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1.
PLoS Negl Trop Dis ; 14(7): e0008427, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628671

RESUMO

A major impediment to eliminate lymphatic filariasis and onchocerciasis is the lack of effective short-course macrofilaricidal drugs or regimens that are proven to be safe for both infections. In this study we tested oxfendazole, an anthelmintic shown to be well tolerated in phase 1 clinical trials. In vitro, oxfendazole exhibited modest to marginal motility inhibition of adult worms of Onchocerca gutturosa, pre-adult worms of Onchocerca volvulus and Onchocerca lienalis microfilariae. In vivo, five days of oral treatments provided sterile cure with up to 100% macrofilaricidal efficacy in the murine Litomosoides sigmodontis model of filariasis. In addition, 10 days of oral treatments with oxfendazole inhibited filarial embryogenesis in patent L. sigmodontis-infected jirds and subsequently led to a protracted but complete clearance of microfilaremia. The macrofilaricidal effect observed in vivo was selective, as treatment with oxfendazole of microfilariae-injected naïve mice was ineffective. Based on pharmacokinetic analysis, the driver of efficacy is the maintenance of a minimal efficacious concentration of approximately 100 ng/ml (based on subcutaneous treatment at 25 mg/kg in mice). From animal models, the human efficacious dose is predicted to range from 1.5 to 4.1 mg/kg. Such a dose has already been proven to be safe in phase 1 clinical trials. Oxfendazole therefore has potential to be efficacious for treatment of human filariasis without causing adverse reactions due to drug-induced microfilariae killing.


Assuntos
Benzimidazóis/farmacologia , Filariose Linfática/tratamento farmacológico , Filarioidea/efeitos dos fármacos , Animais , Anti-Helmínticos/uso terapêutico , Modelos Animais de Doenças , Filariose Linfática/parasitologia , Feminino , Filarioidea/embriologia , Gerbillinae/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microfilárias/efeitos dos fármacos , Onchocerca/efeitos dos fármacos , Onchocerca volvulus/efeitos dos fármacos , Oncocercose/tratamento farmacológico
2.
PLoS One ; 15(7): e0223633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701951

RESUMO

BACKGROUND: Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development. METHODS: We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures. RESULTS: Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 µm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 µm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 µM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 µM, but not 100 µM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 µM 1-EBIO, and partially reduced by 100 µM 1-EBIO. CONCLUSION: The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/agonistas , Animais , Benzimidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/toxicidade
3.
Parasitol Res ; 119(9): 2851-2862, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32651637

RESUMO

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 ß-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.


Assuntos
Albendazol/farmacologia , Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Resistência a Medicamentos/genética , Haemonchus/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Cromatografia Líquida , Glutamato Desidrogenase/metabolismo , Hemoncose/tratamento farmacológico , Haemonchus/genética , Reação em Cadeia da Polimerase , Proteômica , Ovinos , Doenças dos Ovinos/parasitologia , Espectrometria de Massas em Tandem , Tubulina (Proteína)/genética
4.
Chem Biol Interact ; 327: 109163, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32534988

RESUMO

The aim of the study was to synthesize a new series of benzimidazole derivatives and to investigate the underlying molecular mechanisms of the potential cell cycle inhibition and apoptotic effects against a panel of selected human cancer cell lines along with HEK-293 human embryonic kidney cells. MTT assay was used to evaluate cytotoxic effects. Muse™ Cell Analyzer was used to assess cell cycle progression. Annexin-V/PI staining assay was used for detecting apoptosis. All the synthesized compounds showed a significant cytotoxic effect against cancer cells with the IC50 values between 9.2 and 166.1 µg/mL. Among the tested derivatives, compound 5 showed significant cytotoxic activity against MCF-7, DU-145 and H69AR cancer cells with the IC50 values of 17.8 ± 0.24, 10.2 ± 1.4 and 49.9 ± 0.22 µg/mL respectively. The compounds 5 was also tested on HEK-293 human embryonic kidney cells and found to be safer with lesser cytotoxicity. The results revealed that compound 5 significantly increased cell population in the G2/M-phase which is modulated by a p53 independent mechanism. Compound 5 caused an increase in the percentage of late apoptotic cells in all tested cancer cells in a concentration-dependent manner. Among all synthesized derivatives, compound 5 the bromo-derivative, showed the highest cytotoxic potential, induced G2/M cell cycle arrest and apoptotic cell death in genotypically different human cancer cells. These results suggest that compound 5 might be a promising agent for cancer therapy and further structural modifications of benzimidazole derivatives may create promising anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Antineoplásicos/síntese química , Benzimidazóis/síntese química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos
5.
Chem Biol Interact ; 327: 109186, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32590071

RESUMO

In this study, we scrutinized the anticancer effects of FB-15 on human gastric carcinoma MGC-803 cells in vitro and vivo, and its preliminary effect on tubulin and HIF-1α. We confirmed that FB-15 not only inhibited the proliferation of a large number of cells in a concentration and time-dependent manner but also inhibited proliferation of a single cell to form clones. FB-15 manifested little cytotoxicity for normal stomach cells GES-1. The flow cytometry analysis displayed that FB-15 induced apoptosis MGC-803 cells and mainly arrested cells in the S phase in a concentration-dependent manner. The results of the wound healing assay indicated that FB-15 suppressed cell migration. Furthermore, the western blotting showed that FB-15 down-regulated the expression of ß3-tubulin and HIF-1α, consistent with Immunohistochemical assay. The binding modes of FB-15 with tubulin were clarified by molecular docking. FB-15 significantly suppressed the growth of MGC-803 gastric cancer tumors. The inhibitory effect of FB-15 on tumor growth was superior to 5-Fu. Taken together, these results provided evidence for FB-15 to be used as an effective anticancer drug candidate for gastric cancer.


Assuntos
Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Metabolismo Energético/efeitos dos fármacos , Flavonoides/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Flavonoides/metabolismo , Flavonoides/farmacologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Ligação Proteica , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Neoplasias Gástricas/patologia , Tubulina (Proteína)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nucleic Acids Res ; 48(14): 7973-7980, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32597964

RESUMO

Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.


Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Exodesoxirribonuclease V/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Benzimidazóis/química , Sítios de Ligação , Inibidores Enzimáticos/química , Exodesoxirribonuclease V/química , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Mutação
7.
Cancer Immunol Immunother ; 69(11): 2259-2273, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32504246

RESUMO

AKT-inhibition is a promising approach to improve T cell therapies; however, its effect on CD4+ T cells is insufficiently explored. Previously, we and others showed that AKT-inhibition during ex vivo CD8+ T cell expansion facilitates the generation of polyfunctional T cells with stem cell memory-like traits. However, most therapeutic T cell products are generated from lymphocytes, containing CD4+ T cells that can affect CD8+ T cells dependent on the Th-subset. Here, we investigated the effect of AKT-inhibition on CD4+ T cells, during separate as well as total T cell expansions. Interestingly, ex vivo AKT-inhibition preserved the early memory phenotype of CD4+ T cells based on higher CD62L, CXCR4 and CCR7 expression. However, in the presence of AKT-inhibition, Th-differentiation was skewed toward more Th2-associated at the expense of Th1-associated cells. Importantly, the favorable effect of AKT-inhibition on the functionality of CD8+ T cells drastically diminished in the presence of CD4+ T cells. Moreover, also the expansion method influenced the effect of AKT-inhibition on CD8+ T cells. These findings indicate that the effect of AKT-inhibition on CD8+ T cells is dependent on cell composition and expansion strategy, where presence of CD4+ T cells as well as polyclonal stimulation impede the favorable effect of AKT-inhibition.


Assuntos
Benzimidazóis/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Quinoxalinas/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/imunologia , Células Cultivadas , Humanos
8.
Am J Physiol Cell Physiol ; 319(2): C345-C358, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520608

RESUMO

The maturity of osteoblasts by proliferation and differentiation in preosteoblasts is essential for maintaining bone homeostasis. The beneficial effects of vitamin D on bone homeostasis in mammals have been demonstrated experimentally and clinically. However, the direct actions of vitamin D on preosteoblasts remain to be fully elucidated. In this study, we found that the functional activity of intermediate-conductance Ca2+-activated K+ channels (KCa3.1) positively regulated cell proliferation in MC3T3-E1 cells derived from mouse preosteoblasts by enhancing intracellular Ca2+ signaling. We examined the effects of treatment with vitamin D receptor (VDR) agonist on the expression and activity of KCa3.1 by real-time PCR examination, Western blotting, Ca2+ imaging, and patch clamp analyses in mouse MC3T3-E1 cells. Following the downregulation of KCa3.1 transcriptional modulators such as Fra-1 and HDAC2, KCa3.1 activity was suppressed in MC3T3-E1 cells treated with VDR agonists. Furthermore, application of the KCa3.1 activator DCEBIO attenuated the VDR agonist-evoked suppression of cell proliferation rate. These findings suggest that a decrease in KCa3.1 activity is involved in the suppression of cell proliferation rate in VDR agonist-treated preosteoblasts. Therefore, KCa3.1 plays an important role in bone formation by promoting osteoblastic proliferation under physiological conditions.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Osteoblastos/metabolismo , Receptores de Calcitriol/genética , Vitamina D/genética , Células 3T3 , Animais , Benzimidazóis/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Histona Desacetilase 2/genética , Humanos , Camundongos , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-fos/genética , Receptores de Calcitriol/agonistas , Transdução de Sinais/efeitos dos fármacos
9.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G227-G237, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32597706

RESUMO

Antibiotic treatment is a standard therapy for Clostridioides difficile infection, but dysbiosis of the gut microbiota due to antibiotic exposure is also a major risk factor for the disease. Following an initial episode of C. difficile infection, a relentless cycle of recurrence can occur, where persistent treatment-related dysbiosis predisposes the patient to subsequent relapse. This study uses a longitudinal study design to compare the effects of a narrow-spectrum (ridinilazole) or broad-spectrum antibiotic (vancomycin) on intestinal bile acid profiles and their associations with gut bacteria over the course of C. difficile infection treatment. At the end of treatment (day 10), subjects receiving vancomycin showed a nearly 100-fold increase in the ratio of conjugated to secondary bile acids in their stool compared with baseline, whereas subjects receiving ridinilazole maintained this ratio near baseline levels. Correlation analysis detected significant positive associations between secondary bile acids and several Bacteroidales and Clostridiales families. These families were depleted in the vancomycin group but preserved at near-baseline abundance in the ridinilazole group. Enterobacteriaceae, which expanded to a greater extent in the vancomycin group, correlated negatively and positively with secondary and conjugated primary bile acids, respectively. Bile acid ratios at the end of treatment were significantly different between those who recurred and those who did not. These results indicate that a narrow-spectrum antibiotic maintains an intestinal bile acid profile associated with a lowered risk of recurrence.NEW & NOTEWORTHY This is the first study to demonstrate in humans the relationships between Clostridioides difficile antibiotic treatment choice and bile acid metabolism both during therapy and after treatment cessation. The results show a microbiota- and metabolome-preserving property of a novel narrow-spectrum agent that correlates with the agent's favorable sustained clinical response rates compared with broad-spectrum antibiotic treatment.


Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Ácidos e Sais Biliares/química , Clostridiales/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Piridinas/farmacologia , Ácidos e Sais Biliares/metabolismo , Fezes/química , Microbioma Gastrointestinal/fisiologia , Humanos
10.
Cancer Invest ; 38(6): 349-355, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32441531

RESUMO

Background: Meningiomas represent ∼30% of primary central nervous system (CNS) tumors. Although advances in surgery and radiotherapy have significantly improved survival, there remains an important subset of patients whose tumors have more aggressive behavior and are refractory to conventional therapy. Recent advances in molecular genetics and epigenetics suggest that this aggressive behavior may be due to the deletion of the DNA repair and tumor suppressor gene, CHEK2, neurofibromatosis Type 2 (NF2) mutation on chromosome 22q12, and genetic abnormalities in multiple RTKs including FGFRs. Management of higher-grade meningiomas, such as anaplastic meningiomas (AM: WHO grade III), is truly challenging and there isn't an established chemotherapy option. We investigate the effect of active multi tyrosine receptor kinase inhibitor Dovitinib at stopping AM cell growth in in vitro with either frequent codeletion or mutated CHEK2 and NF2 gene.Methods: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot analysis, caspases assay, and DNA fragmentation assay.Results: Treatment of CH157MN and IOMM-Lee cells with Dovitinib suppressed multiple angiokinases-mainly FGFRs, leading to suppression of downstream signaling by RAS-RAF-MAPK molecules and PI3K-AKT molecules which are involved in cell proliferation, cell survival, and tumor invasion. Furthermore, Dovitinib induced apoptosis via downregulation of survival proteins (Bcl-XL), and over-expression of apoptotic factors (Bax and caspase-3) regardless of CHEK2 and NF2 mutation status.Conclusions: This study establishes the groundwork for the development of Dovitinib as a therapeutic agent for high-grade AM with either frequent codeletion or mutated CHEK2 and NF2, an avenue with high translational potential.


Assuntos
Benzimidazóis/farmacologia , Quinase do Ponto de Checagem 2/genética , Meningioma/tratamento farmacológico , Neurofibromina 2/genética , Quinolonas/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meningioma/genética , Meningioma/patologia , Mutação/genética , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína bcl-X/genética
11.
PLoS One ; 15(4): e0232140, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353010

RESUMO

BACKGROUND: Congenital cytomegalovirus infection can lead to severe sequelae. When fetal infection is confirmed, we hypothesize that fetal treatment could improve the outcome. Maternal oral administration of an effective drug crossing the placenta could allow fetal treatment. Letermovir (LMV) and Maribavir (MBV) are new CMV antivirals, and potential candidates for fetal treatment. METHODS: The objective was to investigate the placental transfer of LMV and MBV in the ex vivo method of the human perfused cotyledon. Term placentas were perfused, in an open-circuit model, with LMV or MBV at concentrations in the range of clinical peak plasma concentrations. Concentrations were measured using ultraperformance liquid chromatography coupled with tandem mass spectrometry. Mean fetal transfer rate (FTR) (fetal (FC) /maternal concentration), clearance index (CLI), accumulation index (AI) (retention of each drug in the cotyledon tissue) were measured. Mean FC were compared with half maximal effective concentrations of the drugs (EC50(LMV) and EC50(MBV)). RESULTS: For LMV, the mean FC was (± standard deviation) 1.1 ± 0.2 mg/L, 1,000-fold above the EC50(LMV). Mean FTR, CLI and AI were 9 ± 1%, 35 ± 6% and 4 ± 2% respectively. For MBV, the mean FC was 1.4 ± 0.2 mg/L, 28-fold above the EC50(MBV). Mean FTR, CLI and AI were 10 ± 1%, 50 ± 7% and 2 ± 1% respectively. CONCLUSIONS: Drugs' concentrations in the fetal side should be in the range for in utero treatment of fetuses infected with CMV as the mean FC was superior to the EC50 for both molecules.


Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Troca Materno-Fetal/efeitos dos fármacos , Placenta/efeitos dos fármacos , Acetatos/farmacologia , Adulto , Antivirais/farmacologia , Benzimidazóis/farmacologia , Cromatografia Líquida/métodos , Feminino , Humanos , Cinética , Modelos Biológicos , Perfusão , Gravidez , Quinazolinas/farmacologia , Ribonucleosídeos/farmacologia , Espectrometria de Massas em Tandem/métodos
12.
F1000Res ; 9: 129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32194944

RESUMO

We prepared the three-dimensional model of the SARS-CoV-2 (aka 2019-nCoV) 3C-like protease (3CL pro) using the crystal structure of the highly similar (96% identity) ortholog from the SARS-CoV. All residues involved in the catalysis, substrate binding and dimerisation are 100% conserved. Comparison of the polyprotein PP1AB sequences showed 86% identity. The 3C-like cleavage sites on the coronaviral polyproteins are highly conserved. Based on the near-identical substrate specificities and high sequence identities, we are of the opinion that some of the previous progress of specific inhibitors development for the SARS-CoV enzyme can be conferred on its SARS-CoV-2 counterpart.  With the 3CL pro molecular model, we performed virtual screening for purchasable drugs and proposed 16 candidates for consideration. Among these, the antivirals ledipasvir or velpatasvir are particularly attractive as therapeutics to combat the new coronavirus with minimal side effects, commonly fatigue and headache.  The drugs Epclusa (velpatasvir/sofosbuvir) and Harvoni (ledipasvir/sofosbuvir) could be very effective owing to their dual inhibitory actions on two viral enzymes.


Assuntos
Benzimidazóis/farmacologia , Betacoronavirus/efeitos dos fármacos , Carbamatos/farmacologia , Infecções por Coronavirus , Cisteína Endopeptidases/química , Fluorenos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Pandemias , Pneumonia Viral , Proteínas não Estruturais Virais/química , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Humanos , Pneumonia Viral/tratamento farmacológico
13.
Science ; 368(6486)2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241924

RESUMO

The success of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors (PARPi) to treat cancer relates to their ability to trap PARP-1 at the site of a DNA break. Although different forms of PARPi all target the catalytic center of the enzyme, they have variable abilities to trap PARP-1. We found that several structurally distinct PARPi drive PARP-1 allostery to promote release from a DNA break. Other inhibitors drive allostery to retain PARP-1 on a DNA break. Further, we generated a new PARPi compound, converting an allosteric pro-release compound to a pro-retention compound and increasing its ability to kill cancer cells. These developments are pertinent to clinical applications where PARP-1 trapping is either desirable or undesirable.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerase-1/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Benzimidazóis/química , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Humanos , Isoindóis/química , Isoindóis/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Domínios Proteicos
14.
Artigo em Inglês | MEDLINE | ID: mdl-32251964

RESUMO

Benzimidazoles (BZ) have been the anthelmintic of choice for controlling Nematodirus battus infections since their release in the 1950s. Despite heavy reliance on this single anthelmintic drug class, resistance was not identified in this nematode until 2010 (Mitchell et al., 2011). The study aimed to explore the prevalence of BZ-resistance mutations in N. battus from UK sheep flocks using deep amplicon sequencing and pyrosequencing platforms. Based on evidence from other gastrointestinal nematodes, resistance in N. battus is likely to be conferred by single nucleotide polymorphisms (SNP) within the ß-tubulin isotype 1 locus at codons 167, 198 and 200. Pyrosequencing and deep amplicon sequencing assays were designed to identify the F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) SNPs. Nematodirus battus populations from 253 independent farms were analysed by pyrosequencing; 174 farm populations were included in deep amplicon sequencing and 170 were analysed using both technologies. F200Y was the most prevalent SNP identified throughout the UK, in 12-27% of the populations tested depending on assay, at a low overall individual frequency of 2.2 ±â€¯0.6% (mean ±â€¯SEM, based on pyrosequencing results). Four out of the five populations with high frequencies (>20%) of the F200Y mutation were located in NW England. The F167Y SNP was identified, for the first time in this species, in four of the populations tested at a low frequency (1.2% ±â€¯0.01), indicating the early emergence of the mutation. E198A or E198L were not identified in any of the isolates. Results obtained were comparable between both techniques for F200Y (Lins' CCC, rc = 0.96) with discrepancies being limited to populations with low frequencies. The recent emergence of resistance in this species will provide a unique opportunity to study the early stages of anthelmintic resistance within a natural setting and track its progress in the future.


Assuntos
Benzimidazóis/farmacologia , Resistência a Medicamentos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nematodirus/genética , Doenças dos Ovinos/parasitologia , Infecções por Strongylida/veterinária , Animais , Anti-Helmínticos/farmacologia , Fazendas , Fezes/parasitologia , Frequência do Gene , Genótipo , Mutação , Nematodirus/efeitos dos fármacos , Análise de Sequência de DNA , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/epidemiologia , Infecções por Strongylida/tratamento farmacológico , Infecções por Strongylida/epidemiologia , Reino Unido/epidemiologia
15.
Cancer Sci ; 111(6): 2132-2145, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32304130

RESUMO

In the cell cycle, the G1 /S transition is controlled by the cyclin-dependent kinase (CDK) 4/6-cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1 /S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non-small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell-death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell-death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar-type ATPase (V-ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live-cell imaging revealed that the abemaciclib-induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes.


Assuntos
Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Lisossomos/efeitos dos fármacos , Vacúolos/efeitos dos fármacos
16.
Parasit Vectors ; 13(1): 114, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122383

RESUMO

BACKGROUND: Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms. METHODS: Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 ß-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur. RESULTS: Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5-90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples. CONCLUSIONS: To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.


Assuntos
Benzimidazóis/farmacologia , Códon , Resistência a Medicamentos/genética , Doenças das Cabras/parasitologia , Haemonchus/genética , Polimorfismo de Nucleotídeo Único , Tubulina (Proteína)/genética , Albendazol/farmacologia , Animais , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Feminino , Frequência do Gene , Genótipo , Doenças das Cabras/tratamento farmacológico , Cabras , Haemonchus/efeitos dos fármacos , Masculino , Análise de Sequência de DNA , Sudão , Trichostrongyloidea/genética , Trichostrongylus/genética
17.
J Med Chem ; 63(6): 3047-3065, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32150677

RESUMO

In this study, a successful medicinal chemistry campaign that exploited virtual, biophysical, and biological investigations led to the identification of a novel class of IDO1 inhibitors based on a benzimidazole substructure. This family of compounds is endowed with an extensive bonding network in the protein active site, including the interaction with pocket C, a region not commonly exploited by previously reported IDO1 inhibitors. The tight packing of selected compounds within the enzyme contributes to the strong binding interaction with IDO1, to the inhibitory potency at the low nanomolar level in several tumoral settings, and to the selectivity toward IDO1 over TDO and CYPs. Notably, a significant reduction of L-Kyn levels in plasma, together with a potent effect on abrogating immunosuppressive properties of MDSC-like cells isolated from patients affected by pancreatic ductal adenocarcinoma, was observed, pointing to this class of molecules as a valuable template for boosting the antitumor immune system.


Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Animais , Benzimidazóis/sangue , Linhagem Celular Tumoral , Células Cultivadas , Inibidores Enzimáticos/sangue , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
18.
Hum Cell ; 33(3): 528-536, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32170715

RESUMO

Excessive vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling and stroke during hypertension. Blockade of Angiotensin (AngII) type 1 receptor (AT1R) is shown to effectively attenuate VSMC proliferation and vascular remodeling, while the mechanisms underlying these protective effects are unclear. Here, we investigated whether the amelioration of VSMC proliferation mediated by candesartan, an AT1R blocker, could be associated with miRNA regulation. Based on the published data in rat aortic smooth muscle cells (RASMCs), we discovered that candesartan specifically reversed the AngII-induced decrease of miR-301b level in RASMCs and human aortic smooth muscle cells (HASMCs). Knockdown of miR-301b abolished candesartan-mediated inhibition of HASMC proliferation via promoting cell cycle transition. Computational analysis showed that miR-301b targets at 3'UTR of STAT3. MiR-301b upregulation inhibited the luciferase activity and protein expression of STAT3, whereas miR-301b knockdown increased STAT3 luciferase activity and expression. Furthermore, downregulation of STAT3 markedly abrogated the effects of miR-301b inhibition on candesartan-mediated HASMC proliferation, invasion, and migration. Collectively, this study suggests that miR-301b may be a novel molecular target of candesartan and provides a new understanding for the mechanisms underlying the cardiovascular effects of candesartan.


Assuntos
Benzimidazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/citologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Tetrazóis/farmacologia , Fármacos Cardiovasculares , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Humanos
19.
Biochim Biophys Acta Rev Cancer ; 1873(2): 188355, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32135169

RESUMO

The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.


Assuntos
Carcinogênese/patologia , Canal de Potássio ERG1/metabolismo , Integrina beta1/metabolismo , Neoplasias/patologia , Fator de Transcrição STAT1/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Carcinogênese/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/química , Canal de Potássio ERG1/genética , Epigênese Genética/efeitos dos fármacos , Fluoxetina/farmacologia , Fluoxetina/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Síndrome do QT Longo/genética , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Mutação , Miócitos Cardíacos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Conformação Proteica em alfa-Hélice , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Sulfanilamidas/farmacologia , Sulfanilamidas/uso terapêutico
20.
PLoS One ; 15(3): e0229801, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163428

RESUMO

We report the synthesis and preliminary characterization of IODVA1, a potent small molecule that is active in xenograft mouse models of Ras-driven lung and breast cancers. In an effort to inhibit oncogenic Ras signaling, we combined in silico screening with inhibition of proliferation and colony formation of Ras-driven cells. NSC124205 fulfilled all criteria. HPLC analysis revealed that NSC124205 was a mixture of at least three compounds, from which IODVA1 was determined to be the active component. IODVA1 decreased 2D and 3D cell proliferation, cell spreading and ruffle and lamellipodia formation through downregulation of Rac activity. IODVA1 significantly impaired xenograft tumor growth of Ras-driven cancer cells with no observable toxicity. Immuno-histochemistry analysis of tumor sections suggests that cell death occurs by increased apoptosis. Our data suggest that IODVA1 targets Rac signaling to induce death of Ras-transformed cells. Therefore, IODVA1 holds promise as an anti-tumor therapeutic agent.


Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas ras/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Benzimidazóis/síntese química , Benzimidazóis/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Células NIH 3T3 , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
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