RESUMEN
The mechanisms underlying endothelial dysfunction in Type 1 and Type 2 diabetes (T1DM and T2DM) are unresolved. The red blood cells (RBCs) with increased arginase activity induce endothelial dysfunction in T2DM, but the implications of RBCs and the role of arginase inhibition in T1DM are unexplored. We aimed to investigate the differences in endothelial function in patients with T1DM and T2DM, with focus on RBCs and arginase. Thirteen patients with T1DM and twenty-six patients with T2DM, matched for HbA1c and sex were included. In vivo endothelium-dependent and -independent vasodilation (EDV and EIDV) were assessed by venous occlusion plethysmography before and after administration of an arginase inhibitor. RBCs were co-incubated with rat aortic segments for 18h followed by evaluation of endothelium-dependent (EDR) and -independent relaxation (EIDR) in isolated organ chambers. In vivo EDV, but not EIDV, was significantly impaired in patients with T2DM compared with patients with T1DM. Arginase inhibition resulted in improved EDV only in T2DM. RBCs from patients with T2DM induced impaired EDR but not EIDR in isolated aortic segments, whereas RBCs from patients with T1DM did not affect EDR nor EIDR. The present study demonstrates markedly impaired EDV in patients with T2DM in comparison with T1DM. In addition, it highlights the divergent roles of RBCs and arginase in mediating endothelial dysfunction in T1DM and T2DM. While endothelial dysfunction is mediated via RBCs and arginase in T2DM, these phenomena are not prominent in T1DM thereby indicating distinct differences in underlying mechanisms.
Asunto(s)
Arginasa , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Endotelio Vascular , Eritrocitos , Vasodilatación , Humanos , Arginasa/metabolismo , Arginasa/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/fisiopatología , Diabetes Mellitus Tipo 2/sangre , Masculino , Eritrocitos/enzimología , Eritrocitos/metabolismo , Femenino , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 1/sangre , Persona de Mediana Edad , Endotelio Vascular/fisiopatología , Animales , Adulto , Anciano , Aorta/fisiopatología , Inhibidores Enzimáticos/farmacologíaRESUMEN
Macrophages polarize into alternatively activated M2 macrophages through interleukin (IL)-4, and they express high levels of arginase-1, which promotes anti-inflammatory responses. Several studies have confirmed the anti-inflammatory effects of cyclin-dependent kinase (CDK) 8/19 inhibition, and hence, numerous CDK8/19 inhibitors, such as BRD6989, have been developed. However, the effects of CDK8/19 inhibitors on arginase-1 expression in macrophages have not yet been elucidated. This study investigated the effects of CDK8/19 inhibitor on arginase-1 expression in IL-4-activated macrophages. The results showed that BRD6989 increased arginase-1 expression transcriptionally in murine peritoneal macrophages and the murine macrophage cell line RAW264.7 in an IL-4-dependent manner. In addition, the results indicated that BRD6989 enhances signal transducer and activator of transcription (STAT) 6 phosphorylation. Meanwhile, BRD6989 exhibited the capability to activate p38 mitogen-activated protein kinase (MAPKï¼ even in the absence of IL-4 stimulation. Moreover, we observed that a p38 MAPK inhibitor suppressed the BRD6989-induced increase in arginase-1 expression. Besides, BRD6989 increased the surface expression of CD206, an M2 macrophage marker. Thus, this study demonstrated for the first time that CDK8/19 inhibition increases arginase-1 expression, suggesting that this mechanism involves the activation of STAT6 and p38 MAPK. This finding implies that CDK8/19 inhibition may facilitate the production of anti-inflammatory M2 macrophages.
Asunto(s)
Arginasa , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Interleucina-4 , Factor de Transcripción STAT6 , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Arginasa/metabolismo , Arginasa/antagonistas & inhibidores , Factor de Transcripción STAT6/metabolismo , Ratones , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Células RAW 264.7 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Interleucina-4/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fosforilación/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Activación Enzimática/efectos de los fármacos , Flavonoides , Piperidinas , Quinasa 9 Dependiente de la CiclinaRESUMEN
Venous leg ulcers (VLUs) pose a growing healthcare challenge due to aging, obesity, and sedentary lifestyles. Despite various treatments available, addressing the complex nature of VLUs remains difficult. In this context, this study investigates repurposing boronated drugs to inhibit arginase 1 activity for VLU treatment. The molecular docking study conducted by Schrodinger GLIDE targeted the binuclear manganese cluster of arginase 1 enzyme (2PHO). Further, the ligand-protein complex was subjected to molecular dynamic studies at 500â¯ns in Gromacs-2019.4. Trajectory analysis was performed using the GROMACS simulation package of protein RMSD, RMSF, RG, SASA, and H-Bond. The docking study revealed intriguing results where the tavaborole showed a better docking score (-3.957 Kcal/mol) compared to the substrate L-arginine (-3.379 Kcal/mol) and standard L-norvaline (-3.141 Kcal/mol). Tavaborole interaction with aspartic acid ultimately suggests that the drug molecule binds to the catalytic site of arginase 1, potentially influencing the enzyme's function. The dynamics study revealed the compounds' stability and compactness of the protein throughout the simulation. The RMSD, RMSF, SASA, RG, inter and intra H-bond, PCA, FEL, and MMBSA studies affirmed the ligand-protein and protein complex flexibility, compactness, binding energy, van der waals energy, and solvation dynamics. These results revealed the stability and the interaction of the ligand with the catalytic site of arginase 1 enzyme, triggering the study towards the VLU treatment.
Asunto(s)
Arginasa , Simulación del Acoplamiento Molecular , Arginasa/antagonistas & inhibidores , Arginasa/metabolismo , Arginasa/química , Humanos , Úlcera Varicosa/tratamiento farmacológico , Compuestos de Boro/química , Compuestos de Boro/farmacología , Reposicionamiento de Medicamentos , Simulación de Dinámica Molecular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Estructura MolecularRESUMEN
Three neo-clerodane diterpenoids, including two new tinocordifoliols A (1) and B (2) and one known tinopanoid R (3), were isolated from the ethyl acetate-soluble fraction of the 70% ethanol extract of Tinospora cordifolia stems. The structures were elucidated by various spectroscopic methods, including one dimensional (1D) and 2D-NMR, high resolution-electrospray ionization (HR-ESI)-MS, and electronic circular dichroism (ECD) data. The T. cordifolia extract and all isolated compounds 1-3 possessed arginase I inhibitory activities. Among them, 3 exhibited moderate competitive inhibition of human arginase I (IC50 = 61.9 µM). Furthermore, docking studies revealed that the presence of a ß-substituted furan in 3 may play a key role in the arginase I inhibitory activities.
Asunto(s)
Arginasa , Diterpenos de Tipo Clerodano , Inhibidores Enzimáticos , Simulación del Acoplamiento Molecular , Tallos de la Planta , Tinospora , Tinospora/química , Arginasa/antagonistas & inhibidores , Arginasa/metabolismo , Diterpenos de Tipo Clerodano/farmacología , Diterpenos de Tipo Clerodano/química , Diterpenos de Tipo Clerodano/aislamiento & purificación , Humanos , Tallos de la Planta/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/aislamiento & purificación , Relación Estructura-Actividad , Estructura Molecular , Conformación Molecular , Relación Dosis-Respuesta a DrogaRESUMEN
Leishmaniasis is a disease caused by a protozoan of the genus Leishmania, affecting millions of people, mainly in tropical countries, due to poor social conditions and low economic development. First-line chemotherapeutic agents involve highly toxic pentavalent antimonials, while treatment failure is mainly due to the emergence of drug-resistant strains. Leishmania arginase (ARG) enzyme is vital in pathogenicity and contributes to a higher infection rate, thus representing a potential drug target. This study helps in designing ARG inhibitors for the treatment of leishmaniasis. Py-CoMFA (3D-QSAR) models were constructed using 34 inhibitors from different chemical classes against ARG from L. (L.) amazonensis (LaARG). The 3D-QSAR predictions showed an excellent correlation between experimental and calculated pIC50 values. The molecular docking study identified the favorable hydrophobicity contribution of phenyl and cyclohexyl groups as substituents in the enzyme allosteric site. Molecular dynamics simulations of selected protein-ligand complexes were conducted to understand derivatives' interaction modes and affinity in both active and allosteric sites. Two cinnamide compounds, 7g and 7k, were identified, with similar structures to the reference 4h allosteric site inhibitor. These compounds can guide the development of more effective arginase inhibitors as potential antileishmanial drugs.
Asunto(s)
Arginasa , Inhibidores Enzimáticos , Leishmania , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Arginasa/antagonistas & inhibidores , Arginasa/química , Arginasa/metabolismo , Leishmania/enzimología , Leishmania/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Relación Estructura-Actividad Cuantitativa , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Sitio Alostérico , Antiprotozoarios/farmacología , Antiprotozoarios/química , Dominio CatalíticoRESUMEN
Arginase is an enzyme responsible for converting arginine, a semi-essential amino acid, to ornithine and urea. Arginine depletion suppresses immunity via multiple mechanisms including inhibition of T-cell and NK cell proliferation and activity. Arginase inhibition is therefore an attractive mechanism to potentially reverse immune suppression and thus has been explored as a therapy for oncology and respiratory indications. Small molecules targeting arginase present significant bioanalytical challenges for in vitro and in vivo characterization as inhibitors of arginase are typically hydrophilic in nature. The resulting low or negative LogD characteristics are incompatible with common analytical methods such as RP-ESI-MS/MS. Accordingly, a sensitive, high-throughput bioanalytical method was developed by incorporating benzoyl chloride derivatization to increase the hydrophobic characteristics of these polar analytes. Samples were separated by reversed phase chromatography on a Waters XBridge BEH C18 3.5 µm, 30 × 3â¯mm column using gradient elution. The mass spec was operated in positive mode using electrospray ionization. The m/z 434.1â176.1, 439.4â181.2, 334.9â150.0 and 339.9â150.0 for AZD0011, AZD0011 IS, AZD0011-PL and AZD0011-PL IS respectively were used for quantitation. The linear calibration range of the assay was 1.00-10,000â¯ng/mL with QC values of 5, 50 and 500â¯ng/mL. The qualified method presented herein exhibits a novel, robust analytical performance and was successfully applied to evaluate the in vivo ADME properties of boronic acid-based arginase inhibitor prodrug AZD0011 and its active payload AZD0011-PL.
Asunto(s)
Arginasa , Espectrometría de Masas en Tándem , Arginasa/antagonistas & inhibidores , Espectrometría de Masas en Tándem/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía de Fase Inversa/métodos , Animales , Cromatografía Liquida/métodos , Humanos , Cromatografía Líquida de Alta Presión/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Reproducibilidad de los Resultados , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Elevation of arginase enzyme activity in the lung contributes to the pathogenesis of various chronic inflammatory diseases and infections. Inhibition of arginase expression and activity is able to alleviate those effects. Here, we investigated the immunomodulatory effect of arginase inhibitor in C. neoformans infection. In the pulmonary cryptococcosis model that was shown to recapitulate human infection, we found arginase expression was excessively induced in the lung during the late stage of infection. To inhibit the activity of arginase, we administered a specific arginase inhibitor, nor-NOHA, during C. neoformans infection. Inhibition of arginase reduced eosinophil infiltration and level of IL-13 secretion in the lungs. Whole lung transcriptome RNA-sequencing analysis revealed that treatment with nor-NOHA resulted in shifting the Th2-type gene expression patterns induced by C. neoformans infection to the Th1-type immune profile, with higher expression of cytokines Ifng, Il6, Tnfa, Csf3, chemokines Cxcl9 and Cxcl10 and transcription factor Stat1. More importantly, mice treated with arginase inhibitor had more infiltrating brain leukocytes and enhanced gene expression of Th1-associated cytokines and chemokines that are known to be essential for protection against C. neoformans infection. Inhibition of arginase dramatically attenuated spleen and brain infection, with improved survival. Taken together, these studies demonstrated that inhibiting arginase activity induced by C. neoformans infection can modulate host immune response by enhancing protective type-1 immune response during C. neoformans infection. The inhibition of arginase activity could be an immunomodulatory target to enhance protective anti-cryptococcal immune responses.
Asunto(s)
Arginasa , Arginina/análogos & derivados , Criptococosis , Cryptococcus neoformans , Ratones Endogámicos C57BL , Animales , Arginasa/metabolismo , Arginasa/antagonistas & inhibidores , Arginasa/genética , Criptococosis/inmunología , Criptococosis/tratamiento farmacológico , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/efectos de los fármacos , Ratones , Pulmón/inmunología , Pulmón/patología , Pulmón/efectos de los fármacos , Citocinas/metabolismo , Citocinas/inmunología , Femenino , Modelos Animales de Enfermedad , Enfermedades Pulmonares Fúngicas/inmunología , Enfermedades Pulmonares Fúngicas/tratamiento farmacológico , Humanos , Células Th2/inmunología , Células Th2/efectos de los fármacos , Células TH1/inmunología , Células TH1/efectos de los fármacos , Encéfalo/inmunología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéuticoRESUMEN
BACKGROUND: Retifanlimab is a humanized monoclonal antibody targeting programmed death protein-1, and INCB001158 is an oral arginase inhibitor. This phase Ib study investigated retifanlimab, INCB001158, and their combination in Japanese patients with advanced solid tumors. METHODS: Patients received retifanlimab (500 mg every 4 weeks [Q4W] i.v.) or escalating doses of INCB001158 (75 or 100 mg twice daily [BID]) monotherapy in Part 1 and combination of retifanlimab (500 mg Q4W) and INCB001158 (100 mg BID) in Part 2. Primary endpoints were safety, tolerability, dose-limiting toxicities (DLTs), and determination of recommended phase II doses in Japanese patients. RESULTS: Eighteen patients (retifanlimab or INCB001158 monotherapy and combination; n = 6 each) were enrolled at 2 sites in Japan. There were no DLTs, fatal adverse events (AEs), or discontinuations due to AEs. Rash (all grade 1) was the most common treatment-emergent AE with retifanlimab (n = 6). Treatment-related AEs were reported with retifanlimab (n = 4) or INCB001158 (n = 2) monotherapy and with combination (n = 4); an immune-related AE (thyroid disorder, grade 2) was reported with combination. Two responses were observed with retifanlimab monotherapy (1 complete, 1 partial) and 1 stable disease (SD), for an overall response rate of 33.3% (95% confidence interval [CI], 4.3-77.7) and disease control rate (DCR) of 50% (95% CI, 11.8-88.2). Three patients had SD with INCB001158 monotherapy (DCR 50%; 95% CI, 11.8-88.2). No responses or SD were observed with combination therapy. CONCLUSION: Retifanlimab, INCB001158, and their combination had acceptable safety profiles. Promising retifanlimab antitumor activity warrants further investigation in Japanese patients.
Asunto(s)
Arginasa , Neoplasias , Humanos , Femenino , Masculino , Neoplasias/tratamiento farmacológico , Persona de Mediana Edad , Anciano , Arginasa/antagonistas & inhibidores , Adulto , Japón , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Pueblos del Este de AsiaRESUMEN
Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.
Asunto(s)
Antiprotozoarios/farmacología , Arginasa/antagonistas & inhibidores , Bencimidazoles/farmacología , Leishmania mexicana/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antiprotozoarios/química , Arginasa/metabolismo , Bencimidazoles/química , Línea Celular , Descubrimiento de Drogas , Humanos , Leishmania mexicana/enzimología , Leishmania mexicana/fisiología , Leishmaniasis Cutánea/tratamiento farmacológico , Ratones , Modelos Moleculares , Proteínas Protozoarias/metabolismoRESUMEN
The influenza virus infection poses a serious health threat worldwide. Myeloid cells play pivotal roles in regulating innate and adaptive immune defense. A disintegrin and metalloproteinase (ADAM) family of proteins contributes to various immune responses; however, the role of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in influenza virus infection remains largely unknown. Herein, we investigated its role, focusing on myeloid cells, during influenza virus infection in mice. ADAM10 gene (Adam10)flox/flox/Lyz2-Cre (Adam10ΔLyz2) and control Adam10flox/flox mice were intranasally infected with 200 plaque-forming units of influenza virus A/H1N1/PR8/34. Adam10ΔLyz2 mice exhibited a significantly higher mortality rate, stronger lung inflammation, and a higher virus titer in the lungs than control mice. Macrophages and inflammatory cytokines, such as TNF-α, IL-1ß, and CCL2, were increased in bronchoalveolar lavage fluid from Adam10ΔLyz2 mice following infection. CD11b+Ly6G-F4/80+ myeloid cells, which had an inflammatory monocyte/macrophage-like phenotype, were significantly increased in the lungs of Adam10ΔLyz2 mice. Adoptive transfer experiments suggested that these cells likely contributed to the poorer prognosis in Adam10ΔLyz2 mice. Seven days after infection, CD11b+Ly6G-F4/80+ lung cells exhibited significantly higher arginase-1 expression levels in Adam10ΔLyz2 mice than in control mice, whereas an arginase-1 inhibitor improved the prognosis of Adam10ΔLyz2 mice. Enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF)/GM-CSF receptor signaling likely contributed to this process. Collectively, these results indicate that myeloid ADAM10 protects against influenza virus pneumonia and may be a promising therapeutic target.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Arginasa/biosíntesis , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Células Mieloides/inmunología , Infecciones por Orthomyxoviridae/patología , Proteína ADAM10/genética , Traslado Adoptivo/métodos , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Arginasa/antagonistas & inhibidores , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/análisis , Inmunidad Innata/inmunología , Macrófagos/trasplante , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/trasplante , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/prevención & control , Pronóstico , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismoRESUMEN
COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19 that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.
Asunto(s)
COVID-19/inmunología , Granulocitos/inmunología , Células Supresoras de Origen Mieloide/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antivirales/administración & dosificación , Arginasa/antagonistas & inhibidores , Arginasa/metabolismo , Arginina/administración & dosificación , Arginina/sangre , Arginina/metabolismo , Infecciones Asintomáticas , COVID-19/sangre , COVID-19/diagnóstico , Estudios de Casos y Controles , Quimioterapia Combinada/métodos , Inhibidores Enzimáticos/administración & dosificación , Femenino , Granulocitos/metabolismo , Voluntarios Sanos , Humanos , Interferón Tipo I/metabolismo , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/metabolismo , Índice de Severidad de la Enfermedad , Transducción de Señal/inmunología , Linfocitos T/inmunología , Tratamiento Farmacológico de COVID-19RESUMEN
Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca2+-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca2+ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca2+ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca2+ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1-/-) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca2+ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation. [BMB Reports 2021; 54(10): 516-521].
Asunto(s)
Arginasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estilbenos/farmacología , Animales , Arginasa/antagonistas & inhibidores , Arginasa/efectos de los fármacos , Arginina/genética , Arginina/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Estilbenos/metabolismoRESUMEN
Cutaneous squamous cell carcinomas (cSCC) are among the most commonly diagnosed malignancies, causing significant morbidity and mortality. Tumor-associated macrophage (TAM) expression of arginase is implicated in tumor progression, and therapeutic use of arginase inhibitors has been studied in various cancers. However, investigating potential cSCC immunotherapies including arginase inhibition in pre-clinical models is hampered by the lack of appropriate tumor models in immunocompetent mice. PDV is a cSCC cell line derived from chemical carcinogenesis of mouse keratinocytes. PDVC57 cells were derived from a PDV tumor in C57BL/6 (B6) mice. Unlike PDV, PDVC57 tumors grow consistently in B6 mice, and have increased TAMs, decreased dendritic and T cell intra-tumor infiltration. Arginase inhibition in cSCC tumors using Nω-hydroxy-nor-arginine (nor-NOHA) reduced tumor growth in B6 mice but not immunodeficient Rag1-deficient mice. nor-NOHA administration increased dendritic and T cell tumor-infiltration and PD-1 expression. The combination of nor-NOHA and anti-PD-1 therapy with nivolumab enhanced anti-PD-1 therapeutic efficacy. This study demonstrates the therapeutic potential of transcutaneous arginase inhibition in cSCC. A competent immune microenvironment is required for tumor growth inhibition using this arginase inhibitor. Synergistic co-inhibition of tumor growth in these results, supports further examination of transcutaneous arginase inhibition as a therapeutic modality for cSCC.
Asunto(s)
Antineoplásicos/administración & dosificación , Arginasa/antagonistas & inhibidores , Arginina/análogos & derivados , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Administración Cutánea , Animales , Antineoplásicos/farmacología , Arginina/administración & dosificación , Arginina/farmacología , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunologíaRESUMEN
A simple and rapid Nano LC method has been developed for the screening of arginase inhibitors. The method is based on the immobilization of biotinylated arginase on a neutravidin functionalized nano HPLC capillary column. The arginase immobilization step performed by frontal analysis is very fast and only takes a few minutes. The miniaturized capillary column of 170 nL (length 5 cm, internal diameter 75 µm) significantly decreased the required amount of used enzyme (25 pmol). This was of significance importance when working with less available or expensive purified enzyme. Non-selective adsorption of the organic monolith matrix was reduced (<6%) and the arginase efficient yield was high (92%). The resultant affinity capillary columns showed excellent repeatability and long lifetime. The arginase reaction product was achieved within 60 s and the immobilized arginase retained 97% of the initial activity beyond 90 days. This novel approach can thus be used for the fast evaluation of recognition assay induced bya series of inhibitor molecules (caffeic acid phenylamide, chlorogenic acid, piceatannol, nor-NOHA acetate) and plant extracts.
Asunto(s)
Arginasa/antagonistas & inhibidores , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Inhibidores Enzimáticos/análisis , Extractos Vegetales/químicaRESUMEN
Polyphenolic enriched extracts from two species of Cyperus, Cyperus glomeratus and Cyperus thunbergii, possess mammalian arginase inhibitory capacities, with the percentage inhibition ranging from 80% to 95% at 100 µg/mL and 40% to 64% at 10 µg/mL. Phytochemical investigation of these species led to the isolation and identification of two new natural stilbene oligomers named thunbergin A-B (1-2), together with three other stilbenes, trans-resveratrol (3), trans-scirpusin A (4), trans-cyperusphenol A (6), and two flavonoids, aureusidin (5) and luteolin (7), which were isolated for the first time from C.thunbergii and C. glomeratus. Structures were established on the basis of the spectroscopic data from MS and NMR experiments. The arginase inhibitory activity of compounds 1-7 was evaluated through an in vitro arginase inhibitory assay using purified liver bovine arginase. As a result, five compounds (1, 4-7) showed significant inhibition of arginase, with IC50 values between 17.6 and 60.6 µM, in the range of those of the natural arginase inhibitor piceatannol (12.6 µM). In addition, methanolic extract from Cyperus thunbergii exhibited an endothelium and NO-dependent vasorelaxant effect on thoracic aortic rings from rats and improved endothelial dysfunction in an adjuvant-induced arthritis rat model.
Asunto(s)
Arginasa/antagonistas & inhibidores , Cyperus/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/fisiopatología , Benzofuranos/química , Benzofuranos/aislamiento & purificación , Benzofuranos/farmacología , Calamus , Bovinos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/aislamiento & purificación , Flavonoides/química , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Masculino , Metanol , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Ratas , Resveratrol/química , Resveratrol/aislamiento & purificación , Resveratrol/farmacología , Estilbenos/química , Estilbenos/aislamiento & purificación , Estilbenos/farmacología , Vasodilatadores/química , Vasodilatadores/aislamiento & purificación , Vasodilatadores/farmacologíaRESUMEN
Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid-metabolizing enzymes.
Asunto(s)
Arginasa/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Arginasa/análisis , Arginasa/antagonistas & inhibidores , Arginina/metabolismo , Bioensayo , Biotina/metabolismo , Dimerización , Descubrimiento de Drogas/métodos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Ornitina/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Arginine metabolism mediated by arginases plays a critical role in cell and tissue function. The arginine hydrolysis is deeply involved in the urea cycle, which helps the kidney excrete ammonia from blood. Upregulation of arginases affects microenvironment stability due to the presence of excess urea in blood. To regulate the arginase activities properly, a synthetic peptide based on the structure of human arginase I was designed and assessed. Preliminary data shows it inhibits human arginase I and II with an IC50 of 2.4 ± 0.3 and 1.8 ± 0.1 mmol, respectively. Our kinetic analysis indicates the inhibition is not competitive with substrate - suggesting an allosteric mechanism. This result provides a step towards specific inhibitors design.
Asunto(s)
Arginasa/antagonistas & inhibidores , Péptidos/química , Regulación Alostérica , Arginasa/química , Arginasa/metabolismo , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.
Asunto(s)
Arginasa/química , Proteínas Bacterianas/química , Helicobacter pylori/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/química , Animales , Arginasa/antagonistas & inhibidores , Arginasa/genética , Arginina/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Catálisis , Cobalto/metabolismo , Secuencia Conservada , Polarización de Fluorescencia , Gastritis/microbiología , Gastritis/veterinaria , Helicobacter/enzimología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/veterinaria , Helicobacter pylori/genética , Humanos , Hidrólisis , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación Missense , Mutación Puntual , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
PURPOSE: Radiation therapy (RT) is widely used in the treatment of cancer. Unfortunately, RT alone is insufficient to control the disease in most cases, as regrowth after irradiation still occur. Thus, it would be meaningful to explore the underlying mechanism of tumor regrowth after irradiation. Myeloid-derived suppressor cells (MDSCs) contribute to the immunosuppressive tumor microenvironment and hinder the therapeutic efficacy of RT. However, it is unclear whether MDSCs-mediated immune suppression contributes to local relapse after irradiation. In this article, we tried to figure out how MDSCs sabotage the therapeutic effect of RT, and tried to determine the potential synergistic effect of combination between targeting MDSCs and RT. METHODS AND MATERIALS: A syngeneic murine model of Lewis lung cancer was used. The abundance of tumor infiltrating MDSCs and tumor growth after irradiation was assessed. The percentage and functional state of CD8+ T cells were measured by flow cytometry, with or without polymorphonuclear (PMN)-MDSCs depletion. Arginase 1 (ARG1) expression and activity of MDSCs were examined by hematoxylin and eosin staining and flow cytometry. ARG1 inhibitor and phosphodiesterase 5 inhibitor sildenafil were administered after RT to figure out the underlying mechanism of MDSCs-mediated immunosuppression. RESULTS: We demonstrated that irradiation recruited MDSCs, especially the polymorphonuclear subset, into the tumor microenvironment. PMN-MDSCs inhibited the CD8+ T cell response by elevating ARG1 expression. Selective depletion of PMN-MDSCs or inhibition on ARG1 promoted the infiltration and activation of intratumoral CD8+ T cells, and delayed tumor regrowth after irradiation. We showed that sildenafil reduced the accumulation and ARG1 expression of PMN-MDSCs after irradiation, thus abrogating the MDSCs-mediated immunosuppression. CONCLUSIONS: Our results have suggested that PMN-MDSCs participate in the irradiation-induced immune suppression through ARG1 activation. We have also found that sildenafil has the potential to facilitate antitumor immunity, which provides a new alternative to delay tumor recurrence after RT.