DESIGN of NOVEL and HIGHLY SELECTIVE SARS-CoV-2 MAIN PROTEASE INHIBITORS

Background: SARS-CoV-2 has caused a global pandemic, yet despite vaccine availability, it continues to inflict morbidity and mortality worldwide. The viral main protease (Mpro) is highly conserved across multiple coronaviruses and has a unique viral substrate specificity. Thus, highly selective Mpro inhibitors are expected to be safe, effective, and elude drug resistance for future coronaviruses. Methods: We used a conformationally restricted peptidomimetic to mimic the bioactive conformation of the Mpro-substrate complex to identify potent, selective Mpro inhibitors. We evaluated protease inhibition in biochemical assays, and cellular efficacy in Vero-E6 cells challenged with live virus representing parental (USA-WA1/2020), beta (B.1.351), and delta (B.1.617.2) variants by monitoring infection at day 2 post-infection measuring nucleocapsid-positive cells by high content imaging, and cytopathic effect (CPE) at day 4 post-infection using resazurin viability dye. Results were compared to reference compounds. Group differences were analyzed by two-sided, paired t-test. Results: AP-8-013 required a 2-hour incubation to achieve maximal dose-dependent Mpro inhibition with an IC50 = 230 ± 18 nM, reflecting its highly constrained conformation, compared to the more flexible Cpd 22 (AP-8-001;IC50 = 11 ± 0.7 nM) or GC-376 (IC50 = 18 ± 1.5 μM). Importantly, AP-8-013 showed exquisite selectivity for Mpro with no inhibition at key mammalian cysteine proteases, cathepsin B and L, or the serine protease thrombin, while Cpd 22 (Cat B IC50 = 24 ± 7.5 nM, Cat L IC50 = 1.8 ± 0.3 nM) or GC-376 (Cat B IC50 = 37 ± 1.5 nM, Cat L IC50 = < 1 nM) showed poor selectivity towards mammalian cysteine proteases. AP-8-013 was active in CPE cell-based assays with comparable potency to reference compounds, with EC50 = 4.7 μM compared to Cmp 22 (EC50 = 1.4 μM) or GC-376 (EC50 = 1.1 μM). Using intact SARS-CoV-2 infection-based assays, AP-8-013 significantly inhibited parental virus as well as beta and delta VOC (EC50s = 2.7, 2.5, and 6.0 μM, respectively). Finally, a 3:1 molar mixture of AP-8-013 and remdesivir significantly enhanced antiviral activity in CPE assays (EC50 = 1.3 μM;p < 0.05) when compared against either compound alone (EC50s = 4.7 and 3.3 μM, respectively). Conclusion: We have identified a novel drug-like Mpro inhibitor lead series which is highly selective over cysteine and serine proteases that can inhibit multiple SARS-CoV-2 VOC and increase the antiviral activity of remdesivir.

Impact of Remdesivir on SARS-CoV-2 Clearance in a Real-Life Setting: A Matched-Cohort Study

Background. Evidence regarding the impact of remdesivir (RDV) on SARSCoV-2 viral clearance (VC) is scarce. Aim of this study was to compare VC timing in COVID-19 patients who received RDV with those who did not. Methods. Matched-cohort study conducted (25 February 2020-15 April 2021) at the IRCSS San Raffaele, Milan, Italy. The study enrolled hospitalized patients with pneumonia and a SARS-CoV-2 positive nasopharyngeal swab (NPS) at admission and at least one NPS during follow-up. Follow-up started at hospital admission and ended at the date of the first negative NPS (within 30 days after discharge). Patients who received RDV (cases) and patients who did not (controls) were matched based on age (±5 years), sex and PaO2/FiO2 (P/F;±10 mmHg) values at admission. NPS were analyzed with RT-PCR. Results described as median (IQR) or frequency (%). Time to VC was estimated with Kaplan-Meier curve and compared with log-rank test. Results. 648 patients were enrolled: 216 cases and 432 controls. Patients' characteristics at admission are reported in Table 1. VC was observed in 490 patients (75.6%) in a median time of 25 (16-34) days. Overall, time to VC was similar in patients receiving or not receiving remdesivir (p=0.519). However, time to VC was different when considering both the use of RDV (yes vs no) and age (≤ or > 63 years), as shown in Figure 1A. A significant finding was also observed considering the use of RDV and P/F values at admission (≤ or > 200 mmHg), as reported in Figure 1B. Among the 490 patients who reached VC during follow-up, overall time to VC was similar in patients receiving or not receiving RDV (p=0.075;Figure 2A);however, RDV use was associated with a higher probability of VC in the subgroup of patients with P/F admission values ≤ 200mmHg (p=0.035;Figure 2B), in the age group 55-65 years (p=0.025;Figure 2C) and in patients with comorbidities (p=0.028). Time to viral clearance among the 490 patients who reached VC during follow-up. Panel A: time to VC according to RDV use. Panel B: time to VC according to RDV and P/F ratio value at admission. Panel C: time to VC according to RDV in the age group 55-65 years. Conclusion. Time to viral clearance was similar in patients receiving or not receiving remdesivir;however the use of RDV was associated with a benefit on time to viral clearance in younger patients and in those with a P/F ratio at admission ≤200 mmHg.

Estimating COVID-19 recovery time in a cohort of Italian healthcare workers who underwent surveillance swab testing.

OBJECTIVES: The COVID-19 pandemic is putting a huge strain on the provision and continuity of care. The length of sickness absence of the healthcare workers as a result of SARS-CoV-2 infection plays a pivotal role in hospital staff management. Therefore, the aim of this study was to explore the timing of COVID-19 recovery and viral clearance, and its predictive factors, in a large sample of healthcare workers. STUDY DESIGN: This is a retrospective cohort study. METHODS: The analysis was conducted on data collected during the hospital health surveillance programme for healthcare staff at the University Hospital of Verona; healthcare workers were tested for SARS-CoV-2 through RT-PCR with oronasopharyngeal swab samples. The health surveillance programme targeted healthcare workers who either had close contact with SARS-CoV-2-infected patients or were tested as part of the screening-based strategy implemented according to national and regional requirements. Recovery time was estimated from the first positive swab to two consecutive negative swabs, collected 24 h apart, using survival analysis for both right-censored and interval-censored data. Cox proportional hazard was used for multivariate analysis. RESULTS: During the health surveillance programme, 6455 healthcare workers were tested for SARS-CoV-2 and 248 (3.8%, 95% confidence interval [CI]: 3.4-4.3) reported positive results; among those who tested positive, 49% were asymptomatic, with a median age of 39.8 years, which is significantly younger than symptomatic healthcare workers (48.2 years, P < 0.001). Screening tests as part of the health surveillance programme identified 31 (12.5%) of the positive cases. Median recovery time was 24 days (95% CI: 23-26) and 21.5 days (95% CI: 15.5-30.5) in right- and interval-censoring analysis, respectively, with no association with age, sex or presence of symptoms. Overall, 63% of participants required >20 days to test negative on two consecutive swabs. Hospitalised healthcare workers (4.8%) were older and had a significantly longer recovery time compared with non-hospitalised healthcare workers in both analyses (33.5 vs 24 days, P = 0.005). CONCLUSIONS: Recovery from COVID-19 and viral clearance may take a long time, especially in individuals who are hospitalised. To detect asymptomatic cases, screening programmes for healthcare workers is recommended.

Viral clearance after early corticosteroid treatment in patients with moderate or severe covid-19.

The aim of this study was to evaluate the impact of early treatment with corticosteroids on SARS-CoV-2 clearance in hospitalized COVID-19 patients. Retrospective analysis on patients admitted to the San Raffaele Hospital (Milan, Italy) with moderate/severe COVID-19 and availability of at least two nasopharyngeal swabs. The primary outcome was the time to nasopharyngeal swab negativization. A multivariable Cox model was fitted to determine factors associated with nasopharyngeal swab negativization. Of 280 patients included, 59 (21.1%) patients were treated with steroids. Differences observed between steroid users and non-users included the proportion of patients with a baseline PaO2/FiO2 ≤ 200 mmHg (45.8% vs 34.4% in steroids and non-steroids users, respectively; p = 0.023) or ≤ 100 mmHg (16.9% vs 12.7%; p = 0.027), and length of hospitalization (20 vs 14 days; p < 0.001). Time to negativization of nasopharyngeal swabs was similar in steroid and non-steroid users (p = 0.985). According to multivariate analysis, SARS-CoV-2 clearance was associated with age ≤ 70 years, a shorter duration of symptoms at admission, a baseline PaO2/FiO2 > 200 mmHg, and a lymphocyte count at admission > 1.0 × 109/L. SARS-CoV-2 clearance was not associated with corticosteroid use. Our study shows that delayed SARS-CoV-2 clearance in moderate/severe COVID-19 is associated with older age and a more severe disease, but not with an early use of corticosteroids.