BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

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.

Synthesis, evaluation, and mechanism of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones as potent reversible SARS-CoV-2 entry inhibitors.

A class of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones were designed and synthesized via Michael addition, cyclization, aldol condensation, and deprotonation to inhibit the human transmembrane protease serine 2 (TMPRSS2) and Furin, which are involved in priming the SARS-CoV-2 Spike for virus entry. The most potent inhibitor 2f (81) was found to efficiently inhibit the replication of various SARS-CoV-2 delta and omicron variants in VeroE6 and Calu-3 cells, with EC50 range of 0.001-0.026 µM by pre-incubation with the virus to avoid the virus entry. The more potent antiviral activities than the proteases inhibitory activities led to discovery that the synthesized compounds also inhibited Spike's receptor binding domain (RBD):angiotensin converting enzyme 2 (ACE2) interaction as a main target, and their antiviral activities were enhanced by inhibiting TMPRSS2 and/or Furin. To further confirm the blocking effect of 2f (81) on virus entry, SARS-CoV-2 Spike pseudovirus was used in the entry assay and the results showed that the compound inhibited the pseudovirus entry in a ACE2-dependent pathway, via mainly inhibiting RBD:ACE2 interaction and TMPRSS2 activity in Calu-3 cells. Finally, in the in vivo animal model of SARS-CoV-2 infection, the oral administration of 25 mg/kg 2f (81) in hamsters resulted in reduced bodyweight loss and 5-fold lower viral RNA levels in nasal turbinate three days post-infection. Our findings demonstrated the potential of the lead compound for further preclinical investigation as a potential treatment for SARS-CoV-2.

Inhalable chitosan-based hydrogel as a mucosal adjuvant for hydroxychloroquine in the treatment for SARS-CoV-2 infection in a hamster model.

BACKGROUND: Effective therapy for COVID-19 remains limited. Hydroxychloroquine (HCQ) has been considered, but safety and efficacy concerns remain. Chitosan exhibits antiviral and immunomodulatory effects, yet how the combination of HCQ and chitosan performs in treating COVID-19 is unknown. METHODS: Male Syrian hamsters were inoculated intranasally with standardized stocks of the SARS-CoV-2 virus. Hamsters were allocated to saline (PBS), chitosan oligosaccharide (COS), HCQ, or COS + HCQ groups and received corresponding drugs. On days 1, 7, and 14 post-infection, two animals from each group were euthanized for sample collection. Viral loads were measured in lung homogenates. Biochemistry markers, cytokines, and immunoglobulins were analyzed from hamster sera. HCQ concentrations were compared between the blood, bronchoalveolar lavage, and lung tissues. All groups underwent histopathology exams of the lungs. Additional hamsters were treated with the same drugs to assess for toxicities to the heart and liver. RESULTS: Among all groups, viral loads in the COS + HCQ group were the lowest by day 8. The COS + HCQ group produced the highest interleukin (IL)-6 levels on day 4, and the highest IL-10, IgA and IgG levels on day 8. HCQ concentrations were higher in the COS + HCQ group's lungs than the HCQ group, despite having received half the dose of HCQ. Histopathology demonstrated earlier inflammation resolution and swifter viral clearance in the COS + HCQ group. There was no evidence of cardiac or hepatic injury in hamsters that received HCQ. CONCLUSION: In hamsters infected with the SARS-CoV-2 virus, the combination of intranasal COS and HCQ was associated with increased HCQ absorption in the lungs, more effective immune responses, without increasing the risk of hepatic or cardiac injuries.

Dynamic Changes of the Blood Chemistry in Syrian Hamsters Post-Acute COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that causes coronavirus disease 2019 (COVID-19). However, the long-term health consequences of COVID-19 are not fully understood. We aimed to determine the long-term lung pathology and blood chemistry changes in Syrian hamsters infected with SARS-CoV-2. Syrian hamsters (Mesocricetus auratus) were inoculated with 105 PFU of SARS-CoV-2, and changes post-infection (pi) were observed for 20 days. On days 5 and 20 pi, the lungs were harvested and processed for pathology and viral load count. Multiple blood samples were collected every 3 to 5 days to observe dynamic changes in blood chemistry. Infected hamsters showed consistent weight loss until day 7 pi At day 5 pi, histopathology of the lungs showed moderate to severe inflammation and the virus could be detected. These results indicate that SARS-CoV-2 has an acute onset and recovery course in the hamster infection model. During the acute onset, blood triglyceride levels increased significantly at day 3 pi During the recovery course, uric acid and low-density lipoprotein levels increased significantly, but the total protein and albumin levels decreased. Together, our study suggests that SARS-CoV-2 infection in hamsters not only causes lung damage but also causes long-term changes in blood biochemistry during the recovery process. IMPORTANCE COVID-19 is now considered a multiorgan disease with a wide range of manifestations. There are increasing reports of persistent and long-term effects after acute COVID-19, but the long-term health consequences of COVID-19 are not fully understood. This study reported for the first time the use of blood samples collected continuously in a SARS-CoV-2-infected hamster model, which provides more information about the dynamic changes in blood biochemistry during the acute and recovery phases of SARS-CoV-2 infection. Our study suggests that SARS-CoV-2 infection in hamsters not only causes lung damage but also causes long-term changes in blood biochemistry during the recovery process. The study may be used by several researchers and clinicians, especially those who are studying potential treatments for patients with post-acute COVID-19 syndrome.

Methotrexate inhibition of SARS-CoV-2 entry, infection and inflammation revealed by bioinformatics approach and a hamster model.

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.

Perilla (Perilla frutescens) leaf extract inhibits SARS-CoV-2 via direct virus inactivation.

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.

Micafungin is a novel anti-viral agent of chikungunya virus through multiple mechanisms.

The chikungunya virus (CHIKV) is a mosquito-borne virus that belongs to the genus Alphavirus, family Togaviridae. It is the cause of chikungunya fever in humans, which presents a serious global threat due to its high rate of contagion. The clinical symptoms of CHIKV include fever and persistent, severe arthritis. Micafungin has broad-spectrum fungicidal activity against Candida spp. is a promising echinocandin that was recently approved by the U.S. Food and Drug Administration (FDA) and has demonstrated activity against Candida and Aspergillus. Recent studies have demonstrated the antiviral activity of micafungin; however, the inhibitory effects against CHIKV have yet to be investigated. Our objectives in this study were to explore the antiviral effects of micafungin on CHIKV infection and to elucidate the potential molecular mechanisms of inhibition. We determined that micafungin has the ability to counter CHIKV-induced cytopathic effects. We further discovered that micafungin limits virus replication, release, cell-to-cell transmission, and also slightly affected virus stability during high doses treatment. The efficacy of micafungin was further confirmed against two clinical isolates of CHIKV and two alphaviruses: Sindbis virus (SINV) and Semliki Forest virus (SFV). Our findings suggest that micafungin has considerable potential as a novel inhibitor against the viral replication, and intracellular and extracellular transmission of CHIKV, and has a little effect on virus stability. Our findings also suggest that micafungin could have curative effects on other alphavirus infections.

Effect of melanin produced by a recombinant Escherichia coli on antibacterial activity of antibiotics.

A recombinant plasmid, pYL-1, containing a tyrosinase gene whose expression is under the control of a phage T5 promoter and 2 lac operators, was constructed. Escherichia coli JM109 harboring pYL-1 was used for production of bacterial melanin. A simple procedure for the isolation and purification of melanin was developed. The ultraviolet (UV)-visible light absorption spectra of melanin prepared by chemical synthesis and derived from different organisms, including bacteria, a plant and an animal source, were determined. Melanins produced by both bacteria and chemical synthesis showed a steady increase of absorption at wavelengths of UV light ranging from approximately 200-400 nm, while melanin derived either from plant or animal sources showed an additional discrete absorption peak at wavelength 280 nm upon a similar steady increase of absorption. This additional absorption peak could be due to the presence of protein-bound melanins in animal and plant sources while a free form of melanin was obtained from bacteria and chemical synthesis. Analysis of the effect of bacterial melanin on the activity of antibiotics against E. coli revealed that the activities of polymyxin B, kanamycin, tetracycline, and ampicillin were markedly reduced in the presence of melanin, whereas the activity of norfloxacin was not affected. The reduction of the antibacterial activity may result directly from the interaction of antibiotics with melanin. However, the mechanism of this interaction remains to be demonstrated.