RESUMEN
Currently, bifunctional agents with vasodilation and ameliorated vascular remodeling effects provide more advantages for the treatment of pulmonary arterial hypertension (PAH). In this study, we first screened the hit 1 with heat shock protein 110 (Hsp110) inhibition effect from our in-house compound library with soluble guanylate cyclase (sGC) activity. Subsequently, a series of novel bisamide derivatives were designed and synthesized as Hsp110/sGC dual-target regulators based on hit 1. Among them, 17i exhibited optimal Hsp110 and sGC molecular activities as well as remarkable cell malignant phenotypes inhibitory and vasodilatory effects in vitro. Moreover, compared to riociguat, 17i showed superior efficacy in attenuating pulmonary vascular remodeling and right ventricular hypertrophy via Hsp110 suppression in hypoxia-induced PAH rat models (i.g.). Notably, our study successfully demonstrated that the simultaneous regulation of Hsp110 and sGC dual targets was a novel and feasible strategy for PAH therapy, providing a promising lead compound for anti-PAH drug discovery.
Asunto(s)
Proteínas del Choque Térmico HSP110 , Guanilil Ciclasa Soluble , Animales , Guanilil Ciclasa Soluble/metabolismo , Humanos , Ratas , Proteínas del Choque Térmico HSP110/metabolismo , Proteínas del Choque Térmico HSP110/antagonistas & inhibidores , Ratas Sprague-Dawley , Descubrimiento de Drogas , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Hipertensión Arterial Pulmonar/metabolismo , Masculino , Relación Estructura-Actividad , Hipertensión Pulmonar/tratamiento farmacológico , Remodelación Vascular/efectos de los fármacosRESUMEN
The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Asunto(s)
Antivirales , COVID-19 , Inhibidores de Proteasas , SARS-CoV-2 , Humanos , Antivirales/farmacología , Antivirales/química , Tratamiento Farmacológico de COVID-19 , Ensayos Analíticos de Alto Rendimiento , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteínas no Estructurales ViralesRESUMEN
Clustering enzymes in the same metabolic pathway is a natural strategy to enhance productivity. Synthetic protein, RNA and DNA scaffolds have been designed to artificially cluster multiple enzymes in the cell, which require complex construction processes and possess limited slots for target enzymes. We utilized the Escherichia coli inner cell membrane as a native scaffold to cluster four fatty acid synthases (FAS) and achieved to improve the efficiency of fatty acid synthesis in vivo. The construction strategy is as simple as fusing target enzymes to the N-terminus or C-terminus of the membrane anchor protein (Lgt), and the number of anchored enzymes is not restricted. This novel device not only presents a similar efficiency in clustering multiple enzymes to that of other artificial scaffolds but also promotes the product secretion, driving the entire metabolic flux forward and further increasing the gross yield compared with that in a cytoplasmic scaffold system.
RESUMEN
The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.
Asunto(s)
Proteasas 3C de Coronavirus , ARN Polimerasa Dependiente de ARN de Coronavirus , SARS-CoV-2 , Antivirales/química , Proteasas 3C de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , Poliproteínas/química , Conformación Proteica , Proteolisis , SARS-CoV-2/enzimología , Especificidad por Sustrato/genéticaRESUMEN
A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 µmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three "hot" spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.
Asunto(s)
Proteasas Similares a la Papaína de Coronavirus/química , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteasas/química , Antivirales/química , Antivirales/metabolismo , Antivirales/uso terapéutico , Sitios de Unión , COVID-19/virología , Proteasas Similares a la Papaína de Coronavirus/genética , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Humanos , Imidazoles/química , Imidazoles/metabolismo , Imidazoles/uso terapéutico , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Naftoquinonas/química , Naftoquinonas/metabolismo , Naftoquinonas/uso terapéutico , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/uso terapéutico , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , SARS-CoV-2/aislamiento & purificación , Tratamiento Farmacológico de COVID-19RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an unprecedented global health crisis. It is particularly urgent to develop clinically effective therapies to contain the pandemic. The main protease (Mpro) and the RNA-dependent RNA polymerase (RdRP), which are responsible for the viral polyprotein proteolytic process and viral genome replication and transcription, respectively, are two attractive drug targets for SARS-CoV-2. This review summarizes up-to-date progress in the structural and pharmacological aspects of those two key targets above. Different classes of inhibitors individually targeting Mpro and RdRP are discussed, which could promote drug development to treat SARS-CoV-2 infection.
Asunto(s)
Antivirales/química , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Inhibidores de Proteasa de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , ARN Polimerasa Dependiente de ARN de Coronavirus/química , Inhibidores Enzimáticos/química , SARS-CoV-2/enzimología , Antivirales/farmacología , Inhibidores de Proteasa de Coronavirus/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Conformación Proteica , SARS-CoV-2/efectos de los fármacosRESUMEN
The antineoplastic drug carmofur is shown to inhibit the SARS-CoV-2 main protease (Mpro). Here, the X-ray crystal structure of Mpro in complex with carmofur reveals that the carbonyl reactive group of carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 µM) and is a promising lead compound to develop new antiviral treatment for COVID-19.
Asunto(s)
Betacoronavirus/enzimología , Cisteína Endopeptidasas/química , Fluorouracilo/análogos & derivados , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Animales , Betacoronavirus/efectos de los fármacos , COVID-19 , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/virología , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Fluorouracilo/química , Fluorouracilo/farmacología , Modelos Moleculares , Pandemias , Neumonía Viral/virología , SARS-CoV-2 , Células Vero , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the etiological agent responsible for the global COVID-19 (coronavirus disease 2019) outbreak. The main protease of SARS-CoV-2, Mpro, is a key enzyme that plays a pivotal role in mediating viral replication and transcription. We designed and synthesized two lead compounds (11a and 11b) targeting Mpro Both exhibited excellent inhibitory activity and potent anti-SARS-CoV-2 infection activity. The x-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a or 11b, both determined at a resolution of 1.5 angstroms, showed that the aldehyde groups of 11a and 11b are covalently bound to cysteine 145 of Mpro Both compounds showed good pharmacokinetic properties in vivo, and 11a also exhibited low toxicity, which suggests that these compounds are promising drug candidates.
Asunto(s)
Antivirales/química , Betacoronavirus/enzimología , Diseño de Fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , COVID-19 , Dominio Catalítico , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/tratamiento farmacológico , Cisteína Endopeptidasas , Perros , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Ratones , Estructura Molecular , Pandemias , Neumonía Viral/tratamiento farmacológico , Estructura Terciaria de Proteína , Ratas Sprague-Dawley , SARS-CoV-2 , Pruebas de Toxicidad , Células VeroRESUMEN
A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.
Asunto(s)
Betacoronavirus/química , Cisteína Endopeptidasas/química , Descubrimiento de Drogas/métodos , Modelos Moleculares , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Antivirales/química , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , COVID-19 , Células Cultivadas/virología , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/enzimología , Infecciones por Coronavirus/virología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Pandemias , Neumonía Viral/enzimología , Neumonía Viral/virología , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína , SARS-CoV-2RESUMEN
OsMADS32 is a monocot specific MIKC(c) type MADS-box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1-3 and identified its function in controlling rice flower development by bioinformatics and protein-protein interaction analysis. The cfo1-3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull-like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis-splicing of OsMADS32 transcripts in the cfo1-3 mutant resulted in an extra eight amino acids in the K-domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence complementation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA (PI)-like proteins OsMADS2 and OsMADS4. This work provides new insight into the mechanism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI-like proteins via its K domain.