RESUMEN
Cathepsin C (Cat C) participates in inflammation and immune regulation by affecting the activation of neutrophil serine proteases (NSPs). Therefore, cathepsin C is an attractive target for treatment of NSP-related inflammatory diseases. Here, the complete discovery process of the first potent "non-peptidyl non-covalent cathepsin C inhibitor" was described with hit finding, structure optimization, and lead discovery. Starting with hit 14, structure-based optimization and structure-activity relationship study were comprehensively carried out, and lead compound 54 was discovered as a potent drug-like cathepsin C inhibitor both in vivo and in vitro. Also, compound 54 (with cathepsin C Enz IC50 = 57.4 nM) exhibited effective anti-inflammatory activity in an animal model of chronic obstructive pulmonary disease. These results confirmed that the non-peptidyl and non-covalent derivative could be used as an effective cathepsin C inhibitor and encouraged us to continue further drug discovery on the basis of this finding.
Asunto(s)
Antiinflamatorios/uso terapéutico , Catepsina C/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Pirimidinas/uso terapéutico , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/metabolismo , Antiinflamatorios/toxicidad , Catepsina C/metabolismo , Línea Celular Tumoral , Descubrimiento de Drogas , Humanos , Inflamación/etiología , Inflamación/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Ratones Endogámicos ICR , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/toxicidad , Unión Proteica , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/patología , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Pirimidinas/toxicidad , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Cyanobacteria are microorganisms with photosynthetic mechanisms capable of colonizing several distinct environments worldwide. They can produce a vast spectrum of bioactive compounds with different properties, resulting in an improved adaptative capacity. Their richness in secondary metabolites is related to their unique and diverse metabolic apparatus, such as Non-Ribosomal Peptide Synthetases (NRPSs). One important class of peptides produced by the non-ribosomal pathway is anabaenopeptins. These cyclic hexapeptides demonstrated inhibitory activity towards phosphatases and proteases, which could be related to their toxicity and adaptiveness against zooplankters and crustaceans. Thus, this review aims to identify key features related to anabaenopeptins, including the diversity of their structure, occurrence, the biosynthetic steps for their production, ecological roles, and biotechnological applications.
Asunto(s)
Péptidos Cíclicos , Animales , Ecología , Estructura Molecular , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Péptidos Cíclicos/toxicidad , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/toxicidadRESUMEN
Liver malignant tumors (LMTs) represent a serious adverse drug event associated with drug-induced liver injury. Increases in endocrine-disrupting chemicals (EDCs) have attracted attention in recent years, due to their liver function-inhibiting abilities. Exposure to EDCs can induce nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, which are major etiologies of LMTs, through interaction with nuclear receptors (NR) and stress response pathways (SRs). Therefore, exposure to potential EDC drugs could be associated with drug-induced LMTs. However, the drug classes associated with LMTs and the molecular initiating events (MIEs) that are specific to these drugs are not well understood. In this study, using the Food and Drug Administration Adverse Event Reporting System, we detected LMT-inducing drug signals based on adjusted odds ratios. Furthermore, based on the hypothesis that drug-induced LMTs are triggered by NR and SR modulation of potential EDCs, we used the quantitative structure-activity relationship platform for toxicity prediction to identify potential MIEs that are specific to LMT-inducing drug classes. Events related to cell proliferation and apoptosis, DNA damage, and lipid accumulation were identified as potential MIEs, and their relevance to LMTs was supported by the literature. The findings of this study may contribute to drug development and research, as well as regulatory decision making.
Asunto(s)
Sistemas de Registro de Reacción Adversa a Medicamentos/estadística & datos numéricos , Enfermedad Hepática Inducida por Sustancias y Drogas/epidemiología , Bases de Datos Factuales/estadística & datos numéricos , Neoplasias Hepáticas/epidemiología , United States Food and Drug Administration/estadística & datos numéricos , Carbamatos/efectos adversos , Carbamatos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Predicción , Humanos , Imidazoles/efectos adversos , Imidazoles/toxicidad , Isoquinolinas/efectos adversos , Isoquinolinas/toxicidad , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Inhibidores de Proteasas/efectos adversos , Inhibidores de Proteasas/toxicidad , Pirrolidinas/efectos adversos , Pirrolidinas/toxicidad , Receptores de Calcitriol/genética , Receptores de Estrógenos/genética , Sulfonamidas/efectos adversos , Sulfonamidas/toxicidad , Estados Unidos/epidemiología , Valina/efectos adversos , Valina/análogos & derivados , Valina/toxicidadRESUMEN
This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (Mpro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of Mpro. The cyclic peptide contains a [4-(2-aminoethyl)phenyl]-acetic acid (AEPA) linker that is designed to enforce a conformation that mimics a peptide substrate of Mpro. In vitro evaluation of the cyclic peptide inhibitor reveals that the inhibitor exhibits modest activity against Mpro and does not appear to be cleaved by the enzyme. Conformational searching predicts that the cyclic peptide inhibitor is fairly rigid, adopting a favorable conformation for binding to the active site of Mpro. Computational docking to the SARS-CoV-2 Mpro suggests that the cyclic peptide inhibitor can bind the active site of Mpro in the predicted manner. Molecular dynamics simulations provide further insights into how the cyclic peptide inhibitor may bind the active site of Mpro. Although the activity of the cyclic peptide inhibitor is modest, its design and study lays the groundwork for the development of additional cyclic peptide inhibitors against Mpro with improved activities.
Asunto(s)
Proteasas 3C de Coronavirus/antagonistas & inhibidores , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Inhibidores de Proteasas/farmacología , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Diseño de Fármacos , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptidos Cíclicos/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/toxicidad , Conformación ProteicaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes the papain-like protease (PLpro). The protein not only plays an essential role in viral replication but also cleaves ubiquitin and ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from host proteins, making it an important target for developing new antiviral drugs. In this study, we searched for novel, noncovalent potential PLpro inhibitors by employing a multistep in silico screening of a 15 million compound library. The selectivity of the best-scored compounds was evaluated by checking their binding affinity to the human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which, as a deubiquitylating enzyme, exhibits structural and functional similarities to the PLpro. As a result, we identified 387 potential, selective PLpro inhibitors, from which we retrieved the 20 best compounds according to their IC50 values toward PLpro estimated by a multiple linear regression model. The selected candidates display potential activity against the protein with IC50 values in the nanomolar range from approximately 159 to 505 nM and mostly adopt a similar binding mode to the known, noncovalent SARS-CoV-2 PLpro inhibitors. We further propose the six most promising compounds for future in vitro evaluation. The results for the top potential PLpro inhibitors are deposited in the database prepared to facilitate research on anti-SARS-CoV-2 drugs.
Asunto(s)
Antivirales/química , Antivirales/metabolismo , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , SARS-CoV-2/enzimología , Animales , Antivirales/toxicidad , Simulación por Computador , Cristalografía por Rayos X , Bases de Datos de Compuestos Químicos , Bases de Datos de Proteínas , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Dosificación Letal Mediana , Ligandos , Pruebas de Mutagenicidad , Inhibidores de Proteasas/toxicidad , Relación Estructura-Actividad Cuantitativa , Ratas , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Peptidasa Específica de Ubiquitina 7/metabolismo , Animales , Células Cultivadas , Daño del ADN , Células HCT116 , Humanos , Ratones , Células 3T3 NIH , Inhibidores de Proteasas/toxicidad , Proteína Fosfatasa 2/metabolismo , Transporte de Proteínas , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidoresRESUMEN
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Animales , Antivirales/química , Antivirales/uso terapéutico , COVID-19/patología , COVID-19/virología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL10/metabolismo , Modelos Animales de Enfermedad , Diseño de Fármacos , Humanos , Interferón beta/metabolismo , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Ratones , Ratones Transgénicos , Oligopéptidos , Prolina/análogos & derivados , Inhibidores de Proteasas/química , Inhibidores de Proteasas/uso terapéutico , Inhibidores de Proteasas/toxicidad , Ratas , Ratas Sprague-Dawley , Carga Viral/efectos de los fármacos , Replicación ViralRESUMEN
Proteases are industrially important catalysts. They belong to a complex family of enzymes that perform highly focused proteolysis functions. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. In the present study, a novel extracellular neutral protease produced from Arthrospira platensis was detected and characterized. Its proteolytic activity was strongly activated by ß-mercaptoethanol, 5,5-dithio-bis-(2-nitrobenzoic acid) and highly inhibited by Hg2+ and Zn2+ metal ions which support the fact that the studied protease belongs to the cysteine protease family. Using statistical modelling methodology, the logistic model has been selected to predict A. platensis growth-kinetic values. The optimal culture conditions for neutral protease production were found using Box-Behnken Design. The maximum experimental protease activities (159.79 U/mL) was achieved after 13 days of culture in an optimized Zarrouk medium containing 0.625 g/L NaCl, 0.625 g/L K2HPO4 and set on 9.5 initial pH. The extracellular protease of A. platensis can easily be used in the food industry for its important activity at neutral pH and its low production cost since it is a valuation of the residual culture medium after biomass recovery.
Asunto(s)
Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/toxicidad , Spirulina/enzimología , Análisis de Varianza , Biomasa , Proteasas de Cisteína/metabolismo , Concentración de Iones de Hidrógeno , Iones/toxicidad , Mercaptoetanol/farmacología , Mercurio/toxicidad , Modelos Estadísticos , Nitrobenzoatos/farmacología , Proteolisis , Spirulina/crecimiento & desarrollo , Zinc/toxicidadRESUMEN
Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.
Asunto(s)
Catepsina D/antagonistas & inhibidores , Catepsina D/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Sitios de Unión , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Materiales Biomiméticos/toxicidad , Células CACO-2 , Catepsina D/química , Pruebas de Enzimas , Humanos , Cinética , Estructura Molecular , Pepstatinas/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/toxicidad , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/toxicidad , Unión Proteica , Relación Estructura-ActividadRESUMEN
BACKGROUND: 3C-like protease also called the main protease is an essential enzyme for the completion of the life cycle of Middle East Respiratory Syndrome Coronavirus. In our study we predicted compounds which are capable of inhibiting 3C-like protease, and thus inhibit the lifecycle of Middle East Respiratory Syndrome Coronavirus using in silico methods. METHODS: Lead like compounds and drug molecules which are capable of inhibiting 3C-like protease was identified by structure-based virtual screening and ligand-based virtual screening method. Further, the compounds were validated through absorption, distribution, metabolism and excretion filtering. RESULTS: Based on binding energy, ADME properties, and toxicology analysis, we finally selected 3 compounds from structure-based virtual screening (ZINC ID: 75121653, 41131653, and 67266079) having binding energy -7.12, -7.1 and -7.08 Kcal/mol, respectively and 5 compounds from ligandbased virtual screening (ZINC ID: 05576502, 47654332, 04829153, 86434515 and 25626324) having binding energy -49.8, -54.9, -65.6, -61.1 and -66.7 Kcal/mol respectively. All these compounds have good ADME profile and reduced toxicity. Among eight compounds, one is soluble in water and remaining 7 compounds are highly soluble in water. All compounds have bioavailability 0.55 on the scale of 0 to 1. Among the 5 compounds from structure-based virtual screening, 2 compounds showed leadlikeness. All the compounds showed no inhibition of cytochrome P450 enzymes, no blood-brain barrier permeability and no toxic structure in medicinal chemistry profile. All the compounds are not a substrate of P-glycoprotein. CONCLUSION: Our predicted compounds may be capable of inhibiting 3C-like protease but need some further validation in wet lab.
Asunto(s)
Simulación por Computador , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Disponibilidad Biológica , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/toxicidad , Solubilidad , Relación Estructura-ActividadRESUMEN
Targeting secreted aspartic protease 2 (SAP2), a kind of virulence factor, represents a new strategy for antifungal drug discovery. In this report, the first-generation of small molecule SAP2 inhibitors was rationally designed and optimized using a structure-based approach. In particular, inhibitor 23h was highly potent and selective and showed good antifungal potency for the treatment of resistant Candida albicans infections.
Asunto(s)
Antifúngicos/uso terapéutico , Proteasas de Ácido Aspártico/antagonistas & inhibidores , Candidiasis/tratamiento farmacológico , Proteínas Fúngicas/antagonistas & inhibidores , Inhibidores de Proteasas/uso terapéutico , Tiazolidinas/uso terapéutico , Animales , Antifúngicos/síntesis química , Antifúngicos/química , Antifúngicos/toxicidad , Proteasas de Ácido Aspártico/química , Caenorhabditis elegans , Candida albicans/efectos de los fármacos , Femenino , Proteínas Fúngicas/química , Humanos , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/toxicidad , Relación Estructura-Actividad , Tiazolidinas/síntesis química , Tiazolidinas/química , Tiazolidinas/toxicidad , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/químicaRESUMEN
The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.
Asunto(s)
Clorometilcetonas de Aminoácidos/toxicidad , Apoptosis/efectos de los fármacos , Leucil Aminopeptidasa/antagonistas & inhibidores , Necrosis/inducido químicamente , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Proteasas/toxicidad , Linfocitos T/efectos de los fármacos , Clorometilcetonas de Aminoácidos/antagonistas & inhibidores , Biomarcadores/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/química , Caspasas/metabolismo , Forma del Núcleo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Humanos , Células Jurkat , Leucil Aminopeptidasa/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nucleosomas/efectos de los fármacos , Nucleosomas/inmunología , Nucleosomas/metabolismo , Concentración Osmolar , Fragmentos de Péptidos/metabolismo , Inhibidores de Proteasas/química , Proteolisis/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Sacubitril/valsartan (LCZ696) is the first angiotensin receptor neprilysin inhibitor approved to reduce cardiovascular mortality and hospitalization in patients with heart failure with reduced ejection fraction. As neprilysin (NEP) is one of several enzymes known to degrade amyloid-ß (Aß), there is a theoretical risk of Aß accumulation following long-term NEP inhibition. The primary objective of this study was to evaluate the potential effects of sacubitril/valsartan on central nervous system clearance of Aß isoforms in cynomolgus monkeys using the sensitive Stable Isotope Labeling Kinetics (SILK™)-Aß methodology. The in vitro selectivity of valsartan, sacubitril, and its active metabolite sacubitrilat was established; sacubitrilat did not inhibit other human Aß-degrading metalloproteases. In a 2-week study, sacubitril/valsartan (50mg/kg/day) or vehicle was orally administered to female cynomolgus monkeys in conjunction with SILK™-Aß. Despite low cerebrospinal fluid (CSF) and brain penetration, CSF exposure to sacubitril was sufficient to inhibit NEP and resulted in an increase in the elimination half-life of Aß1-42 (65.3%; p=0.026), Aß1-40 (35.2%; p=0.04) and Aßtotal (29.8%; p=0.04) acutely; this returned to normal as expected with repeated dosing for 15days. CSF concentrations of newly generated Aß (AUC(0-24h)) indicated elevations in the more aggregable form Aß1-42 on day 1 (20.4%; p=0.039) and day 15 (34.7%; p=0.0003) and in shorter forms Aß1-40 (23.4%; p=0.009), Aß1-38 (64.1%; p=0.0001) and Aßtotal (50.45%; p=0.00002) on day 15. However, there were no elevations in any Aß isoforms in the brains of these monkeys on day 16. In a second study cynomolgus monkeys were administered sacubitril/valsartan (300mg/kg) or vehicle control for 39weeks; no microscopic brain changes or Aß deposition, as assessed by immunohistochemical staining, were present. Further clinical studies are planned to address the relevance of these findings.
Asunto(s)
Aminobutiratos/toxicidad , Péptidos beta-Amiloides/metabolismo , Antagonistas de Receptores de Angiotensina/toxicidad , Encéfalo/efectos de los fármacos , Neprilisina/antagonistas & inhibidores , Inhibidores de Proteasas/toxicidad , Tetrazoles/toxicidad , Administración Oral , Aminobutiratos/administración & dosificación , Aminobutiratos/farmacocinética , Antagonistas de Receptores de Angiotensina/administración & dosificación , Antagonistas de Receptores de Angiotensina/farmacocinética , Animales , Biotransformación , Compuestos de Bifenilo , Encéfalo/enzimología , Combinación de Medicamentos , Femenino , Humanos , Inmunohistoquímica , Marcaje Isotópico , Macaca fascicularis , Neprilisina/metabolismo , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacocinética , Isoformas de Proteínas , Proteínas Recombinantes/metabolismo , Medición de Riesgo , Tetrazoles/administración & dosificación , Tetrazoles/farmacocinética , Regulación hacia Arriba , ValsartánRESUMEN
Aluminium is a major pollutant due to its constant disposal in aquatic environments through anthropogenic activities. The physiological effects of this metal in fish are still scarce in the literature. This study investigated the in vivo and in vitro effects of aluminium sulfate on the activity of enzymes from Nile tilapia (Oreochromis niloticus): brain acetylcholinesterase (AChE), muscle cholinesterases (AChE-like and BChE-like activities), pepsin, trypsin, chymotrypsin and amylase. Fish were in vivo exposed during 14days when the following experimental groups were assayed: control group (CG), exposure to Al2(SO4)3 at 1µg·mL-1 (G1) and 3µg·mL-1 (G3) (concentrations compatible with the use of aluminium sulfate as coagulant in water treatment). In vitro exposure was performed using animals of CG treatment. Both in vivo and in vitro exposure increased cholinesterase activity in relation to controls. The highest cholinesterase activity was observed for muscle BChE-like enzyme in G3. In contrast, the digestive enzymes showed decreased activity in both in vivo and in vitro exposures. The highest inhibitory effect was observed for pepsin activity. The inhibition of serine proteases was also quantitatively analyzed in zymograms using pixel optical densitometry as area under the peaks (AUP) and integrated density (ID). These results suggest that the inhibition of digestive enzymes in combination with activation of cholinesterases in O. niloticus is a set of biochemical effects that evidence the presence of aluminium in the aquatic environment. Moreover, these enzymatic alterations may support further studies on physiological changes in this species with implications for its neurological and digestive metabolisms.
Asunto(s)
Compuestos de Alumbre/toxicidad , Encéfalo/efectos de los fármacos , Cíclidos/metabolismo , Proteínas de Peces/metabolismo , Tracto Gastrointestinal/efectos de los fármacos , Hidrolasas/metabolismo , Músculos/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Acetilcolinesterasa/metabolismo , Amilasas/antagonistas & inhibidores , Amilasas/metabolismo , Animales , Encéfalo/enzimología , Butirilcolinesterasa/metabolismo , Densitometría , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Activadores de Enzimas/toxicidad , Proteínas de Peces/antagonistas & inhibidores , Tracto Gastrointestinal/enzimología , Hidrolasas/antagonistas & inhibidores , Músculos/enzimología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/toxicidad , Factores de TiempoRESUMEN
Human noroviruses are the primary cause of epidemic and sporadic acute gastroenteritis. The worldwide high morbidity and mortality associated with norovirus infections, particularly among the elderly, immunocompromised patients and children, constitute a serious public health concern. There are currently no approved human vaccines or norovirus-specific small-molecule therapeutics or prophylactics. Norovirus 3CL protease has recently emerged as a potential therapeutic target for the development of anti-norovirus agents. We hypothesized that the S4 subsite of the enzyme may provide an effective means of designing potent and cell permeable inhibitors of the enzyme. We report herein the structure-guided exploration and exploitation of the S4 subsite of norovirus 3CL protease in the design and synthesis of effective inhibitors of the protease.
Asunto(s)
Diseño de Fármacos , Norovirus/enzimología , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Línea Celular , Humanos , Modelos Moleculares , Norovirus/efectos de los fármacos , Norovirus/fisiología , Permeabilidad , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/toxicidad , Conformación Proteica , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival.
Asunto(s)
Inhibidores de Proteasas/química , Venenos de Escorpión/química , Escorpiones/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , Inhibidores de Proteasas/toxicidad , Venenos de Escorpión/genética , Venenos de Escorpión/toxicidad , Escorpiones/genética , Tripsina/químicaRESUMEN
Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 µM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified.
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Toxinas Botulínicas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Bibliotecas de Moléculas Pequeñas/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Ratones , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/toxicidad , Pirazoles/química , Piridinas/química , Quinolinas/química , Relación Estructura-Actividad , Tiadiazoles/químicaRESUMEN
The protease ß-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is required for the production of the amyloid-ß (Aß) peptide, which is central to the pathogenesis of Alzheimer's disease (AD). Chronic inhibition of this protease may temper amyloid production and cure or prevent AD. However, while BACE1 inhibitors are being pushed forward as drug candidates, a remarkable gap in knowledge on the physiological functions of BACE1 and its close homolog BACE2 becomes apparent. Here we discuss the major discoveries of the past 3 years concerning BACE1 biology and to what extent these could limit the use of BACE1 inhibitors in the clinic.
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Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Inhibidores de Proteasas/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/toxicidadRESUMEN
Application of safety lead optimization screening strategies during the early stage of drug discovery led to the identification of a series of CNS-active small molecule inhibitors with opioid off-target effects, as evidenced by potent agonistic activity in functional cell-based assays for mu (MOP), kappa (KOP) and delta (DOP) opioid receptors. The translation of these effects was confirmed in vivo with the following observations: hypoactivity and decreased fecal production in rats (characteristic of MOP agonism); increased urine production in rats (characteristic of KOP agonism); and decreased intestinal transit time in mice, which was partially blocked by the MOP antagonist naloxone, demonstrating that the in vivo effects were specific for MOP. Based on the confirmation of in vitro-in vivo translatability, an in vitro screening strategy was implemented that resulted in the identification of an optimized backup molecule, devoid of in vivo off-target opioid effects. In addition, in silico modeling by docking of the various molecules to the opioid receptors allowed the identification of the structural drivers of these off-target effects, which can be applied to future chemical-design criteria. Thus, implementation of the safety lead optimization strategy described in this article demonstrates the utility and impact of such approaches on risk mitigation and identification of lead small molecules with improved safety profiles.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores de Proteasas/farmacología , Receptores Opioides/agonistas , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Células CHO , Cricetulus , Defecación/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Tránsito Gastrointestinal/efectos de los fármacos , Cobayas , Íleon/efectos de los fármacos , Íleon/metabolismo , Técnicas In Vitro , Ratones , Simulación del Acoplamiento Molecular , Antagonistas de Narcóticos/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/toxicidad , Unión Proteica , Conformación Proteica , Ratas Sprague-Dawley , Receptores Opioides/genética , Receptores Opioides/metabolismo , Relación Estructura-Actividad , Factores de Tiempo , Transfección , Micción/efectos de los fármacosRESUMEN
BACKGROUND: Dengue has emerged as the most significant of arboviral diseases in the 21st century. It is endemic to >100 tropical and sub-tropical countries around the world placing an estimated 3.6 billion people at risk. It is caused by four genetically similar but antigenically distinct, serotypes of dengue viruses. There is neither a vaccine to prevent nor a drug to treat dengue infections, at the present time. The major objective of this work was to explore the possibility of identifying a small molecule inhibitor of the dengue virus protease and assessing its ability to suppress viral replication in cultured cells. METHODS: We cloned, expressed and purified recombinant dengue virus type 2 protease. Using an optimized and validated fluorogenic peptide substrate cleavage assay to monitor the activity of this cloned dengue protease we randomly screened ~1000 small molecules from an 'in-house' library to identify potential dengue protease inhibitors. RESULTS: A benzimidazole derivative, named MB21, was found to be the most potent in inhibiting the cloned protease (IC50 = 5.95 µM). In silico docking analysis indicated that MB21 binds to the protease in the vicinity of the active site. Analysis of kinetic parameters of the enzyme reaction suggested that MB21 presumably functions as a mixed type inhibitor. Significantly, this molecule identified as an inhibitor of dengue type 2 protease was also effective in inhibiting each one of the four serotypes of dengue viruses in infected cells in culture, based on analysis of viral antigen synthesis and infectious virus production. Interestingly, MB21 did not manifest any discernible cytotoxicity. CONCLUSIONS: This work strengthens the notion that a single drug molecule can be effective against all four dengue virus serotypes. The molecule MB21 could be a potential candidate for 'hit-to-lead' optimization, and may pave the way towards developing a pan-dengue virus antiviral drug.