RESUMO
Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , COVID-19 , Pirazóis , Quinolinas , Humanos , SARS-CoV-2/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Simulação de Acoplamento Molecular , Tratamento Farmacológico da COVID-19 , Antivirais/químicaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.
Assuntos
COVID-19/complicações , Doenças Cardiovasculares/imunologia , Síndrome da Liberação de Citocina/imunologia , SARS-CoV-2/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Doenças Cardiovasculares/virologia , Diferenciação Celular , Linhagem Celular , Biologia Computacional , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Síndrome da Liberação de Citocina/patologia , Síndrome da Liberação de Citocina/virologia , Humanos , Células-Tronco Pluripotentes Induzidas , Miocárdio/citologia , Miocárdio/imunologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/virologia , Fosfoproteínas/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Regulação para Cima/imunologia , Internalização do Vírus/efeitos dos fármacosRESUMO
(Background) The coronavirus disease 2019 (COVID-19) that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries high infectivity and mortality. Efficient intervention strategies are urgently needed. Avian immunoglobulin Y (IgY) showed efficacy against viral infection whereas the in vivo efficacy remains unclear. (Methods) We immunized laying hens with S1, S1 receptor-binding domain (S1-RBD), or S2 subunits of the SARS-CoV-2 spike (S) protein. After immunization, IgYs were collected and extracted from the egg yolks. The neutralization potential of IgYs was examined by the plaque reduction neutralization test (PRNT). The bioutility of IgYs was examined in Syrian hamsters in vivo. (Results) IgYs exhibited typical banding patterns in SDS-PAGE and Western blot and were immunoreactive against S1, S1-RBD, and S2 subunits. The plaque reduction neutralization test (PRNT) showed that all purified IgYs potently neutralized different SARS-CoV-2 strains in vitro. In Syrian hamsters, the combination of IgYs for S1-RBD and S2 subunits administered before or after SARS-CoV-2 infection effectively restored body weight loss and reduced intrapulmonary lesions and the amount of immunoreactive N protein-positive cells, which were caused by SARS-CoV-2 infection. (Conclusions) Collectively, IgYs specific for S protein subunits effectively neutralized SARS-CoV-2 in vitro and in vivo and may serve as prophylactic or therapeutic antibodies in the prevention or treatment of COVID-19.
RESUMO
The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants has altered the trajectory of the COVID-19 pandemic and raised some uncertainty on the long-term efficiency of vaccine strategy. The development of new therapeutics against a wide range of SARS-CoV-2 variants is imperative. We, here, have designed an inhalable siRNA, C6G25S, which covers 99.8% of current SARS-CoV-2 variants and is capable of inhibiting dominant strains, including Alpha, Delta, Gamma, and Epsilon, at picomolar ranges of IC50 in vitro. Moreover, C6G25S could completely inhibit the production of infectious virions in lungs by prophylactic treatment, and decrease 96.2% of virions by cotreatment in K18-hACE2-transgenic mice, accompanied by a significant prevention of virus-associated extensive pulmonary alveolar damage, vascular thrombi, and immune cell infiltrations. Our data suggest that C6G25S provides an alternative and effective approach to combating the COVID-19 pandemic.
Assuntos
COVID-19 , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Pandemias , RNA Interferente Pequeno/genética , SARS-CoV-2/genéticaRESUMO
Background: Drug repurposing is a fast and effective way to develop drugs for an emerging disease such as COVID-19. The main challenges of effective drug repurposing are the discoveries of the right therapeutic targets and the right drugs for combating the disease. Methods: Here, we present a systematic repurposing approach, combining Homopharma and hierarchal systems biology networks (HiSBiN), to predict 327 therapeutic targets and 21,233 drug-target interactions of 1,592 FDA drugs for COVID-19. Among these multi-target drugs, eight candidates (along with pimozide and valsartan) were tested and methotrexate was identified to affect 14 therapeutic targets suppressing SARS-CoV-2 entry, viral replication, and COVID-19 pathologies. Through the use of in vitro (EC50 = 0.4 µM) and in vivo models, we show that methotrexate is able to inhibit COVID-19 via multiple mechanisms. Results: Our in vitro studies illustrate that methotrexate can suppress SARS-CoV-2 entry and replication by targeting furin and DHFR of the host, respectively. Additionally, methotrexate inhibits all four SARS-CoV-2 variants of concern. In a Syrian hamster model for COVID-19, methotrexate reduced virus replication, inflammation in the infected lungs. By analysis of transcriptomic analysis of collected samples from hamster lung, we uncovered that neutrophil infiltration and the pathways of innate immune response, adaptive immune response and thrombosis are modulated in the treated animals. Conclusions: We demonstrate that this systematic repurposing approach is potentially useful to identify pharmaceutical targets, multi-target drugs and regulated pathways for a complex disease. Our findings indicate that methotrexate is established as a promising drug against SARS-CoV-2 variants and can be used to treat lung damage and inflammation in COVID-19, warranting future evaluation in clinical trials.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Inflamação/tratamento farmacológico , Biologia ComputacionalRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe "flu-like" symptoms that can progress to acute respiratory distress syndrome (ARDS), pneumonia, renal failure, and death. From the therapeutic perspective, 3-chymotrypsin-like protein (3CLpro) is a plausible target for direct-acting antiviral agents because of its indispensable role in viral replication. The flavonoid ugonin J (UJ) has been reported to have antioxidative and anti-inflammatory activities. However, the potential of UJ as an antiviral agent remains unexplored. In this study, we investigated the therapeutic activity of UJ against SARS-CoV-2 infection. Importantly, UJ has a distinct inhibitory activity against SARS-CoV-2 3CLpro, compared to luteolin, kaempferol, and isokaempferide. Specifically, UJ blocks the active site of SARS-CoV-2 3CLpro by forming hydrogen bonding and van der Waals interactions with H163, M165 and E166, G143 and C145, Q189, and P168 in subsites S1, S1', S2, and S4, respectively. In addition, UJ forms strong, stable interactions with core pharmacophore anchors of SARS-CoV-2 3CLpro in a computational model. UJ shows consistent anti-inflammatory activity in inflamed human alveolar basal epithelial A549 cells. Furthermore, UJ has a 50% cytotoxic concentration (CC50) and a 50% effective concentration (EC50) values of about 783 and 2.38 µM, respectively, with a selectivity index (SI) value of 329, in SARS-CoV-2-infected Vero E6 cells. Taken together, UJ is a direct-acting antiviral that obstructs the activity of a fundamental protease of SARS-CoV-2, offering the therapeutic potential for SARS-CoV-2 infection.
RESUMO
BACKGROUND: While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents with mild or no symptoms in most cases, a significant number of patients become critically ill. Remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19) in several countries, but its use as monotherapy has not substantially lowered mortality rates. Because agents from traditional Chinese medicine (TCM) have been successfully utilized to treat pandemic and endemic diseases, we designed the current study to identify novel anti-SARS-CoV-2 agents from TCM. METHODS: We initially used an antivirus-induced cell death assay to screen a panel of herbal extracts. The inhibition of the viral infection step was investigated through a time-of-drug-addition assay, whereas a plaque reduction assay was carried out to validate the antiviral activity. Direct interaction of the candidate TCM compound with viral particles was assessed using a viral inactivation assay. Finally, the potential synergistic efficacy of remdesivir and the TCM compound was examined with a combination assay. RESULTS: The herbal medicine Perilla leaf extract (PLE, approval number 022427 issued by the Ministry of Health and Welfare, Taiwan) had EC50 of 0.12 ± 0.06 mg/mL against SARS-CoV-2 in Vero E6 cells - with a selectivity index of 40.65. Non-cytotoxic PLE concentrations were capable of blocking viral RNA and protein synthesis. In addition, they significantly decreased virus-induced cytokine release and viral protein/RNA levels in the human lung epithelial cell line Calu-3. PLE inhibited viral replication by inactivating the virion and showed additive-to-synergistic efficacy against SARS-CoV-2 when used in combination with remdesivir. CONCLUSION: Our results demonstrate for the first time that PLE is capable of inhibiting SARS-CoV-2 replication by inactivating the virion. Our data may prompt additional investigation on the clinical usefulness of PLE for preventing or treating COVID-19.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Perilla frutescens , Extratos Vegetais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Inativação de Vírus , Animais , COVID-19 , Chlorocebus aethiops , Humanos , Perilla frutescens/químicaRESUMO
Chikungunya disease results from an infection with the arbovirus, chikungunya virus (CHIKV). Symptoms of CHIKV include fever and persistent, severe arthritis. In recent years, several antiviral drugs have been evaluated in clinical trials; however, no registered antivirals have been approved for clinical therapy. In this study, we established a high-throughput screening (HTS) system based on CHIKV 26S mediated insect cell fusion inhibition assay. Our screening system was able to search potential anti-CHIKV drugs in vitro. Using this system, four compounds (niclosamide, nitazoxanide, niflumic acid, tolfenamic acid) were identified. These compounds were then further analyzed using a microneutralization assay. We determined that niclosamide and nitazoxanide exhibit ability to against CHIKV-induced CPE. The anti-CHIKV abilities of these compounds were further confirmed by RT-qPCR and IFA. Moreover, niclosamide and nitazoxanide were found to (1) limit virus entry, (2) inhibit both viral release and cell-to-cell transmission, and (3) possess broad anti-alphavius activities, including against two clinical CHIKV isolates and two alphaviruses: Sindbis virus (SINV) and Semliki forest virus (SFV). In conclusion, our findings suggested that niclosamide and nitazoxanide were able to inhibit CHIKV entry and transmission, which might provide a basis for the development of novel human drug therapies against CHIKV and other alphavirus infections.