A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

Aminopeptidase N is an entry co-factor triggering porcine deltacoronavirus entry via an endocytotic pathway

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the role of porcine APN (pAPN) during PDCoV infection of non-susceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter non-susceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly co-localized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters non-susceptible cells, but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate anti-viral drugs.

Phytochemical Characterization of Olea europea Leaf Extracts and Assessment of Their Anti-Microbial and Anti-HSV-1 Activity

Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 μg/mL and between 15.61 and 31.25 μg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC50) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.

Susceptibility of global HIV-1 clinical isolates to fostemsavir using the PhenoSense® Entry assay

Background Fostemsavir is a prodrug of a first-in-class HIV-1 attachment inhibitor, temsavir, that binds to gp120 and blocks attachment to the host-cell CD4 receptor, preventing entry and infection of the target cell. Previous studies using a limited number of clinical isolates showed that there was intrinsic variability in their susceptibility to temsavir. Objectives Here, an analysis was performed using all clinical isolates analysed in the Monogram Biosciences PhenoSense® Entry assay as part of the development programme. Methods In total, 1337 individual envelopes encompassing 20 different HIV-1 subtypes were examined for their susceptibility to temsavir. However, only seven subtypes (B, C, F1, A, [B, F1], BF and A1) were present more than five times, with subtype B (881 isolates) and subtype C (156 isolates) having the largest numbers. Results As expected, variability in susceptibility was observed within all subtypes. However, for the great majority of these viruses, temsavir was highly potent, with most viruses exhibiting IC50s <10 nM. One exception was CRF01_AE viruses, where all five isolates exhibited IC50s >100 nM. For the 607 isolates where tropism data were available, geometric mean temsavir IC50 values were remarkably similar for CCR5-, CXCR4- and dual mixed-tropic envelopes from infected individuals. Conclusions These data show that HIV-1 viruses from most subtypes are highly susceptible to temsavir and that temsavir susceptibility is independent of tropism.

Coronavirus Antiviral Research Database (CoV-RDB): An Online Database Designed to Facilitate Comparisons between Candidate Anti-Coronavirus Compounds

Background: To prioritize the development of antiviral compounds, it is necessary to compare their relative preclinical activity and clinical efficacy. Methods: We reviewed in vitro, animal model, and clinical studies of candidate anti-coronavirus compounds and placed extracted data in an online relational database. Results: As of August 2020, the Coronavirus Antiviral Research Database (CoV-RDB; covdb.stanford.edu) contained over 2800 cell culture, entry assay, and biochemical experiments, 259 animal model studies, and 73 clinical studies from over 400 published papers. SARS-CoV-2, SARS-CoV, and MERS-CoV account for 85% of the data. Approximately 75% of experiments involved compounds with known or likely mechanisms of action, including monoclonal antibodies and receptor binding inhibitors (21%), viral protease inhibitors (17%), miscellaneous host-acting inhibitors (10%), polymerase inhibitors (9%), interferons (7%), fusion inhibitors (5%), and host protease inhibitors (5%). Of 975 compounds with known or likely mechanism, 135 (14%) are licensed in the U.S. for other indications, 197 (20%) are licensed outside the U.S. or are in human trials, and 595 (61%) are pre-clinical investigational compounds. Conclusion: CoV-RDB facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies prioritize the most promising compounds and repurposed drugs for further development.

Coronavirus Antiviral Research Database (CoV-RDB): An Online Database Designed to Facilitate Comparisons Between Candidate Anti-Coronavirus Compounds

Background: To prioritize the development of antiviral compounds, it is necessary to compare their relative preclinical activity and clinical efficacy. Methods: We reviewed in vitro, animal model, and clinical studies of candidate anti-coronavirus compounds and placed extracted data in an online relational database. Results: As of July 2020, the Coronavirus Antiviral Research Database (CoV-RDB; covdb.stanford.edu) contained >2,400 cell culture, entry assay and biochemical experiments, 240 animal model studies, and 56 clinical studies from >300 published papers. SARS-CoV-2, SARS-CoV, and MERS-CoV account for approximately 85% of the data. Approximately 75% of experiments involved compounds with a known or likely mechanism of action, including receptor binding inhibitors and monoclonal antibodies (20%); viral protease inhibitors (18%); polymerase inhibitors (9%); interferons (8%); fusion inhibitors (8%); host endosomal trafficking inhibitors (7%); and host protease inhibitors (5%). For 724 compounds with a known or likely mechanism, 95 (13%) are licensed in the US for other indications, 72 (10%) are licensed outside the US or are in human trials, and 557 (77%) are pre-clinical investigational compounds. Conclusion: CoV-RDB facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies prioritize the most promising compounds and repurposed drugs for further development.

Receptor utilization of angiotensin converting enzyme 2 (ACE2) indicates a narrower host range of SARS-CoV-2 than that of SARS-CoV

Coronavirus pandemics have become a huge threat to the public health worldwide in the recent decades. Typically, SARS-CoV caused SARS pandemic in 2003 and SARS-CoV-2 caused the COVID-19 pandemic recently. Both viruses have been reported to originate from bats. Thus, direct or indirect interspecies transmission from bats to humans is required for the viruses to cause pandemics. Receptor utilization is a key factor determining the host range of viruses which is critical to the interspecies transmission. Angiotensin converting enzyme 2 (ACE2) is the receptor of both SARS-CoV and SARS-CoV-2, but only ACE2s of certain animals can be utilized by the viruses. Here, we employed pseudovirus cell-entry assay to evaluate the receptor-utilizing capability of ACE2s of 20 animals by the two viruses and found that SARS-CoV-2 utilized less ACE2s than SARS-CoV, indicating a narrower host range of SARS-CoV-2. Meanwhile, pangolin CoV, another SARS-related coronavirus highly homologous to SARS-CoV-2 in its genome, yet showed similar ACE2 utilization profile with SARS-CoV rather than SARS-CoV-2. To clarify the mechanism underlying the receptor utilization, we compared the amino acid sequences of the 20 ACE2s and found 5 amino acid residues potentially critical for ACE2 utilization, including the N-terminal 20th and 42nd amino acids that may determine the different receptor utilization of SARS-CoV, SARS-CoV-2 and pangolin CoV. Our studies promote the understanding of receptor utilization of pandemic coronaviruses, potentially contributing to the virus tracing, intermediate host screening and epidemic prevention for pathogenic coronaviruses.

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.