RESUMEN
Glioma is a primary brain tumor with limited treatment approaches and glioblastoma stem cells (GSCs) are manifested with the self-renewal capability and high tumorigenic capacity. This study was performed to investigate the regulatory effect of the SUMO-specific protease 1 (SENP1)/methyltransferase-like 3 (METTL3)/MYC axis on the self-renewal of GSCs mediated by transcription factor Yin Yang 1 (YY1). Following bioinformatics analysis and clinical and cellular experiments, we found that YY1 was highly expressed in GBM tissues and cells, while silencing its expression reduced the self-renewal ability of GSCs. Functionally, YY1 promoted the transcriptional expression of SENP1 by binding to the promoter region of SENP1, while the deSUMOase SENP1 facilitated the methylase activity of m6A through deSUMOylation of the methylase METTL3, thereby promoting the m6A modification of MYC mRNA via METL3 and promoting the expression of MYC. A nude mouse xenograft model of GBM was also constructed to examine the tumorigenicity of GSCs. The obtained findings demonstrated that YY1 promoted tumorigenicity of GSCs by promoting the expression of MYC in vivo. Conclusively, YY1 can transcriptionally upregulate the SUMOylase SENP1 and enhance the methylase activity of METTL3, resulting in the increased m6A modification level of MYC mRNA, thereby promoting the self-renewal of GSCs.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Animales , Ratones , Humanos , Glioblastoma/patología , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismo , Péptido Hidrolasas/metabolismo , Células Madre Neoplásicas/patología , ARN Mensajero/metabolismo , Neoplasias Encefálicas/genética , Proliferación Celular/genética , Línea Celular Tumoral , Metiltransferasas/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismoRESUMEN
Genetic association between E3 ubiquitin ligase SMURF2 and colorectal cancer (CRC) has been identified, while the mechanism remains undefined. Tumor-promoting gene YY1 represents a downstream factor of SMURF2. The study was designed to evaluate the effect of SMURF2 on the malignant phenotypes of CRC cells and the underlying mechanism. The expression pattern of SMURF2 and YY1 in CRC clinical tissues and cells was characterized by immunohistochemistry (IHC) and Western blot. Gain- and loss-of-function experiments were conducted to assess the effect of SMURF2 and YY1 on the behaviors of CRC cells. After bioinformatics analysis, the relationship between YY1 and SENP1 as well as between SENP1 and c-myc was determined by luciferase reporter and ChIP assays. Rescue experiments were performed to show their involvement during CRC progression. Finally, in vivo models of tumor growth were established for validation. SMURF2 was lowly expressed and YY1 was highly expressed in CRC tissues and cells. YY1 overexpression resulted in promotion of CRC cell proliferation, migration, and invasion, which could be reversed by SMURF2. Furthermore, SMURF2 could induce ubiquitination-mediated degradation of YY1, which bound to the SENP1 promoter and upregulated SENP1 expression, leading to enhancement of c-myc expression. The in vivo data revealed the suppressive role of SMURF2 gain-of-function in tumor growth through downregulation of YY1, SENP1, or c-myc. Altogether, our data demonstrate the antitumor activity of SMURF2 in CRC and the anti-tumor mechanism associated with degradation of YY1 and downregulation of SENP1/c-myc.
Asunto(s)
Neoplasias Colorrectales , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proliferación Celular/genética , Regulación hacia Abajo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismoRESUMEN
The actinidin proteinase family has a striking sequence diversity; isoelectric points range from 3.9 to 9.3. The biological drive for this variation is thought to be actinidin's role as a defense-related protein. In this study we map mutations in the primary sequence onto the 3D structure of the protein and show that the region with the highest diversity is close to the substrate binding groove. Non-conservative substitutions in the active site determine substrate preference and therefore create problems for quantification of actinidin activity. Here we use a peptide substrate library to compare two actinidin isoforms, one from the kiwiberry cultivar 'Hortgem Tahi' (Actinidia arguta), and the other from the familiar kiwifruit cultivar 'Hayward' (Actinidia chinensis var. deliciosa). Among 360 octamer substrates we find one substrate (RVAAGSPI) with the useful property of being readily cleaved by all the functionally active actinidins in a set of A. arguta and A. chinensis var. deliciosa isoforms. In addition, we find that two substrates (LPPKSQPP & ILRDKDNT) have the ability to differentiate different isoforms from a single fruit. We compare actinidins from 'Hayward' and A. arguta for their ability to digest the allergenic gluten peptide (PFPQPQLPY) but find the peptide to be indigestible by all sources of actinidin. The ability to inactivate salivary amylase is shown to be a common trait in Actinidia cultivars due to proteolysis by actinidin and is particularly strong in 'Hortgem Tahi'. A mixture of 10% 'Hortgem Tahi' extract with 90% saliva inactivates 100% of amylase activity within 5 minutes. Conceivably, 'Hortgem Tahi' might lower the glycaemic response in a meal rich in cooked starch.
Asunto(s)
Actinidia , Actinidia/química , Actinidia/metabolismo , Amilasas , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Glútenes , Extractos Vegetales , Isoformas de Proteínas/genética , AlmidónRESUMEN
The effect of paeoniflorin on apoptosis and cell cycle in human B-cell acute lymphoblastic leukemia(B-ALL) and its underlying mechanism were investigated in this study. Nalm-6 and SUP-B15 cells were cultured in vitro and divided into control group(0 µg·mL~(-1)) and experimental groups(200, 400, and 800 µg·mL~(-1) paeoniflorin). Cell counting kit-8(CCK-8) was used to measure the viability of Nalm-6 and SUP-B15 cells, and cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blot was used to detect the protein levels of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase(cleaved PARP), c-Myc, and small ubiquitin-like modifier-specific protease 1(SENP1). The mRNA levels of c-Myc and SENP1 in acute lymphoblastic leukemia(ALL) patients were analyzed based on the Oncomine database. AutoDock was used for molecular docking to analyze the interaction of paeoniflorin with c-Myc and SENP1 proteins. RESULTS:: showed that paeoniflorin inhibited the viability of Nalm-6 and SUP-B15 cells in concentration and time-dependent manners. Compared with the control group, paeoniflorin significantly up-regulated the expression of apoptosis-related proteins cleaved caspase-3 and cleaved PARP to induce apoptosis, evidently increased the proportion of G_2/M phase cells and induced G_2/M phase arrest, and obviously down-regulated the expression of c-Myc and SENP1 proteins in Nalm-6 and SUP-B15 cells. The mRNA levels of c-Myc and SENP1 in ALL patients were higher than those in the normal cell. Molecular docking demonstrated that paeoniflorin had good binding to c-Myc and SENP1 proteins. In summary, paeoniflorin inhibits the proliferation of Nalm-6 and SUP-B15 cells by inducing apoptosis and G_2/M phase arrest, which may be related to the down-regulation of c-Myc and SENP1 proteins.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Transducción de Señal , Apoptosis , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/farmacología , Cisteína Endopeptidasas/uso terapéutico , Glucósidos , Humanos , Simulación del Acoplamiento Molecular , Monoterpenos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN MensajeroRESUMEN
Malaria is a huge global health burden with resistance to currently available medicines resulting in the search for newer antimalarial compounds from traditional medicinal plants in malaria-endemic regions. Previous studies on two chalcones, homobutein and 5-prenylbutein, present in E. abyssinica, have shown moderate antiplasmodial activity. Here, we describe results from experimental and computational investigations of four structurally related chalcones, butein, 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDM), homobutein and 5-prenylbutein to elucidate possible molecular mechanisms by which these compounds clear malaria parasites. The crystal structures of butein and DHDM show that butein engages in more hydrogen bonding and consequently, more intermolecular interactions than DHDM. Rotating ring-disk electrode (RRDE) voltammetry results show that butein has a higher antioxidant activity towards the superoxide radical anion compared to DHDM. Computational docking experiments were conducted to examine the inhibitory potential of all four compounds on falcipain-2, a cysteine protease that is involved in the degradation of hemoglobin in plasmodium-infected red blood cells of the host. Overall, this work suggests butein as a better antimalarial compound due to its structural features which allow it to have greater intermolecular interactions, higher antioxidant activity and to create a covalent complex at the active site of falcipain-2.
Asunto(s)
Antimaláricos/química , Antimaláricos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Chalconas/química , Chalconas/farmacología , Sitios de Unión , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus.
Asunto(s)
Quitosano/análogos & derivados , Quitosano/uso terapéutico , Gentamicinas/uso terapéutico , Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/uso terapéutico , Penaeidae/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Quitosano/síntesis química , Cisteína Endopeptidasas/metabolismo , Suplementos Dietéticos , Gentamicinas/síntesis química , Hemocitos/metabolismo , Hepatopáncreas/efectos de los fármacos , Hepatopáncreas/microbiología , Hepatopáncreas/patología , Factores Inmunológicos/síntesis química , Penaeidae/inmunología , Penaeidae/metabolismo , Penaeidae/microbiología , Fagocitos/metabolismo , Esterol Esterasa/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vibrio parahaemolyticus/patogenicidadRESUMEN
OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.
Asunto(s)
Agar/química , Bacterias/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Pectinas/química , Bacillus/enzimología , Bacillus/crecimiento & desarrollo , Bacillus/aislamiento & purificación , Bacillus clausii/enzimología , Bacillus clausii/crecimiento & desarrollo , Bacillus clausii/aislamiento & purificación , Bacterias/enzimología , Bacterias/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Técnicas Bacteriológicas , Frío , Medios de Cultivo/química , Concentración de Iones de Hidrógeno , Paenibacillus/enzimología , Paenibacillus/crecimiento & desarrollo , Paenibacillus/aislamiento & purificación , Proteolisis , Microbiología del SueloRESUMEN
In recent years, several recombinant strains of potato virus Y, notably PVYNTN and PVYN:O have displaced the ordinary strain, PVYO, and emerged as the predominant strains affecting the USA potato crop. Previously we reported that recombinant strains were transmitted more efficiently than PVYO when they were acquired sequentially, regardless of acquisition order. In another recent study, we showed that PVYNTN binds preferentially to the aphid stylet over PVYO when aphids feed on a mixture of PVYO and PVYNTN. To understand the mechanism of this transmission bias as well as preferential virus binding, we separated virus and active helper component proteins (HC), mixed them in homologous and heterologous combinations, and then fed them to aphids using Parafilm sachets. Mixtures of PVYO HC with either PVYN:O or PVYNTN resulted in efficient transmission. PVYN:O HC also facilitated the transmission of PVYO and PVYNTN, albeit with reduced efficiency. PVYNTN HC failed to facilitate transmission of either PVYO or PVYN:O. When PVYO HC or PVYN:O HC was mixed with equal amounts of the two viruses, both viruses in all combinations were transmitted at high efficiencies. In contrast, no transmission occurred when combinations of viruses were mixed with PVYNTN HC. Further study evaluated transmission using serial dilutions of purified virus mixed with HCs. While PVYNTN HC only facilitated the transmission of the homologous virus, the HCs of PVYO and PVYN:O facilitated the transmission of all strains tested. This phenomenon has likely contributed to the increase in the recombinant strains affecting the USA potato crop.
Asunto(s)
Áfidos/virología , Cisteína Endopeptidasas/metabolismo , Enfermedades de las Plantas/virología , Potyvirus/genética , Potyvirus/fisiología , Solanum tuberosum/virología , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Animales , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Recombinación Genética , Nicotiana/virología , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Momordin Ic (MI) is a natural pentacyclic triterpenoid enriched in various Chinese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. Studies have shown that MI presents antitumor properties in liver and prostate cancers. However, the activity and potential mechanisms of MI against colorectal cancer remain elusive. Here, we showed that MI inhibited cell proliferation with G0/1 phase cell cycle arrest in colon cancer cells. Moreover, it was observed that MI increased apoptosis compared to untreated cells. Further investigation showed that the SUMOylation of c-Myc was enhanced by MI and led to the down-regulated protein level of c-Myc, which is involved in regulating cell proliferation and apoptosis. SENP1 has been demonstrated to be critical for the SUMOylation of c-Myc. Meanwhile, knockdown of SENP1 by siRNA abolished the effects of MI on c-Myc level and cell viability in colon cancer cells. Together, these results revealed that MI exerted an anti-tumor activity in colon cancer cells via SENP1/c-Myc signaling pathway. These finding provide an insight into the potential of MI for colon cancer therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Cisteína Endopeptidasas/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Ácido Oleanólico/análogos & derivados , Proteínas Proto-Oncogénicas c-myc/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología , Antineoplásicos Fitogénicos , Bassia scoparia/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias del Colon/tratamiento farmacológico , Humanos , Ácido Oleanólico/aislamiento & purificación , Ácido Oleanólico/farmacología , Ácido Oleanólico/uso terapéutico , FitoterapiaRESUMEN
BACKGROUND: The oncogenic transcript factor c-Maf is stabilized by the deubiquitinase Otub1 and promotes myeloma cell proliferation and confers to chemoresistance. Inhibition of the Otub1/c-Maf axis is a promising therapeutic target, but there are no inhibitors reported on this specific axis. METHODS: A luciferase assay was applied to screen potential inhibitors of Otub1/c-Maf. Annexin V staining/flow cytometry was applied to evaluate cell apoptosis. Immunoprecipitation was applied to examine protein ubiquitination and interaction. Xenograft models in nude mice were used to evaluate anti-myeloma activity of AVT. RESULTS: Acevaltrate (AVT), isolated from Valeriana glechomifolia, was identified based on a bioactive screen against the Otub1/c-Maf/luciferase system. AVT disrupts the interaction of Otub1/c-Maf thus inhibiting Otub1 activity and leading to c-Maf polyubiquitination and subsequent degradation in proteasomes. Consistently, AVT inhibits c-Maf transcriptional activity and downregulates the expression of its target genes key for myeloma growth and survival. Moreover, AVT displays potent anti-myeloma activity by triggering myeloma cell apoptosis in vitro and impairing myeloma xenograft growth in vivo but presents no marked toxicity. CONCLUSIONS: The natural product AVT inhibits the Otub1/c-Maf axis and displays potent anti-myeloma activity. Given its great safety and efficacy, AVT could be further developed for MM treatment. Video Abstract.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/uso terapéutico , Iridoides/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-maf/antagonistas & inhibidores , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cisteína Endopeptidasas/genética , Inhibidores de Cisteína Proteinasa/farmacología , Femenino , Humanos , Iridoides/farmacología , Ratones Endogámicos BALB C , Ratones Desnudos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteínas Proto-Oncogénicas c-maf/genética , Proteínas Proto-Oncogénicas c-maf/metabolismoRESUMEN
This study investigated the effect of actinidin, a cysteine protease in kiwifruit, on the hydrolysis of gluten proteins and digestion-resistant gluten peptides (synthetic 33-mer peptide and pentapeptide epitopes) under static simulated gastrointestinal conditions. Actinidin efficacy in hydrolysing gliadin was compared with that of other gluten-degrading enzymes. Actinidin hydrolysed usually resistant peptide bonds adjacent to proline residues in the 33-mer peptide. The gastric degree of hydrolysis of gluten proteins was influenced by an interaction between pH and actinidin concentration (P < 0.05), whereas the pentapeptide epitopes hydrolysis was influenced only by the actinidin concentration (P < 0.05). The rate of gastric degree of hydrolysis of gliadin was greater (P < 0.05) by actinidin (0.8%/min) when compared to papain, bromelain, and one commercial enzyme (on average 0.4%/min), while all exogenous enzymes were able to hydrolyse the pentapeptide epitopes effectively. Actinidin is able to hydrolyse gluten proteins under simulated gastric conditions.
Asunto(s)
Actinidia/enzimología , Biomimética , Cisteína Endopeptidasas/metabolismo , Digestión , Tracto Gastrointestinal/fisiología , Glútenes/metabolismo , HidrólisisRESUMEN
The current outbreak of Covid-19 infection due to SARS-CoV-2, a virus from the coronavirus family, has become a major threat to human healthcare. The virus has already infected more than 44 M people and the number of deaths reported has reached more than 1.1 M which may be attributed to lack of medicine. The traditional drug discovery approach involves many years of rigorous research and development and demands for a huge investment which cannot be adopted for the ongoing pandemic infection. Rather we need a swift and cost-effective approach to inhibit and control the viral infection. With the help of computational screening approaches and by choosing appropriate chemical space, it is possible to identify lead drug-like compounds for Covid-19. In this study, we have used the Drugbank database to screen compounds against the most important viral targets namely 3C-like protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp) and the spike (S) protein. These targets play a major role in the replication/transcription and host cell recognition, therefore, are vital for the viral reproduction and spread of infection. As the structure based computational screening approaches are more reliable, we used the crystal structures for 3C-like main protease and spike protein. For the remaining targets, we used the structures based on homology modeling. Further, we employed two scoring methods based on binding free energies implemented in AutoDock Vina and molecular mechanics-generalized Born surface area approach. Based on these results, we propose drug cocktails active against the three viral targets namely 3CLpro, PLpro and RdRp. Interestingly, one of the identified compounds in this study i.e. Baloxavir marboxil has been under clinical trial for the treatment of Covid-19 infection. In addition, we have identified a few compounds such as Phthalocyanine, Tadalafil, Lonafarnib, Nilotinib, Dihydroergotamine, R-428 which can bind to all three targets simultaneously and can serve as multi-targeting drugs. Our study also included calculation of binding energies for various compounds currently under drug trials. Among these compounds, it is found that Remdesivir binds to targets, 3CLpro and RdRp with high binding affinity. Moreover, Baricitinib and Umifenovir were found to have superior target-specific binding while Darunavir is found to be a potential multi-targeting drug. As far as we know this is the first study where the compounds from the Drugbank database are screened against four vital targets of SARS-CoV-2 and illustrates that the computational screening using a double scoring approach can yield potential drug-like compounds against Covid-19 infection.
Asunto(s)
Infecciones por Coronavirus/tratamiento farmacológico , Bases de Datos Farmacéuticas , Evaluación Preclínica de Medicamentos/métodos , Terapia Molecular Dirigida , Neumonía Viral/tratamiento farmacológico , COVID-19 , Proteasas 3C de Coronavirus , Análisis Costo-Beneficio , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Evaluación Preclínica de Medicamentos/economía , Humanos , Simulación del Acoplamiento Molecular , Pandemias , Conformación Proteica , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic with very limited specific treatments. To fight COVID-19, various traditional antiviral medicines have been prescribed in China to infected patients with mild to moderate symptoms and received unexpected success in controlling the disease. However, the molecular mechanisms of how these herbal medicines interact with the SARS-CoV-2 virus that causes COVID-19 have remained elusive. It is well known that the main protease (Mpro) of SARS-CoV-2 plays an important role in maturation of many viral proteins such as the RNA-dependent RNA polymerase. Here, we explore the underlying molecular mechanisms of the computationally determined top candidate, namely, rutin which is a key component in many traditional antiviral medicines such as Lianhuaqinwen and Shuanghuanlian, for inhibiting the viral target-Mpro. Using in silico methods (docking and molecular dynamics simulations), we revealed the dynamics and energetics of rutin when interacting with the Mpro of SARS-CoV-2, suggesting that the highly hydrophilic rutin molecule can be bound inside the Mpro's pocket (active site) and possibly inhibit its biological functions. In addition, we optimized the structure of rutin and designed two more hydrophobic analogs, M1 and M2, which satisfy the rule of five for western medicines and demonstrated that they (M2 in particular) possess much stronger binding affinities to the SARS-COV-2s Mpro than rutin, due to the enhanced hydrophobic interaction as well as more hydrogen bonds. Therefore, our results provide invaluable insights into the mechanism of a ligand's binding inside the Mpro and shed light on future structure-based designs of high-potent inhibitors for SARS-CoV-2 Mpro.
Asunto(s)
Betacoronavirus/enzimología , Cisteína Endopeptidasas/metabolismo , Inhibidores de Proteasas/química , Rutina/química , Proteínas no Estructurales Virales/metabolismo , Betacoronavirus/aislamiento & purificación , Sitios de Unión , COVID-19 , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Cisteína Endopeptidasas/química , Medicina de Hierbas , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Pandemias , Neumonía Viral/patología , Neumonía Viral/virología , Inhibidores de Proteasas/metabolismo , Dominios Proteicos , Rutina/metabolismo , SARS-CoV-2 , Termodinámica , Proteínas no Estructurales Virales/químicaRESUMEN
The Ananas comosus stem extract is a complex mixture containing various cysteine ââproteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 µM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.
Asunto(s)
Ananas/química , Bromelaínas/química , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Bromelaínas/antagonistas & inhibidores , Bromelaínas/metabolismo , Bromelaínas/farmacología , Dominio Catalítico , Línea Celular Tumoral , Cisteína/química , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/farmacología , Inhibidores de Cisteína Proteinasa/metabolismo , Disulfuros/química , Humanos , Leucina/análogos & derivados , Leucina/química , Leucina/metabolismo , Modelos Moleculares , Tallos de la Planta/química , Conformación Proteica , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Clorometilcetona Tosilisina/química , Clorometilcetona Tosilisina/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Diterpenos/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Betacoronavirus/enzimología , Dominio Catalítico , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/química , Diterpenos/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Simulación del Acoplamiento Molecular , Conformación Proteica , Multimerización de Proteína , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , SARS-CoV-2 , Proteínas no Estructurales Virales/químicaRESUMEN
The widespread problem of a 2019-novel coronavirus (SARS-CoV-2) strain outbreak in Wuhan, China has prompted a search for new drugs to protect against and treat this disease. It is necessary to immediately investigate this due to the mutation of the viral genome and there being no current protective vaccines or therapeutic drugs. Molecular modelling and molecular docking based on in silico screening strategies were employed to determine the potential activities of seven HIV protease (HIV-PR) inhibitors, two flu drugs, and eight natural compounds. The computational approach was carried out to discover the structural modes with a high binding affinity for these drugs on the homology structure of the Wuhan coronavirus protease (SARS-CoV-2 PR). From the theoretical calculations, all the drugs and natural compounds demonstrated various favorable binding affinities. An interesting finding was that the natural compounds tested had a higher potential binding activity with the pocket sites of SARS-CoV-2 PR compared to the groups of HIV-PR inhibitors. The binding modes of each complex illustrated between the drugs and compounds interacted with the functional group of amino acids in the binding pocket via hydrophilic, hydrophobic, and hydrogen bond interactions using the molecular dynamics simulation technique. This result supports the idea that existing protease inhibitors and natural compounds could be used to treat the new coronavirus. This report sought to provide fundamental knowledge as preliminary experimental data to propose an existing nutraceutical material against viral infection. Collectively, it is suggested that molecular modelling and molecular docking are suitable tools to search and screen for new drugs and natural compounds that can be used as future treatments for viral diseases.
Asunto(s)
Antivirales/farmacología , Cisteína Endopeptidasas/química , Suplementos Dietéticos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Antivirales/química , Sitios de Unión , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/metabolismo , Dioxoles/química , Dioxoles/farmacología , Diterpenos/química , Diterpenos/farmacología , Enlace de Hidrógeno , Lignanos/química , Lignanos/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Conformación Proteica , Proteínas no Estructurales Virales/metabolismoRESUMEN
BACKGROUND: Glioma is the most common tumor of the central nervous system. Hericium erinaceus, which has been reported to have a variety of pharmacological activities, is a widely used Traditional Chinese Medicine (TCM), and also a kind of delicious food accepted by the public. METHODS AND RESULTS: In this study, two new natural products, compounds 1 and 2, were isolated and identified from Hericium erinaceus. They were named erinacerin O and erinacerin P, respectively, after the structural identification, and their effects on human glioma cell line U87 were evaluated. Erinacerin P (2) exhibited obvious cytotoxicity on human glioma cell line U87. The IC50 value of 2 was 19.32µg/mL. The results showed that the apoptosis of U87 cells treated with 2 increased and the morphology of U87 cells altered significantly. Flow cytometry experiment showed that 2 could significantly increase the apoptosis rate of U87 cells and reduce DNA replication. Western blot results suggested the Bax/capase-3 pathway was involved in the U87 cell apoptosis induced by 2. CONCLUSION: Erinacerin O and Erinacerin P are novel compounds obtained from Hericium erinaceus and Erinacerin P could be a potential novel glioma inhibitor.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Glioma/tratamiento farmacológico , Hericium/química , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Caspasa 2/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/patología , Cisteína Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glioma/metabolismo , Glioma/patología , Humanos , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Four new indolyl diketopiperazines, aspamides A-E (1-4) and two new diketopiperazines, aspamides F-G (5-6), along with 11 known diketopiperazines and intermediates were isolated from the solid culture of Aspergillus versicolor, which is an endophyte with the sea crab (Chiromantes haematocheir). Further chiral high-performance liquid chromatography resolution gave enantiomers (+)- and (-)-4, respectively. The structures and absolute configurations of compounds 1-6 were determined by the comprehensive analyses of nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-MS), and electronic circular dichroism (ECD) calculation. All isolated compounds were selected for the virtual screening on the coronavirus 3-chymoretpsin-like protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the docking scores of compounds 1-2, 5, 6, 8 and 17 were top among all screened molecules, may be helpful in fighting with Corona Virus Disease-19 (COVID-19) after further studies.
Asunto(s)
Antivirales , Organismos Acuáticos/química , Aspergillus/química , Cisteína Endopeptidasas/metabolismo , Dicetopiperazinas/química , Dicetopiperazinas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Antivirales/química , Antivirales/metabolismo , Betacoronavirus/metabolismo , Cromatografía Líquida de Alta Presión , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/química , Evaluación Preclínica de Medicamentos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Simulación del Acoplamiento Molecular , SARS-CoV-2 , Estereoisomerismo , Interfaz Usuario-Computador , Proteínas no Estructurales Virales/químicaRESUMEN
The global emergency caused by COVID-19 makes the discovery of drugs capable of inhibiting SARS-CoV-2 a priority, to reduce the mortality and morbidity of this disease. Repurposing approved drugs can provide therapeutic alternatives that promise rapid and ample coverage because they have a documented safety record, as well as infrastructure for large-scale production. The main protease of SARS-CoV-2 (Mpro) is an excellent therapeutic target because it is critical for viral replication; however, Mpro has a highly flexible active site that must be considered when performing computer-assisted drug discovery. In this work, potential inhibitors of the main protease (Mpro) of SARS-Cov-2 were identified through a docking-assisted virtual screening procedure. A total of 4384 drugs, all approved for human use, were screened against three conformers of Mpro. The ligands were further studied through molecular dynamics simulations and binding free energy analysis. A total of nine currently approved molecules are proposed as potential inhibitors of SARS-CoV-2. These molecules can be further tested to speed the development of therapeutics against COVID-19.
Asunto(s)
Betacoronavirus/enzimología , Infecciones por Coronavirus/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Neumonía Viral/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , COVID-19 , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Humanos , Simulación de Dinámica Molecular , Pandemias , Inhibidores de Proteasas/química , Conformación Proteica , SARS-CoV-2 , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
The prevalence of respiratory illness caused by the novel SARS-CoV-2 virus associated with multiple organ failures is spreading rapidly because of its contagious human-to-human transmission and inadequate globalhealth care systems. Pharmaceutical repurposing, an effective drug development technique using existing drugs, could shorten development time and reduce costs compared to those of de novo drug discovery. We carried out virtual screening of antiviral compounds targeting the spike glycoprotein (S), main protease (Mpro), and the SARS-CoV-2 receptor binding domain (RBD)-angiotensin-converting enzyme 2 (ACE2) complex of SARS-CoV-2. PC786, an antiviral polymerase inhibitor, showed enhanced binding affinity to all the targets. Furthermore, the postfusion conformation of the trimeric S protein RBD with ACE2 revealed conformational changes associated with PC786 drug binding. Exploiting immunoinformatics to identify T cell and B cell epitopes could guide future experimental studies with a higher probability of discovering appropriate vaccine candidates with fewer experiments and higher reliability.