedgehog signaling mediates embryologic development of the central nervous system and other tissues and is frequently hijacked by neoplasia to facilitate uncontrolled cellular proliferation. Meningiomas, the most common primary brain tumor, exhibit Hedgehog signaling activation in 6.5% of cases, triggered by recurrent mutations in pathway mediators such as SMO. In this study, we find 35.6% of meningiomas that lack previously known drivers acquired various types of somatic structural variations affecting chromosomes 2q35 and 7q36.3. These cases exhibit ectopic expression of Hedgehog ligands, IHH and SHH, respectively, resulting in Hedgehog signaling activation. Recurrent tandem duplications involving IHH permit de novo chromatin interactions between super-enhancers within DIRC3 and a locus containing IHH. Our work expands the landscape of meningioma molecular drivers and demonstrates enhancer hijacking of Hedgehog ligands as a route to activate this pathway in neoplasia.
Assuntos
Neoplasias Meníngeas , Meningioma , Humanos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meningioma/genética , Ligantes , Transdução de Sinais , Neoplasias Meníngeas/genéticaRESUMO
BACKGROUND: The complex pathogenesis of multiple sclerosis, combined with an unpredictable prognosis, requires identification of disease-specific diagnostic and prognostic biomarkers. OBJECTIVE: To determine whether inflammatory proteins, such as neurofilament light chain, myelin oligodendrocyte glycoprotein and myelin basic protein, and neurodegenerative proteins, such as tau and glial fibrillary acidic protein, can serve as biomarkers for predicting the clinical subtype and prognosis of MS. METHODS: Cerebrospinal fluid and serum samples were collected from patients with a diagnosis of clinically isolated syndrome (n = 46), relapsing-remitting MS (n = 67) or primary-progressive MS (n = 22) along with controls having other non-inflammatory neurological disease (n = 22). Western blot analyses were performed for the listed proteins. Protein levels were compared among different clinical subtypes using one-way analysis of variance analysis. The k-nearest neighbour algorithm was further used to assess the predictive use of these proteins for clinical subtype classification. RESULTS: The results showed that each of tau, GFAP, MOG and NFL protein concentrations differed significantly (p < 0.001) in multiple sclerosis clinical subtypes compared with the controls. Levels of the proteins also differed between the multiple sclerosis clinical subtypes, which may be associated with the underlying disease process. Classification studies revealed that these proteins might be useful for identifying multiple sclerosis clinical subtypes. CONCLUSIONS: We showed that select biomarkers may have potential in identifying multiple sclerosis clinical subtypes. We also showed that the predictive value of the prognosis increased when using a combination of the proteins versus using them individually.
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Doenças Desmielinizantes/diagnóstico , Esclerose Múltipla Crônica Progressiva/diagnóstico , Esclerose Múltipla Recidivante-Remitente/diagnóstico , Adulto , Análise de Variância , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Western Blotting , Estudos de Casos e Controles , Proteínas do Líquido Cefalorraquidiano/sangue , Doenças Desmielinizantes/sangue , Doenças Desmielinizantes/líquido cefalorraquidiano , Diagnóstico Diferencial , Proteína Glial Fibrilar Ácida/sangue , Proteína Glial Fibrilar Ácida/líquido cefalorraquidiano , Humanos , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/sangue , Esclerose Múltipla Crônica Progressiva/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Glicoproteína Mielina-Oligodendrócito/sangue , Glicoproteína Mielina-Oligodendrócito/líquido cefalorraquidiano , Proteínas de Neurofilamentos/sangue , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Turquia , Adulto Jovem , Proteínas tau/sangue , Proteínas tau/líquido cefalorraquidianoRESUMO
AIM: To investigate the angiogenic effects of bevacizumab and imatinib on different meningioma tissue grades. MATERIAL AND METHODS: In this study, in silico analysis of angiogenesis-related gene expression was carried out using previously reported datasets. Messenger ribonucleic acid expressions of VEGFA, VEGFB, PDGFRA, and PDGFRB genes were obtained from two different meningioma transcriptome datasets. The effect of antiangiogenic drugs, bevacizumab and imatinib, on meningiomainduced vascularization was assessed by using rat corneal angiogenesis assay (CAA). RESULTS: Bevacizumab and imatinib both significantly reduced meningioma-induced neovascularization in the CAA model. CONCLUSION: The angiogenic characteristics of meningiomas may be suppressed by using antiangiogenic drugs to prevent neovascularization, thus improving prognosis.
Assuntos
Neovascularização da Córnea , Neoplasias Meníngeas , Meningioma , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Neovascularização da Córnea/tratamento farmacológico , Mesilato de Imatinib/uso terapêutico , Neoplasias Meníngeas/tratamento farmacológico , Neoplasias Meníngeas/metabolismo , Meningioma/tratamento farmacológico , Meningioma/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , RNA/uso terapêutico , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/uso terapêuticoRESUMO
The COVID-19 outbreak caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to have high incidence and mortality rate globally. To meet the increasingly growing demand for new therapeutic drugs and vaccines, researchers are developing different diagnostic techniques focused on screening new drugs in clinical use, developing an antibody targeting a SARS-CoV-2 receptor, or interrupting infection/replication mechanisms of SARS-CoV-2. Although many prestigious research publications are addressing this subject, there is no open access platform where all experimental techniques for COVID-19 research can be seen as a whole. Many researchers have accelerated the development of in silico methods, high-throughput screening techniques, and in vitro assays. This development has played an important role in the emergence of improved, innovative strategies, including different antiviral drug development, new drug discovery protocols, combinations of approved drugs, and setting up new drug classes during the COVID-19 outbreak. Hence, the present review discusses the current literature on these modalities, including virtual in silico methods for instant ligand- and target-driven based techniques, nucleic acid amplification tests, and in vitro models based on sensitive cell cultures, tissue equivalents, organoids, and SARS-CoV-2 neutralization systems (lentiviral pseudotype, viral isolates, etc.). This pack of complementary tests informs researchers about the accurate, most relevant emerging techniques available and in vitro assays allow them to understand their strengths and limitations. This review could be a pioneer reference guide for the development of logical algorithmic approaches for new drugs and vaccine strategies against COVID-19.
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Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , Técnicas de Cultura de Células , Ensaios de Triagem em Larga Escala/métodos , Humanos , LigantesRESUMO
Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma (GBM), the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall GBM cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in GBM A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. Our results indicate that BAU-243 activates autophagic cell death by disrupting the Beclin 1:Bcl-2 complex and may serve as a potential small molecule for treating GBM.
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Caulerpa cylindracea Sonder is a green alga belonging to the Caulerpaceae family. This is the first chemical investigation of C. cylindracea in the Dardanelles which resulted in the isolation of four compounds, caulerpin (1), monomethyl caulerpinate (2), beta-sitosterol (3), and palmitic acid (4). Their structures were elucidated by spectroscopic analyses including 1D- and 2D NMR and mass. The isolated compounds 1 and 2 were tested against the SARS-CoV-2 viral targets spike protein and main protease (3CL) enzyme, and both compounds significantly inhibit the interaction of spike protein and ACE2, while the main protease activity was not significantly reduced. Docking studies suggested that compounds 1 and 2 may bind to the ACE2 binding pocket on spike, and compound 2 may also bind to an allosteric site on spike. As such, these compounds may inhibit the spike-ACE2 complex formation competitively and/or allosterically and have the potential to be used against SARS-CoV-2 virus infection. In addition, compounds 1 and 2 showed at least two-fold higher cytotoxicity against breast cancer cell lines MCF7 and MDA-MB-231 compared to the CCD fibroblast control cell line.
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In the current study, we used 7922 FDA approved small molecule drugs as well as compounds in clinical investigation from NIH's NPC database in our drug repurposing study. SARS-CoV-2 main protease as well as Spike protein/ACE2 targets were used in virtual screening and top-100 compounds from each docking simulations were considered initially in short molecular dynamics (MD) simulations and their average binding energies were calculated by MM/GBSA method. Promising hit compounds selected based on average MM/GBSA scores were then used in long MD simulations. Based on these numerical calculations following compounds were found as hit inhibitors for the SARS-CoV-2 main protease: Pinokalant, terlakiren, ritonavir, cefotiam, telinavir, rotigaptide, and cefpiramide. In addition, following 3 compounds were identified as inhibitors for Spike/ACE2: Denopamine, bometolol, and rotigaptide. In order to verify the predictions of in silico analyses, 4 compounds (ritonavir, rotigaptide, cefotiam, and cefpiramide) for the main protease and 2 compounds (rotigaptide and denopamine) for the Spike/ACE2 interactions were tested by inâ vitro experiments. While the concentration-dependent inhibition of the ritonavir, rotigaptide, and cefotiam was observed for the main protease; denopamine was effective at the inhibition of Spike/ACE2 binding.
Assuntos
Antivirais , Reposicionamento de Medicamentos , Drogas em Investigação/farmacologia , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2 , Antivirais/farmacologia , Cefotiam/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Ritonavir/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Tratamento Farmacológico da COVID-19RESUMO
Cuprizone, copper chelator, treatment of mouse is a toxic model of multiple sclerosis (MS) in which oligodendrocyte death, demyelination and remyelination can be observed. Understanding T and B cell subset as well as their cytokines involved in MS pathogenesis still requires further scrutiny to better understand immune component of MS. The study presented here, aimed to evaluate relevant cytokines, lymphocytes, and gene expressions profiles during demyelination and remyelination in the cuprizone mouse model of MS. Eighty male C57BL/6J mice fed with 0.2% cuprizone for eight weeks. Cuprizone has been removed from the diet in the following eight weeks. Cuprizone treated and control mice sacrificed biweekly, and corpus callosum of the brain was investigated by staining. Lymphocyte cells of mice analyzed by flow cytometry with CD3e, CD11b, CD19, CD80, CD86, CD4, CD25 and FOXP3 antibodies. IFN-gamma, IL-1alpha, IL-2, IL-5, IL-6, IL-10, IL-17, TNF-alpha cytokines were analyzed in plasma samples. Neuregulin 1 (Nrg1), ciliary neurotrophic factor (Cntf) and C-X-C chemokine receptor type 4 (Cxcr4) gene expressions in corpus callosum sections of the mice brain were quantified. Histochemistry analysis showed that demyelination began at the fourth week of cuprizone administration and total demyelination occurred at the twelfth week in chronic model. Remyelination occurred at the fourth week of following withdrawal of cuprizone from diet. The level of mature and activated T cells, regulatory T cells, T helper cells and mature B cells increased during demyelination and decreased when cuprizone removed from diet. Further, both type 1 and type 2 cytokines together with the proinflammatory cytokines increased. The level of oligodendrocyte maturation and survival genes showed differential gene expression in parallel to that of demyelination and remyelination. In conclusion, for the first-time, involvement of both cellular immune response and antibody response as well as oligodendrocyte maturation and survival factors having role in demyelination and remyelination of cuprizone mouse model of MS have been shown.
RESUMO
B-Cell lymphoma 2 (BCL-2) regulates cell death in humans. In this study, combined multiscale in silico approaches and in vitro studies were employed. A small-molecule library that includes more than 210â¯000 compounds was used. The predicted therapeutic activity value (TAV) of the compounds in this library was computed with the binary cancer quantitative structure-activity relationships (QSAR) model. The molecules with a high calculated TAV were used in 26 individual toxicity QSAR models. As a result of this screening protocol, 288 nontoxic molecules with high predicted TAV were identified. These selected hits were then screened against the BCL-2 target protein using hybrid docking and molecular dynamics (MD) simulations. The interaction energies of identified compounds were compared with two known BCL-2 inhibitors. Then, the short MD simulations were carried out by initiating the best docking poses of 288 molecules. Average MM/GBSA energies were computed, and long MD simulations were employed to selected hits. The same calculations were also applied for two known BCL-2 inhibitors. Moreover, a five-site (AHRRR) structure-based pharmacophore model was constructed, and this model was used in the screening of the same database. On the basis of hybrid data-driven ligand identification study, final hits were selected and used in in vitro studies. Based on results of the time-resolved fluorescence resonance energy transfer (TR-FRET) analysis, further filtration was carried out for the U87-MG cell line tests. MTT cell proliferation assay analysis results showed that selected three potent compounds were significantly effective on glioma cells.
RESUMO
AIM: To elucidate the association of the MTHFR, MTRR, and RAD54L gene variations with meningioma in Turkish cohort. MATERIAL AND METHODS: DNAs were isolated from 87 retrospective meningioma samples. The MTHFR, MTRR, and RAD54L gene hotspot regions were amplified with specific primers via polymerase chain reaction (PCR), and next-generation sequencing (NGS) was performed. All the detected variations and single-nucleotide polymorphisms (SNPs) were listed and compared with healthy control frequencies in different genomic databases. The histopathological characteristics of meningiomas and genomic variations were compared. Pearson?s chi-squared test was used to detect the statistical differences of SNPs, and correlation analysis was conducted. RESULTS: rs1801131, rs1801133, and rs4846051 on MTHFR, rs1801394 on MTRR, and rs1048771 on RAD54L gene frequencies were found to be significantly altered in the overall cohort of 87 patients with meningioma. The frequency of rs18011031 is 0.09 in the meningioma cohort, which is significantly correlated with WHO tumor grades (p = 0.038). The frequency of rs18011033 is 0.29 in the meningioma cohort, which is significantly correlated with WHO tumor grades (p = 0.045). Furthermore, the frequency of rs4846051 is 0.18 in the meningioma cohort, which is significantly correlated with WHO tumor grades (p = 0.023) and also with low Ki67 proliferation index (p = 0.00455). The frequency of rs1801394 is 0.15 and significantly associated with high Ki67 proliferation index in the meningioma cohort (p = 0.0144). The frequency of rs1048771 is 0.09 in the meningioma cohort and is significantly associated with the non-necrotic histopathological form of the tumor (p = 0.05). CONCLUSION: We reported a significant association between the genetic alterations of folate metabolism (MTHFR, MTRR) and DNA repair mechanism (RAD54L) genes with the histopathological characteristics of meningioma. Five significant SNPs on these genes and four significant correlations of SNPs with histopathological characteristics were identified. This is a preliminary promising study conducted to establish the genetic marker analysis for meningioma diagnosis and prognosis for folate metabolism and DNA repair genes in Turkish cohort.