Resistance mechanisms of SARS-CoV-2 3CLpro to the non-covalent inhibitor WU-04 (preprint)

Drug resistance poses a significant challenge in the development of effective therapies against SARS-CoV-2. Here, we identified two double mutations, M49K/M165V and M49K/S301P, in the 3C-like protease (3CLpro) that confer resistance to a novel non-covalent inhibitor, WU-04. Crystallographic analysis indicates that the M49K mutation destabilizes the WU-04 binding pocket, impacting the binding of WU-04 more significantly than the binding of 3CLpro substrates. The M165V mutation directly interferes with WU-04 binding. The S301P mutation, which is far from the WU-04 binding pocket, indirectly affects WU-04 binding by restricting the rotation of 3CLpros C-terminal tail and impeding 3CLpro dimerization. We further explored 3CLpro mutations that confer resistance to two clinically used inhibitors: ensitrelvir and nirmatrelvir, and revealed a trade-off between the catalytic activity, thermostability, and drug resistance of 3CLpro. We found that mutations at the same residue (M49) can have distinct effects on the 3CLpro inhibitors, highlighting the importance of developing multiple antiviral agents with different skeletons for fighting SARS-CoV-2. These findings enhance our understanding of SARS-CoV-2 resistance mechanisms and inform the development of effective therapeutics.

Relationship between mental health and physical activity among fourth grade students in Shanghai

[Background] Students in early adolescence have a high incidence of mental health problems. Studies have found an association between physical activity and mental health in adults, but the association among children and adolescents is not clear. [Objective] This study investigates the relationship between mental health and physical activity among students in early adolescence, aiming to provide evidence for the prevention and control of mental problems among students in this stage. [Methods] By using stratified cluster random sampling method, a questionnaire survey was conducted among 859 fourth grade students from 12 elementary schools in 3 urban and 3 suburban areas in Shanghai. The survey included students' general information, the total and mental health problems, and physical activity time, and calculated the total and dimensional (emotional symptoms, conduct problems, hyperactivity, peer problems, and prosocial behaviors) positive rates of mental health and the daily moderate-to-vigorous-intensity physical activity time. Chi-square test was used to compare the differences in mental health status among students with different family economic, educational backgrounds and family relationships. Logistic regression models were used to analyze the associations between mental health problems and physical activity. [Results] The total positive rate of mental health problems among the participants was 10.7%, and the rate of boys was higher than that of girls (13.4% vs. 8.0%, X 2=6.58, P < 0.05). The dimensional positive rates from high to low were peer problems (22.9%), hyperactivity (15.8%), emotional symptoms (9.0%), conduct problems (7.6%), and social impairment (5.2%). The median time[M (P25, P75)] of daily moderate-tovigorous-intensity physical activity of the students was 76.9 (8.4, 107.1) min;48.7% of the students spent less than 60 min on moderateto-vigorous-intensity physical activities per day, and the proportion in boys (52.2%) was higher than that in girls (45.1%) (X 2=4.36, P < 0.05). The rank-sum test results showed that the daily time of moderate-to-vigorous-intensity physical activity was correlated with the positive rate of emotional symptoms, conduct problems, hyperactivity, peer problems, and total mental health problems (P < 0.05). The chi-square test results showed that the factors such as mother's education level, father's and mother's monthly income, and family relationships were related to the positive rate of total mental health problems (P < 0.05). The logistic regression analysis results showed that spending less than 60 min on moderate-to-vigorous-intensity physical activities per day was associated to reporting hyperactivity, total mental health problems, and social impairment, and the ORs (95%CI) were 1.633 (1.095-2.436), 2.811 (1.676-4.714), and 2.307 (1.153-4.618) respectively. [Conclusion] Among the fourth grade students in Shanghai, the positive rate of boys' mental health problems is higher than that of girls', and the overall rate of peer problems is high. Nearly half of the students spend less than 60 min on moderate-to-vigorous-intensity physical activities per day. Doing moderate-to-vigorous-intensity physical activity for less than 60 min per day is possibly associated to the positive rate of students' mental health problems.

Development of highly potent non-covalent inhibitors of SARS-CoV-2 3CLpro (preprint)

The SARS-CoV-2 virus is the causal agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19). There is an urgent need for potent, specific antiviral compounds against SARS-CoV-2. The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses, and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, non-covalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC 50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment. One-Sentence Summary A oral non-covalent inhibitor of 3C-like protease effectively inhibits SARS-CoV-2 replication.

VV-ECMO combined with prone position ventilation in the treatment of Pneumocystis jirovecii pneumonia : A case report

Introduction: Pneumocystis jirovecii pneumonia (PJP) occurs in immunocompromised hosts. It is classified as PJP with human immunodeficiency virus (HIV) infection (HIV-PJP) and PJP without HIV infection (non-HIV PJP). Compared with HIV-PJP, non-HIV PJP is more likely to develop rapidly into respiratory failure, with difficult diagnosis and high mortality. Patient concerns: A 46-year-old male with membranous nephropathy was treated with oral corticosteroids and tacrolimus. He was admitted to our hospital for fever and dyspnea which developed 4 days ago. Laboratory data revealed that leukocytes were 10.99 × 109/L, neutrophils 87.7%, lymphocytes 9.6%, C-reactive protein 252.92 mg/L, New coronavirus nucleic acid detection negative. CT scan of chest revealed ground-glass opacity in both lungs. He was admitted to the respiratory department of our hospital, and then transferred to ICU because of his critical condition. Diagnosis: High throughput gene detection of pathogenic microorganisms in alveolar lavage fluid showed that the detection sequence of Pneumocystis yersiniae increased significantly. The serum HIV-antibody was negative. Therefore, the patient was diagnosed as non-HIV PJP. Interventions: After admission, the patient was assisted by noninvasive ventilator and treated with compound trimethoprim-sulfamethoxazole (SMX-TMP) and caspofungin. The patient's condition continued to deteriorate, and then underwent endotracheal intubation and veno-venous extracorporeal membrane oxygenation (VV-ECMO) combined with prone position ventilation until the lung lesion improved. Outcomes: VV-ECMO was stopped on day 12, tracheal intubation was removed after 2 days. The patient was transferred to the respiratory department on day 15, discharged after 12 days without complications. Two months later, the follow-up showed that the patient was in good condition. Conclusion: VV-ECMO combined with prone position ventilation could be a useful choice for respiratory assistance in non-HIV PJP patients.

BRET-based self-cleaving biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery (preprint)

The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments and more inhibitors are being developed. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors have to be evaluated in a Biosafety Level 3 (BSL-3) or BSL-4 laboratory. Here, we design genetically encoded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After over-expression of the biosensors in HEK293 cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their anti-SARS-CoV-2 activities reported previously. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. Furthermore, our strategy can be used to design biosensors for other viral proteases that share the same activation mechanism as 3CLpro, such as HIV protease PR and HCV protease NS3.