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2.
PLoS One ; 16(10): e0258911, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34673834

RESUMO

Trabecular meshwork constitutes the conventional outflow pathway and controls intraocular pressure by regulating aqueous outflow. Mechanical stimulation has been studied as one of the triggers to regulate aqueous outflow in trabecular meshwork, but it is not well understood. We investigated that how transient receptor potential cation channel subfamily V member 4 (TRPV4) functions in human trabecular meshwork cells (HTMC) and affects intraocular pressure (IOP). HTMC were treated with TRPV4 siRNA, followed by incubation for 24 hours. We confirmed the suppression of TRPV4 mRNA expression and the reduction of Ca2+ influx by the TRPV4 agonist GSK1016790A in TRPV4 siRNA-treated HTMC. TRPV4 siRNA-treated HTMC exhibited a significant reduction in Ca2+ influx and production of arachidonic acid and prostaglandin (PG) E2 induced by mechanical stretch, and direct activation of TRPV4 by GSK1016790A increased production of arachidonic acid, PGE2, and PGD2 and inhibited gel contraction. Furthermore, TRPV4-deficient mice had higher IOP than wild-type mice, and GSK1016790A administration lowered IOP. These results suggest that TRPV4 mediates the cellular response induced by trabecular meshwork stretch, leading to IOP reduction through the production of prostaglandins and inhibition of cell contraction. Targeting TRPV4 may have therapeutic benefits that lead to lowering IOP in glaucoma patients.


Assuntos
Ácido Araquidônico/metabolismo , Dinoprostona/metabolismo , Pressão Intraocular/fisiologia , Canais de Cátion TRPV/metabolismo , Malha Trabecular/metabolismo , Animais , Humanos , Pressão Intraocular/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Camundongos Knockout , Estimulação Física , RNA Interferente Pequeno , Sulfonamidas/farmacologia , Canais de Cátion TRPV/genética , Malha Trabecular/efeitos dos fármacos
3.
Nutrients ; 13(10)2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34684538

RESUMO

Higher daily protein intake, with an emphasis on leucine content, is thought to mitigate age-related anabolic resistance, potentially counteracting age-related morphological and functional declines. The present study investigated potential associations between total daily leucine intake and dependent variables, including quadriceps muscle cross-sectional area (CSA) and maximum dynamic muscle strength (1-RM) in a cohort of healthy free-living older individuals of both sexes (n = 67; 34/33 men/women). Participants performed three 24 h dietary recalls and underwent a magnetic resonance imaging exam followed by 1-RM tests. Our results demonstrate moderate associations between total daily leucine and both quadriceps CSA (r = 0.42; p = 0.004) and 1-RM (r = 0.45; p = 0.001). Furthermore, our exploratory biphasic linear regression analyses, adjusted for sex, age, and protein intake relative to body weight, revealed a plateau for daily leucine intake and muscle mass and muscle strength (~7.6-8.0 g·day-1) in older adults. In conclusion, we demonstrated that total daily leucine intake is associated with muscle mass and strength in healthy older individuals and this association remains after controlling for multiple factors, including overall protein intake. Furthermore, our breakpoint analysis revealed non-linearities and a potential threshold for habitual leucine intake, which may help guide future research on the effects of chronic leucine intake in age-related muscle loss.


Assuntos
Ingestão de Alimentos , Leucina/farmacologia , Extremidade Inferior/fisiologia , Força Muscular/fisiologia , Músculo Esquelético/fisiologia , Idoso , Feminino , Humanos , Masculino , Força Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Análise de Regressão
4.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502033

RESUMO

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CLpro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CLpro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CLpro-PF-07321332 and 3CLpro-α-ketoamide complexes remained stable compared with 3CLpro-ritonavir and 3CLpro-lopinavir. Investigating the dynamic behavior of ligand-protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41-Cys145 of 3CLpro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CLpro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Lactamas/farmacologia , Leucina/farmacologia , Nitrilas/farmacologia , Prolina/farmacologia , Antivirais/uso terapêutico , Domínio Catalítico/efeitos dos fármacos , Proteases 3C de Coronavírus/metabolismo , Inibidores de Protease de Coronavírus/uso terapêutico , Humanos , Lactamas/uso terapêutico , Leucina/uso terapêutico , Lopinavir/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrilas/uso terapêutico , Prolina/uso terapêutico , Ritonavir/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia
5.
Biomed Res Int ; 2021: 6614000, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337036

RESUMO

Chloroquine (CQ) and hydroxychloroquine (HCQ) have shown the ability to inhibit in vitro viral replications of coronaviridae viruses such as SARS-CoV and SARS-CoV-2. However, clinical trial outcomes have been disparate, suggesting that CQ and HCQ antiviral mechanisms are not fully understood. Based on three-dimensional structural similarities between HCQ and the known ACE2 specific inhibitor MLN-4760, we compared their modulation on ACE2 activity. Here we describe, for the first time, in a cell-free in vitro system that HCQ directly and dose-dependently inhibits the activity of recombinant human ACE2, with a potency similar to the MLN-4760. Further analysis suggests that HCQ binds to a noncompetitive site other than the one occupied by MLN-4760. We also determined that the viral spike glycoprotein segment that comprises the RBD segment has no effect on ACE2 activity but unexpectedly was able to partially reverse the inhibition induced by HCQ but not that by MLN-4760. In summary, here we demonstrate the direct inhibitory action of HCQ over the activity of the enzyme ACE2. Then, by determining the activity of ACE2, we reveal that the interaction with the spike protein of SARS-CoV-2 leads to structural changes that at least partially displace the interaction of the said enzyme with HCQ. These results may help to explain why the effectiveness of HCQ in clinical trials has been so variable. Additionally, this knowledge could be used for to develop techniques for the detection of SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais , COVID-19/tratamento farmacológico , Hidroxicloroquina , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Humanos , Hidroxicloroquina/química , Hidroxicloroquina/metabolismo , Hidroxicloroquina/farmacologia , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Leucina/análogos & derivados , Leucina/química , Leucina/metabolismo , Leucina/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
6.
Nutr Metab Cardiovasc Dis ; 31(10): 2979-2986, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34362635

RESUMO

BACKGROUND AND AIMS: Coronary heart disease is a major global health concern. Further, severity of this condition is greatly influenced by myocardial ischemia/reperfusion (I/R) injury. Branched-chain amino acids (BCAAs) have cardioprotective effects against I/R via mammalian target of rapamycin (mTOR) activity, wherein Leu is considered to particularly regulate mTOR activation. However, the mechanism underlying cardioprotective effects of Leu via mTOR activity is not fully elucidated. Here, we aimed to study the signaling pathway of cardioprotection and mitochondrial function induced by Leu treatment. METHODS AND RESULTS: Cardiac myocytes isolated from adult male Wistar rats were incubated and exposed to simulated I/R (SI/R) injury by replacing the air content. Cardiac myocytes were treated with Leu and subsequently, their survival rate was calculated. To elucidate the signaling pathway and mitochondrial function, immunoblots and mitochondrial permeability transition pore were examined. Cell survival rate was decreased with SI/R but improved by 160 µM Leu (38.5 ± 3.6% vs. 64.5 ± 4.2%, respectively, p < 0.001). Although rapamycin (mTOR inhibitor) prevented this cardioprotective effect induced by Leu, wortmannin (PI3K inhibitor) did not interfere with this effect. In addition, we indicated that overexpression of Opa-1 and mitochondrial function are ameliorated via Leu-induced mitochondrial biogenesis. In contrast, knockdown of Opa-1 suppressed Leu-induced cardioprotection. CONCLUSION: Leu treatment is critical in rendering a cardioprotective effect exhibited by BCAAs via mTOR signaling. Furthermore, Leu improved mitochondrial function.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Leucina/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , GTP Fosfo-Hidrolases/genética , Masculino , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Biogênese de Organelas , Ratos Wistar , Transdução de Sinais
7.
Front Immunol ; 12: 656573, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34367129

RESUMO

L-leucine (Leu), as one of the effective amino acids to activate the mTOR signaling pathway, can alleviate transmissible gastroenteritis virus (TGEV) infection. However, the underlying mechanism by which Leu alleviates the virus infection has not been fully characterized. In particular, how Leu impacts TGEV replication through mTOR signaling has yet to be elucidated. In the present study, we found that TGEV proliferated efficiently in intestinal porcine epithelial cells (IPEC-J2 cells) as evidenced by the increase in viral contents by flow cytometry, the inhibition of cell proliferation by CCK-8 assay as well as the reduction of PCNA level by western blot. Besides, western blot analysis showed that STAT1 expression was markedly reduced in TGEV-infected cells. The results of ELISA revealed the inhibition of ISGs (ISG56, MxA, and PKR) expressions by TGEV infection. TGEV-induced mTOR and its downstream p70 S6K and 4E-BP1, STAT1 and ISGs downregulation were blocked by an mTOR activator-MHY1485 but not by an mTOR inhibitor-RAPA. Concurrently, mTOR activation by MHY1485 reduced the contents of TGEV and vice versa. Furthermore, Leu reversed the inhibition of STAT1 and ISGs by activating mTOR and its downstream p70 S6K and 4E-BP1 in TEGV-infected cells. Our findings demonstrated that Leu promoted the expressions of STAT1 and ISGs via activating mTOR signaling in IPEC-J2 cells, aiming to prevent TGEV infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Leucina/farmacologia , Fator de Transcrição STAT1/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Gastroenterite Suína Transmissível , Interações Hospedeiro-Patógeno/genética , Interferon Tipo I/metabolismo , Modelos Biológicos , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Vírus da Gastroenterite Transmissível/fisiologia , Replicação Viral
8.
FASEB J ; 35(9): e21862, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34416035

RESUMO

Loss of muscle mass and strength after disuse followed by impaired muscle recovery commonly occurs with aging. Metformin (MET) and leucine (LEU) individually have shown positive effects in skeletal muscle during atrophy conditions but have not been evaluated in combination nor tested as a remedy to enhance muscle recovery following disuse atrophy in aging. The purpose of this study was to determine if a dual treatment of metformin and leucine (MET + LEU) would prevent disuse-induced atrophy and/or promote muscle recovery in aged mice and if these muscle responses correspond to changes in satellite cells and collagen remodeling. Aged mice (22-24 months) underwent 14 days of hindlimb unloading (HU) followed by 7 or 14 days of reloading (7 or 14 days RL). MET, LEU, or MET + LEU was administered via drinking water and were compared to Vehicle (standard drinking water) and ambulatory baseline. We observed that during HU, MET + LEU resolved whole body grip strength and soleus muscle specific force decrements caused by HU. Gastrocnemius satellite cell abundance was increased with MET + LEU treatment but did not alter muscle size during disuse or recovery conditions. Moreover, MET + LEU treatment alleviated gastrocnemius collagen accumulation caused by HU and increased collagen turnover during 7 and 14 days RL driven by a decrease in collagen IV content. Transcriptional pathway analysis revealed that MET + LEU altered muscle hallmark pathways related to inflammation and myogenesis during HU. Together, the dual treatment of MET and LEU was able to increase muscle function, satellite cell content, and reduce collagen accumulation, thus improving muscle quality during disuse and recovery in aging.


Assuntos
Envelhecimento , Colágeno/metabolismo , Leucina/uso terapêutico , Metformina/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/prevenção & controle , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Fibrose/tratamento farmacológico , Elevação dos Membros Posteriores , Imunoglobulina G/análise , Leucina/farmacologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Tamanho do Órgão/efeitos dos fármacos , RNA-Seq , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/patologia , Transdução de Sinais/efeitos dos fármacos
9.
Cells ; 10(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34440840

RESUMO

Cystatin C is a potent cysteine protease inhibitor that plays an important role in various biological processes including cancer, cardiovascular diseases and neurodegenerative diseases. However, the role of CstC in inflammation is still unclear. In this study we demonstrated that cystatin C-deficient mice were significantly more sensitive to the lethal LPS-induced sepsis. We further showed increased caspase-11 gene expression and enhanced processing of pro-inflammatory cytokines IL-1ß and IL-18 in CstC KO bone marrow-derived macrophages (BMDM) upon LPS and ATP stimulation. Pre-treatment of BMDMs with the cysteine cathepsin inhibitor E-64d did not reverse the effect of CstC deficiency on IL-1ß processing and secretion, suggesting that the increased cysteine cathepsin activity determined in CstC KO BMDMs is not essential for NLRP3 inflammasome activation. The CstC deficiency had no effect on (mitochondrial) reactive oxygen species (ROS) generation, the MAPK signaling pathway or the secretion of anti-inflammatory cytokine IL-10. However, CstC-deficient BMDMs showed dysfunctional autophagy, as autophagy induction via mTOR and AMPK signaling pathways was suppressed and accumulation of SQSTM1/p62 indicated a reduced autophagic flux. Collectively, our study demonstrates that the excessive inflammatory response to the LPS-induced sepsis in CstC KO mice is dependent on increased caspase-11 expression and impaired autophagy, but is not associated with increased cysteine cathepsin activity.


Assuntos
Cistatina C/genética , Lipopolissacarídeos/efeitos adversos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sepse/etiologia , Animais , Autofagia/genética , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Cistatina C/deficiência , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Sepse/mortalidade , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima
10.
J Enzyme Inhib Med Chem ; 36(1): 1646-1650, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34289752

RESUMO

The chemical structure of PF-07321332, the first orally available Covid-19 clinical candidate, has recently been revealed by Pfizer. No information has been provided about the interaction pattern between PF-07321332 and its biomolecular counterpart, the SARS-CoV-2 main protease (Mpro). In the present work, we exploited Supervised Molecular Dynamics (SuMD) simulations to elucidate the key features that characterise the interaction between this drug candidate and the protease, emphasising similarities and differences with other structurally related inhibitors such as Boceprevir and PF-07304814. The structural insights provided by SuMD will hopefully be able to inspire the rational discovery of other potent and selective protease inhibitors.


Assuntos
Antivirais/química , Lactamas/química , Leucina/química , Simulação de Dinâmica Molecular , Nitrilas/química , Prolina/química , Inibidores de Proteases/química , Antivirais/farmacologia , Humanos , Lactamas/farmacologia , Leucina/farmacologia , Ligantes , Nitrilas/farmacologia , Peptídeo Hidrolases/metabolismo , Prolina/farmacologia , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Software
11.
Nutrients ; 13(5)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064996

RESUMO

(1) Background: Whey protein lowers postprandial blood glucose in health and type 2 diabetes, by stimulating insulin and incretin hormone secretion and slowing gastric emptying. The branched-chain amino acids, leucine, isoleucine and valine, abundant in whey, may mediate the glucoregulatory effects of whey. We investigated the comparative effects of intragastric administration of leucine, isoleucine and valine on the plasma glucose, C-peptide and glucagon responses to and gastric emptying of a mixed-nutrient drink in healthy men. (2) Methods: 15 healthy men (27 ± 3 y) received, on four separate occasions, in double-blind, randomised fashion, either 10 g of leucine, 10 g of isoleucine, 10 g of valine or control, intragastrically, 30 min before a mixed-nutrient drink. Plasma glucose, C-peptide and glucagon concentrations were measured before, and for 2 h following, the drink. Gastric emptying of the drink was quantified using 13C-acetate breath-testing. (3) Results: Amino acids alone did not affect plasma glucose or C-peptide, while isoleucine and valine, but not leucine, stimulated glucagon (p < 0.05), compared with control. After the drink, isoleucine and leucine reduced peak plasma glucose compared with both control and valine (all p < 0.05). Neither amino acid affected early (t = 0-30 min) postprandial C-peptide or glucagon. While there was no effect on overall gastric emptying, plasma glucose at t = 30 min correlated with early gastric emptying (p < 0.05). (4) Conclusion: In healthy individuals, leucine and isoleucine lower postprandial blood glucose, at least in part by slowing gastric emptying, while valine does not appear to have an effect, possibly due to glucagon stimulation.


Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Glicemia/metabolismo , Peptídeo C/sangue , Esvaziamento Gástrico/efeitos dos fármacos , Glucagon/sangue , Isoleucina/farmacologia , Leucina/farmacologia , Valina/farmacologia , Adulto , Diabetes Mellitus Tipo 2 , Método Duplo-Cego , Polipeptídeo Inibidor Gástrico/sangue , Humanos , Insulina , Masculino , Pessoa de Meia-Idade , Período Pós-Prandial/efeitos dos fármacos , Proteínas do Soro do Leite/farmacologia , Adulto Jovem
12.
J Pharmacol Sci ; 146(4): 233-243, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34116737

RESUMO

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease, initiated by delayed retinal vascular growth after premature birth. In the majority of cases, ROP resolves spontaneously; however, a history of ROP may increase the risk of long-term visual problems. In this study, we evaluated the endothelial function of retinal blood vessels in adult rats with a history of ROP. ROP was induced in rats by subcutaneous injection of a vascular endothelial growth factor receptor tyrosine kinase inhibitor (KRN633) on postnatal day (P) 7 and P8. On P56, vasodilator responses to acetylcholine, GSK1016790A (an activator of transient receptor potential vanilloid 4 channels), NOR3 (a nitric oxide [NO] donor), and salbutamol (a ß2-adrenoceptor agonist) were assessed. Compared to age-matched controls, retinal vasodilator responses to acetylcholine and GSK1016790A were attenuated in P56 rats with a history of ROP. No attenuation of acetylcholine-induced retinal vasodilator response was observed under inhibition of NO synthase. Retinal vasodilator responses to NOR3 and salbutamol were unaffected. These results suggest that the production of and/or release of NO is impaired in retinal blood vessels in adult rats with a history of ROP. A history of ROP might increase the risk of impaired retinal circulation in adulthood.


Assuntos
Endotélio Vascular/fisiopatologia , Vasos Retinianos/fisiopatologia , Retinopatia da Prematuridade/fisiopatologia , Vasodilatação , Acetilcolina/farmacologia , Albuterol/farmacologia , Animais , Animais Recém-Nascidos , Circulação Sanguínea/efeitos dos fármacos , Feminino , Leucina/análogos & derivados , Leucina/farmacologia , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Gravidez , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Vasodilatação/efeitos dos fármacos
13.
Biochem J ; 478(13): 2697-2713, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34133730

RESUMO

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. Overall, the P. vivax and P. berghei enzymes had a faster substrate turnover rate than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.


Assuntos
Aminopeptidases/metabolismo , Peptídeos/metabolismo , Plasmodium/enzimologia , Proteínas de Protozoários/metabolismo , Aminopeptidases/classificação , Aminopeptidases/genética , Animais , Biocatálise/efeitos dos fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Leucina/análogos & derivados , Leucina/farmacologia , Malária/parasitologia , Camundongos , Plasmodium/genética , Plasmodium/fisiologia , Plasmodium berghei/enzimologia , Plasmodium berghei/genética , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium vivax/enzimologia , Plasmodium vivax/genética , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Especificidade por Substrato
14.
Sci Rep ; 11(1): 9609, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953295

RESUMO

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC-376 is a Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice. Overall outcome of clinical symptoms and survival upon SARS-CoV-2 challenge were not improved in mice treated with GC-376 compared to controls. The treatment with GC-376 slightly improved survival from 0 to 20% in mice challenged with a high virus dose at 105 TCID50/mouse. Most notably, GC-376 treatment led to milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls in mice challenged with a low virus dose at 103 TCID50/mouse. This was particularly the case in the brain where a 5-log reduction in viral titers was observed in GC-376 treated mice compared to vehicle controls. This study supports the notion that GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection and that the K18-hACE2 mouse model is suitable to study antiviral therapies against SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Carbonatos/farmacologia , Leucina/farmacologia , Ácidos Sulfônicos/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , COVID-19/patologia , COVID-19/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Queratina-18/genética , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Camundongos Transgênicos , Células Vero , Carga Viral
15.
J Phys Chem Lett ; 12(20): 4814-4822, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-33999630

RESUMO

Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of the lungs, heart, kidney, and pancreas. It is considered an important drug target for treating SARS-CoV-2 as well as pulmonary diseases, heart failure, hypertension, renal diseases, and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we have addressed this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond time scale LiGaMD simulations have unprecedentedly captured multiple times of spontaneous binding and unbinding of a potent inhibitor MLN-4760 in the ACE2 receptor. With ligand far away in the unbound state, the ACE2 receptor samples distinct Open, Partially Open, Closed, and Fully Closed conformations. Upon ligand binding to the active site, conformational ensemble of the ACE2 receptor is biased toward the Closed state as observed in the X-ray experimental structure. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the highly flexible ACE2 receptor, which is expected to facilitate rational drug design targeting human ACE2 against coronaviruses and other related human diseases.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Antivirais/química , COVID-19/tratamento farmacológico , Imidazóis/química , Leucina/análogos & derivados , Inibidores de Proteases/química , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , COVID-19/metabolismo , Domínio Catalítico , Desenho de Fármacos , Humanos , Imidazóis/farmacologia , Leucina/química , Leucina/farmacologia , Ligantes , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , SARS-CoV-2/metabolismo
16.
Neurochem Res ; 46(8): 2143-2153, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34014488

RESUMO

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation channel that is involved in the development of neuropathic pain. P2X7 receptor (P2X7) belongs to a class of ATP-gated nonselective cation channels that plays an important role in neuropathic pain. Nevertheless, little is known about the interaction between them for neuropathic pain. In this paper, we investigated role of TRPV4-P2X7 pathway in neuropathic pain. We evaluated the effect of TRPV4-P2X7 pathway on neuropathic pain in a chronic compression of the dorsal root ganglion (DRG) (hereafter termed CCD) model. We analyzed the effect of P2X7 on mechanical and thermal hyperalgesia mediated by TRPV4 in CCD. Furthermore, we assessed the effect of TRPV4 on the expression of P2X7 and the release of IL-1ß and IL-6 in DRG after CCD. We found that intraperitoneal injection of TRPV4 agonist GSK-1016790A led to a significant increase of mechanical and thermal hyperalgesia in CCD, which was partially suppressed by P2X7 blockade with antagonist Brilliant Blue G (BBG). Then, we further noticed that GSK-1016790A injection increased the P2X7 expression of CCD, which was decreased by TRPV4 blockade with antagonist RN-1734 and HC-067047. Furthermore, we also discovered that the expressions of IL-1ß and IL-6 were upregulated by GSK-1016790A injection but reduced by RN-1734 and HC-067047. Our results provide evidence that P2X7 contributes to development of neuropathic pain mediated by TRPV4 in the CCD model, which may be the basis for treatment of neuropathic pain relief.


Assuntos
Gânglios Espinais/metabolismo , Síndromes de Compressão Nervosa/fisiopatologia , Neuralgia/fisiopatologia , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais/fisiologia , Canais de Cátion TRPV/metabolismo , Animais , Gânglios Espinais/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Morfolinas/farmacologia , Síndromes de Compressão Nervosa/tratamento farmacológico , Neuralgia/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/farmacologia , Pirróis/farmacologia , Ratos Wistar , Corantes de Rosanilina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores
17.
Bioorg Chem ; 112: 104921, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33933805

RESUMO

l-Type amino acid transporter 1 (LAT1) is an interesting protein due to its peculiar expression profile. It can be utilized not only as a carrier for improved or targeted drug delivery, e.g., into the brain but also as a target protein by which amino acid supply can be restricted, e.g., from the cancer cells. The recognition and binding processes of LAT1-ligands, such as amino acids and clinically used small molecules, including l-dopa, gabapentin, and melphalan, are today well-known. Binding to LAT1 is crucial, particularly when designing the LAT1-inhibitors. However, it will not guarantee effective translocation across the cell membrane via LAT1, which is a definite requirement for LAT1-substrates, such as drugs that elicit their pharmacological effects inside the cells. Therefore, in the present study, the accumulation of known LAT1-utilizing compounds into the selected LAT1-expressing cancer cells (MCF-7) was explored experimentally over a time period. The differences found among the transport efficiency and affinity of the studied compounds for LAT1 were subsequently explained by docking the ligands into the human LAT1 model (based on the recent cryo-electron microscopy structure). Thus, the findings of this study clarify the favorable structural requirements of the size, shape, and polarity of the ligands that support the translocation and effective transport across the cell membrane via LAT1. This knowledge can be applied in future drug design to attain improved or targeted drug delivery and hence, successful LAT1-utilizing drugs with increased therapeutic effects.


Assuntos
Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Leucina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Leucina/química , Células MCF-7 , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas
18.
FASEB J ; 35(5): e21526, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33813773

RESUMO

Discovering safe and effective drugs that promote neuron regeneration is an essential strategy for the recovery of central nervous system injuries. In this study, we found that L-leucine, an essential amino acid obtained from both supplements and food sources, could dramatically boost axonal outgrowth and regeneration. First, the effects of L-leucine on neurons were evaluated by cell apoptosis, survival, and death assays, and the results showed no changes in these processes after treatment. By live cell imaging, L-leucine was found to remarkably increase axonal length and growth velocity after axotomy. We also verified that L-leucine enhanced p-mTOR/p-S6K activation in neurons by testing with an mTOR inhibitor, rapamycin. Thereafter, we investigated the effects of L-leucine on the spinal cord injury in vivo. A mouse model of spinal cord hemi-section was established, and L-leucine was administered by tail intravenous injection. Basso mouse scale values revealed that L-leucine could improve functional recovery after injury. It was also notable that L-leucine treatment promoted axon growth across chondroitin sulfate proteoglycan (CSPG) areas. Furthermore, we used CSPGs as inhibitory environmental cues and clarified that L-leucine significantly enhanced axonal outgrowth and regeneration by promoting p-mTOR and p-S6K activation. Therefore, our study is the first to report that L-leucine promotes axonal regeneration in vitro and in vivo and could be candidate drug for axonal re-growth and nervous functional recovery.


Assuntos
Leucina/farmacologia , Regeneração Nervosa , Crescimento Neuronal , Neurônios/citologia , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/terapia , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Serina-Treonina Quinases TOR/genética
19.
Eur J Pharmacol ; 902: 174113, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-33901460

RESUMO

The transient receptor potential vanilloid channel 4 (TRPV4) is associated with the development of several pathologies, particularly gastric disorders. However, there are no studies associating this receptor with the pathophysiology of gastric erosions. The aim of this study was to investigate the role of TRPV4 in the development of ethanol-induced gastric damage in vivo. Gastric lesions were induced by ethanol in Swiss mice pretreated with TRPV4 antagonists, GSK2193874 (0.1; 0.3 and 0.9 mg/kg) or Ruthenium red (0.03; 0.1 or 0.3 mg/kg) or its agonist, GSK1016790A (0.9 mg/kg). Gastric mucosal samples were taken for histopathology, immunohistochemistry, atomic force microscopy and evaluation of antioxidant parameters. The gastric mucus content and TRPV4 mRNA expression were analyzed. Ethanol exposure induced upregulation of gastric mRNA and protein expression of TRPV4. TRPV4 blockade promoted gastroprotection against ethanol-induced injury on macro- and microscopic levels, leading to reduced hemorrhage, cell loss and edema and enhanced gastric mucosal integrity. Moreover, an increase in superoxide dismutase (SOD) and glutathione (GSH) activity was observed, followed by a decrease in malondialdehyde (MDA) levels. TRPV4 blockade during alcohol challenge reestablished gastric mucus content. The combination of TRPV4 agonist and ethanol revealed macroscopic exacerbation of gastric damage area. Our results confirmed the association of TRPV4 with the development of gastric injury, showing the importance of this receptor for further investigations in the field of gastrointestinal pathophysiology and pharmacology.


Assuntos
Úlcera Gástrica/metabolismo , Úlcera Gástrica/fisiopatologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismo , Animais , Edema/induzido quimicamente , Edema/metabolismo , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/lesões , Mucosa Gástrica/metabolismo , Glutationa/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Leucina/uso terapêutico , Masculino , Malondialdeído/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Rutênio Vermelho/farmacologia , Rutênio Vermelho/uso terapêutico , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/patologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Superóxido Dismutase/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Regulação para Cima/efeitos dos fármacos
20.
Molecules ; 26(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802860

RESUMO

The COVID-19 outbreak continues to spread worldwide at a rapid rate. Currently, the absence of any effective antiviral treatment is the major concern for the global population. The reports of the occurrence of various point mutations within the important therapeutic target protein of SARS-CoV-2 has elevated the problem. The SARS-CoV-2 main protease (Mpro) is a major therapeutic target for new antiviral designs. In this study, the efficacy of PF-00835231 was investigated (a Mpro inhibitor under clinical trials) against the Mpro and their reported mutants. Various in silico approaches were used to investigate and compare the efficacy of PF-00835231 and five drugs previously documented to inhibit the Mpro. Our study shows that PF-00835231 is not only effective against the wild type but demonstrates a high affinity against the studied mutants as well.


Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Indóis/química , Indóis/farmacologia , Leucina/química , Leucina/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Sítios de Ligação , COVID-19/tratamento farmacológico , Simulação por Computador , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Bases de Dados de Proteínas , Diarilquinolinas/química , Diarilquinolinas/farmacologia , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrobenzenos/química , Nitrobenzenos/farmacologia , Nitrofenóis/química , Nitrofenóis/farmacologia , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Prolina/análogos & derivados , Prolina/química , Prolina/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética
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