RESUMEN
The receptor recognition of the novel coronavirus SARS-CoV-2 relies on the "down-to-up" conformational change in the receptor-binding domain (RBD) of the spike (S) protein. Therefore, understanding the process of this change at the molecular level facilitates the design of therapeutic agents. With the help of coarse-grained molecular dynamic simulations, we provide evidence showing that the conformational dynamics of the S protein are globally cooperative. Importantly, an allosteric path was discovered that correlates the motion of the RBD with the motion of the junction between the subdomain 1 (SD1) and the subdomain 2 (SD2) of the S protein. Building on this finding, we designed non-RBD binding modulators to inhibit SARS-CoV-2 by prohibiting the conformational change of the S protein. Their inhibition effect and function stages at inhibiting SARS-CoV-2 were evaluated experimentally. In summary, our studies establish a molecular basis for future therapeutic agent design through allosteric effects.
Asunto(s)
Antivirales/farmacología , Simulación de Dinámica Molecular , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Células Cultivadas , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , SARS-CoV-2/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.
Asunto(s)
Azoles/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Compuestos de Organoselenio/farmacología , SARS-CoV-2/enzimología , Antivirales/farmacología , Azoles/química , Dominio Catalítico , Proteasas 3C de Coronavirus/metabolismo , Cristalografía por Rayos X , Cisteína/química , Hidrólisis , Isoindoles , Modelos Moleculares , Compuestos de Organoselenio/química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Estándares de Referencia , SARS-CoV-2/efectos de los fármacos , Salicilanilidas/química , Salicilanilidas/farmacología , Selenio/metabolismoRESUMEN
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
BACKGROUND: Gallium has demonstrated strong anti-inflammatory activity in numerous animal studies, and has also demonstrated direct antiviral activity against the influenza A H1N1 virus and the human immunodeficiency virus (HIV). Gallium maltolate (GaM), a small metal-organic coordination complex, has been tested in several Phase 1 clinical trials, in which no dose-limiting or other serious toxicity was reported, even at high daily oral doses for several months at a time. For these reasons, GaM may be considered a potential candidate to treat coronavirus disease 2019 (COVID-19), which is caused by the SARS-CoV-2 virus and can result in severe, sometimes lethal, inflammatory reactions. In this study, we assessed the ability of GaM to inhibit the replication of SARS-CoV-2 in a culture of Vero E6 cells. METHODS: The efficacy of GaM in inhibiting the replication of SARS-CoV-2 was determined in a screening assay using cultured Vero E6 cells. The cytotoxicity of GaM in uninfected cells was determined using the Cell Counting Kit-8 (CCK-8) colorimetric assay. RESULTS: The results showed that GaM inhibits viral replication in a dose-dependent manner, with the concentration that inhibits replication by 50% (EC50) being about 14 µM. No cytotoxicity was observed at concentrations up to at least 200 µM. CONCLUSION: The in vitro activity of GaM against SARS-CoV-2, together with GaM's known anti-inflammatory activity, provide justification for testing GaM in COVID-19 patients.
Asunto(s)
Antiinflamatorios/farmacología , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Compuestos Organometálicos/farmacología , Pironas/farmacología , SARS-CoV-2/efectos de los fármacos , Animales , Antiinflamatorios/uso terapéutico , Antiinflamatorios/toxicidad , Antivirales/uso terapéutico , Antivirales/toxicidad , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Hierro/metabolismo , Compuestos Organometálicos/uso terapéutico , Compuestos Organometálicos/toxicidad , Pironas/uso terapéutico , Pironas/toxicidad , SARS-CoV-2/fisiología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.
Asunto(s)
Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus , Medicamentos Herbarios Chinos , Flavanonas , Flavonoides , Pandemias , Neumonía Viral , Replicación Viral/efectos de los fármacos , Administración Oral , Animales , Antivirales/química , Antivirales/farmacología , Betacoronavirus/fisiología , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Pruebas de Enzimas , Flavanonas/química , Flavanonas/farmacocinética , Flavonoides/química , Flavonoides/farmacocinética , Humanos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , SARS-CoV-2 , Células Vero , Replicación Viral/fisiologíaRESUMEN
Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.
Asunto(s)
Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Oxidorreductasas/antagonistas & inhibidores , Pandemias , Neumonía Viral/tratamiento farmacológico , Virus ARN/efectos de los fármacos , Tiazoles/farmacología , Animales , Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , Betacoronavirus/fisiología , Sitios de Unión/efectos de los fármacos , COVID-19 , Línea Celular , Infecciones por Coronavirus/virología , Crotonatos/farmacología , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Dihidroorotato Deshidrogenasa , Evaluación Preclínica de Medicamentos , Técnicas de Inactivación de Genes , Humanos , Hidroxibutiratos , Virus de la Influenza A/efectos de los fármacos , Leflunamida/farmacología , Ratones , Ratones Endogámicos BALB C , Nitrilos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oseltamivir/uso terapéutico , Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Neumonía Viral/virología , Unión Proteica/efectos de los fármacos , Pirimidinas/biosíntesis , Virus ARN/fisiología , SARS-CoV-2 , Relación Estructura-Actividad , Tiazoles/uso terapéutico , Toluidinas/farmacología , Ubiquinona/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
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
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 VeroAsunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antimaláricos , Antivirales , Betacoronavirus/efectos de los fármacos , Cloroquina , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/farmacología , Alanina/uso terapéutico , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Betacoronavirus/patogenicidad , Bioensayo , COVID-19 , Chlorocebus aethiops , Cloroquina/farmacología , Cloroquina/uso terapéutico , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , SARS-CoV-2 , Sincalida , Células Vero , Tratamiento Farmacológico de COVID-19RESUMEN
Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Here, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the New World arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that V36M in the ectodomain of the SSP mutant and V436A in the transmembrane domain of the GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitor design.IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface.