Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants.

We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wild type (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 µM, 8.5 µM, 24.1 µM, 8.2 µM and 13.6 µM, respectively. A separate experiment focusing on a combination of 10 µM stenoparib and 0.5 µM remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.

Analytical sensitivity of the Rapid Antigen Test kits for detection of SARS-CoV-2 Omicron variant BA.2 sublineage.

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) Omicron was classified as a variant of concern in November 2021. The sublineage BA.2 spreads rapidly worldwide. Currently, there is a lack of data for the parallel comparison of Rapid Antigen Test (RAT) Kits to detect SARS-CoV-2 Omicron BA.2. We evaluated the analytical sensitivity of 12 RAT kits to detect Omicron BA.2 in the present study. Analytical sensitivity was determined by means of the limit of detection (LOD). We prepared a dilution set using a respiratory specimen collected from a COVID-19 patient infected by Omicron BA.2. The reverse transcription-polymerase chain reaction was used as a reference method. The LOD results showed that all 12 RAT kits had comparable analytical sensitivity to detect Omicron BA.2. The RAT kits selected in the current study may be used for the first-line screening of the rapid spreading Omicron BA.2.

Detection of SARS-CoV-2 VOC-Omicron using commercial sample-to-answer real-time RT-PCR platforms and melting curve-based SNP assays.

Objectives: The World Health Organization (WHO) had designated the SARS-CoV-2 lineage B.1.1.529 as the new Variant of Concern Omicron (VOC-Omicron) on 26th November 20211. Real-time reverse transcription polymerase chain reaction (RT-PCR), single nucleotide polymorphisms (SNP) and whole genome sequencing (WGS) tests were widely employed to detect SARS-CoV-2 and its variant. Yet, the SARS-CoV-2 Omicron detection performance of commercial real-time RT-PCR platforms and SARS-CoV-2 spike SNP assays remain to be elucidated. Methods: In the first part of this study, we evaluated the VOC-Omicron detection performance of three commercial RT-PCR sample-to-answer platforms i.e. Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems. The detection performances were compared to one commercial conventional real-time RT-PCR assay (TIB MOLBIOL LightMix Modular SARS and Wuhan CoV E-gene) and one in-house real-time RT-PCR assay targeting RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2 in the WHO COVID-19 Reference Laboratory at Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, The Government of the Hong Kong Special Administrative Region. In the second part of this study, we evaluated the SNP detection performance of four TIB MOLBIOL melting curve-based assays (1. Spike S371L/S373P, 2. Spike E484A, 3. Spike E484K and 4. Spike N501Y) in clinical samples obtained from hospitalized COVID-19 patients in Hong Kong. The SNP results were compared to whole genome sequences generated by Illumina platform. Results: The VOC-Omicron detection limits of three commercial sample-to-answer assays were tested to be ≤ 2.35 Log10 dC/ml. The detection performances of the sample-to-answer platforms were comparable to the two tested conventional real-time RT-PCR assays. The test sensitivities of TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P assay and the Spike E484A assays were 100% and 96.6% respectively and the test specificities of both assays were 100%. An aberrant melting peak at Tm 42-44°C was observed when the specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay. Notably, the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike N501Y assay failed to detect the spike N501Y mutation of Omicron variant in the tested specimens. Conclusions: The SARS-CoV-2 detection sensitivity of three commercial platforms, Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems were shown not to be impacted by the large number of mutations of VOC-Omicron. Also, the signature mutations i.e. Spike S371L/Spike S373P and Spike E484A in VOC-Omicron were correctly identified by the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P and VirSNiP SARS-CoV-2 Spike E484A assays. Unexpected findings including a shifted melting peak or absence of amplification curve/melting peak were observed when specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay and Spike N501Y assay, suggesting a potential alert for Omicron variant, prior confirmation by whole genome sequencing.

Evaluation of Rapid Antigen Detection Kits for Detection of SARS-CoV-2 B.1.1.529

Background Currently, there is a lack of studies evaluating rapid antigen detection (RAD) kits to detect SARS-CoV-2 B.1.1.529. Objective To evaluate the analytical sensitivity of seven RAD kits to detect SARS-CoV-2 B.1.1.529. Study design The analytical sensitivity was determined by means of limit of detection (LOD). A dilution set using a respiratory specimen collected from a COVID-19 patient infected with SARS-CoV-2 B.1.1.529 was prepared. RT-PCR was used as a reference method. Results The LOD results showed that all seven RAD kits had comparable analytical sensitivity for detection of SARS-CoV-2 B.1.1.529. Conclusions The RAD kits selected in the current study may be used for first-line screening of the recently emerged SARS-CoV-2 B.1.1.529.

Evaluation of rapid antigen detection kits for detection of SARS-CoV-2 B.1.617.2 virus.

Aim: Currently, there is lack of data regarding rapid antigen detection (RAD) kits to detect SARS-CoV-2 B.1.617.2 virus. Objective: The purpose of this evaluation is to assess analytical sensitivity of 12 RAD kits against SARS-CoV-2 B.1.617.2. Study design: Analytical sensitivity was determined by limit of detection (LOD). A serial tenfold dilution set from a respiratory specimen collected from a COVID-19 patient infected by SARS-CoV-2 B.1.617.2 was used. RT-PCR was used as a reference method. Results: The LOD results showed that 11 and one RAD kits were 100- and 1000-fold less sensitive than RT-PCR respectively. Conclusion: The results showed that the RAD kits evaluated in this study may be used for first-line screening of the SARS-CoV-2 B.1.617.2 variant.

Co-circulation of two SARS-CoV-2 variant strains within imported pet hamsters in Hong Kong.

During the investigation of a pet shop outbreak of severe acute respiratory coronavirus 2 (SARS-CoV-2) with probable hamster-to-human transmission, the environmental and hamster samples in epidemiologically linked pet shops were found positive for SARS-CoV-2 Delta variant AY.127 strains which are phylogenetically closely related to patients and reported European strains. This interspecies' spill-over has triggered transmission in 58 patients epidemiologically linked to three pet shops. Incidentally, three dwarf hamsters imported from the Netherlands and centralized in a warehouse distributing animals to pet shops were positive for SARS-CoV-2 spike variant phylogenetically related to European B.1.258 strains from March 2020. This B.1.258 strain almost disappeared in July 2021. While no hamster-to-human transmission of B.1.258-like strain was found in this outbreak, molecular docking showed that its spike receptor-binding domain (RBD) has a similar binding energy to human ACE2 compared to that of Delta variant AY.127. Therefore, the potential of this B.1.258-related spike variant for interspecies jumping cannot be ignored. The co-circulation of B.1.258-related spike variants with Delta AY.127, which originated in Europe and was not previously found in Hong Kong, suggested that hamsters in our wholesale warehouse and retail pet shops more likely have acquired these viruses in the Netherlands or stopovers during delivery by aviation than locally. The risk of human-to-hamster reverse zoonosis by multiple SARS-CoV-2 variants leading to further adaptive spike mutations with subsequent transmission back to humans cannot be underestimated as an outbreak source of COVID-19. Testing imported pet animals susceptible to SARS-CoV-2 is warranted to prevent future outbreaks.

Outbreak investigation of airborne transmission of Omicron (B.1.1.529) - SARS-CoV-2 variant of concern in a restaurant: Implication for enhancement of indoor air dilution.

Airborne transmission of SARS-CoV-2 has been increasingly recognized in the outbreak of COVID-19, especially with the Omicron variant. We investigated an outbreak due to Omicron variant in a restaurant. Besides epidemiological and phylogenetic analyses, the secondary attack rates of customers of restaurant-related COVID-19 outbreak before (Outbreak R1) and after enhancement of indoor air dilution (Outbreak R2) were compared. On 27th December 2021, an index case stayed in restaurant R2 for 98 min. Except for 1 sitting in the same table, six other secondary cases sat in 3 corners at 3 different zones, which were served by different staff. The median exposure time was 34 min (range: 19-98 min). All 7 secondary cases were phylogenetically related to the index. Smoke test demonstrated that the airflow direction may explain the distribution of secondary cases. Compared with an earlier COVID-19 outbreak in another restaurant R1 (19th February 2021), which occurred prior to the mandatory enhancement of indoor air dilution, the secondary attack rate among customers in R2 was significantly lower than that in R1 (3.4%, 7/207 vs 28.9%, 22/76, p<0.001). Enhancement of indoor air dilution through ventilation and installation of air purifier could minimize the risk of SARS-CoV-2 transmission in the restaurants.

Evaluation of automated antigen detection test for detection of SARS-CoV-2.

RT-PCR is the gold standard to detect SARS-CoV-2, however, its capacity is limited. We evaluated an automated antigen detection (AAD) test, Elecsys SARS-CoV-2 Antigen (Roche, Germany), for detecting SARS-CoV-2. We compared the limit of detection (LOD) between AAD test, rapid antigen detection (RAD) test; SARS-CoV-2 Rapid Antigen Test (SD Biosensor, Korea), and in-house RT-PCR test. LOD results showed that the AAD test was 100 fold more sensitive than the RAD test, while the sensitivity of the AAD test was comparable to the RT-PCR test. The AAD test detected between 85.7% and 88.6% of RT-PCR-positive specimens collected from COVID-19 patients, false negative results were observed for specimens with Ct values >30. Although clinical sensitivity for the AAD test was not superior or comparable to the RT-PCR test in the present study, the AAD test may be an alternative to RT-PCR test in terms of turn-around time and throughput.

Early detection of community acquired of SARS-CoV-2 lineage B.1.351 in Hong Kong.

Background: Prior to this report, variants of concern for SARS-CoV-2 were only detected from imported cases in Hong Kong. Objective: Multiple cases of SARS-CoV-2 lineage B.1.351 have been identified in local community. We reported the phylogenetic relationship of these cases. Study design: SARS-CoV-2 cases were screened for the key non-synonymous substitutions in spike protein by different assays. Preliminary positive cases were further tested by whole genome sequencing. Results: From Dec 2020 to May 2021, 55 SARS-CoV-2 cases belonged to lineage B.1.351. Among them, eight genomes were clustered together, all of them were local cases with epidemiological link. Conclusions: To track variants of SARS-CoV-2 and to allow early implementation of control measures, SARS-CoV-2 genomic surveillance must be consistently performed.

Stenoparib, an Inhibitor of Cellular Poly(ADP-Ribose) Polymerase, Blocks Replication of the SARS-CoV-2 and HCoV-NL63 Human Coronaviruses In Vitro.

By late 2020, the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new poly(ADP-ribose) polymerase (PARP) inhibitor, stenoparib, that recently advanced to phase II clinical trials for treatment of ovarian cancer, for activity against human respiratory coronaviruses, including SARS-CoV-2, in vitro Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to the human seasonal respiratory coronavirus HCoV-NL63. Compared to remdesivir, which inhibits viral replication downstream of cell entry, stenoparib impedes entry and postentry processes, as determined by time-of-addition (TOA) experiments. Moreover, a 10 µM dosage of stenoparib-below the approximated 25.5 µM half-maximally effective concentration (EC50)-combined with 0.5 µM remdesivir suppressed coronavirus growth by more than 90%, indicating a potentially synergistic effect for this drug combination. Stenoparib as a stand-alone or as part of combinatorial therapy with remdesivir should be a valuable addition to the arsenal against COVID-19.IMPORTANCE New therapeutics are urgently needed in the fight against COVID-19. Repurposing drugs that are either already approved for human use or are in advanced stages of the approval process can facilitate more rapid advances toward this goal. The PARP inhibitor stenoparib may be such a drug, as it is currently in phase II clinical trials for the treatment of ovarian cancer and its safety and dosage in humans have already been established. Our results indicate that stenoparib possesses strong antiviral activity against SARS-CoV-2 and other coronaviruses in vitro. This activity appears to be based on multiple modes of action, where both pre-entry and postentry viral replication processes are impeded. This may provide a therapeutic advantage over many current options that have a narrower target range. Moreover, our results suggest that stenoparib and remdesivir in combination may be especially potent against coronavirus infection.

Interaction of small molecules with the SARS-CoV-2 main protease in silico and in vitro validation of potential lead compounds using an enzyme-linked immunosorbent assay.

Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic is ongoing, with no proven safe and effective vaccine to date. Further, effective therapeutic agents for COVID-19 are limited, and as a result, the identification of potential small molecule antiviral drugs is of particular importance. A critical antiviral target is the SARS-CoV-2 main protease (Mpro), and our aim was to identify lead compounds with potential inhibitory effects. We performed an initial molecular docking screen of 300 small molecules, which included phenolic compounds and fatty acids from our OliveNet™ library (224), and an additional group of curated pharmacological and dietary compounds. The prototypical α-ketoamide 13b inhibitor was used as a control to guide selection of the top 30 compounds with respect to binding affinity to the Mpro active site. Further studies and analyses including blind docking were performed to identify hypericin, cyanidin-3-O-glucoside and SRT2104 as potential leads. Molecular dynamics simulations demonstrated that hypericin (ΔG = -18.6 and -19.3 kcal/mol), cyanidin-3-O-glucoside (ΔG = -50.8 and -42.1 kcal/mol), and SRT2104 (ΔG = -8.7 and -20.6 kcal/mol), formed stable interactions with the Mpro active site. An enzyme-linked immunosorbent assay indicated that, albeit, not as potent as the covalent positive control (GC376), our leads inhibited the Mpro with activity in the micromolar range, and an order of effectiveness of hypericin and cyanidin-3-O-glucoside > SRT2104 > SRT1720. Overall, our findings, and those highlighted by others indicate that hypericin and cyanidin-3-O-glucoside are suitable candidates for progress to in vitro and in vivo antiviral studies.

Site mapping and small molecule blind docking reveal a possible target site on the SARS-CoV-2 main protease dimer interface.

The SARS-CoV-2 virus is causing COVID-19 resulting in an ongoing pandemic with serious health, social, and economic implications. Much research is focused in repurposing or identifying new small molecules which may interact with viral or host-cell molecular targets. An important SARS-CoV-2 target is the main protease (Mpro), and the peptidomimetic α-ketoamides represent prototypical experimental inhibitors. The protease is characterised by the dimerization of two monomers each which contains the catalytic dyad defined by Cys145 and His41 residues (active site). Dimerization yields the functional homodimer. Here, our aim was to investigate small molecules, including lopinavir and ritonavir, α-ketoamide 13b, and ebselen, for their ability to interact with the Mpro. The sirtuin 1 agonist SRT1720 was also used in our analyses. Blind docking to each monomer individually indicated preferential binding of the ligands in the active site. Site-mapping of the dimeric protease indicated a highly reactive pocket in the dimerization region at the domain III apex. Blind docking consistently indicated a strong preference of ligand binding in domain III, away from the active site. Molecular dynamics simulations indicated that ligands docked both to the active site and in the dimerization region at the apex, formed relatively stable interactions. Overall, our findings do not obviate the superior potency with respect to inhibition of protease activity of covalently-linked inhibitors such as α-ketoamide 13b in the Mpro active site. Nevertheless, along with those from others, our findings highlight the importance of further characterisation of the Mpro active site and any potential allosteric sites.

Interaction of the prototypical α-ketoamide inhibitor with the SARS-CoV-2 main protease active site in silico: Molecular dynamic simulations highlight the stability of the ligand-protein complex.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes an illness known as COVID-19, which has been declared a global pandemic with over 2 million confirmed cases and 137,000 deaths in 185 countries and regions at the time of writing (16 April 2020), over a quarter of these cases being in the United States. In the absence of a vaccine, or an approved effective therapeutic, there is an intense interest in repositioning available drugs or designing small molecule antivirals. In this context, in silico modelling has proven to be an invaluable tool. An important target is the SARS-CoV-2 main protease (Mpro), involved in processing translated viral proteins. Peptidomimetic α-ketoamides represent prototypical inhibitors of Mpro. A recent attempt at designing a compound with enhanced pharmacokinetic properties has resulted in the synthesis and evaluation of the α-ketoamide 13b analogue. Here, we performed molecular docking and molecular dynamics simulations to further characterize the interaction of α-ketoamide 13b with the active site of the SARS-CoV-2 Mpro. We included the widely used antibiotic, amoxicillin, for comparison. Our findings indicate that α-ketoamide 13b binds more tightly (predicted GlideScore = -8.7 and -9.2 kcal/mol for protomers A and B, respectively), to the protease active site compared to amoxicillin (-5.0 and -4.8 kcal/mol). Further, molecular dynamics simulations highlight the stability of the interaction of the α-ketoamide 13b ligand with the SARS-CoV-2 Mpro (ΔG = -25.2 and -22.3 kcal/mol for protomers A and B). In contrast, amoxicillin interacts unfavourably with the protease (ΔG = +32.8 kcal/mol for protomer A), with unbinding events observed in several independent simulations. Overall, our findings are consistent with those previously observed, and highlight the need to further explore the α-ketoamides as potential antivirals for this ongoing COVID-19 pandemic.