New insights in the mechanism of the SARS-CoV-2 Mpro inhibition by benzisoselenazolones and diselenides (preprint)

Although global vaccination campaigns relieved the SARS-CoV-2 pandemic in terms of morbidity and mortality, the capability of the virus to originate mutants may reduce vaccines efficiency, posing a serious risk to fall into the pandemic again. As a result, there is the need to develop small molecules able to tackle conserved viral targets, such as the main protease (Mpro). Here a series of benzisoselenazolones and diselenides were tested for their ability to inhibit Mpro, then, for the most potent compounds, the antiviral activity was measured in vitro, and the mechanism of action was investigated. Density functional theory and molecular docking procedures were also implemented to shed a light into the protein/compound interaction. Finally, a bioorganic model was set up to investigate the reaction between selenorganic compounds and biologically relevant thiols, to unravel possible metabolic pathways of such compounds. The overall results contribute to identify a series of novel Se-containing molecules active against SARS-CoV-2, and to clarify some important aspects in the mechanisms of action of such inhibitors targeting the SARS-CoV-2 Mpro.

Discovery and evaluation of active compounds from Xuanfei Baidu Formula against COVID-19 via SARS-Cov-2 Mpro (preprint)

Background The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) is still a widespread concern. As one of the effective traditional Chinese medicine (TCM) formula, Xuanfei Baidu formula (XFBD) shows significant efficacy for treatment of COVID-19 patients. However, its antiviral compounds and mechanism are still unclear. Purpose: In this study, we explored the bioactive compounds of XFBD and its antiviral mechanism by integrating computational analysis and experimental testing. Methods Aiming at the SARS-CoV-2 main protease (Mpro), as a key target in virus replication, the fluorescence resonance energy transfer (FRET) assay was built to screen out satisfactory natural inhibitors from XFBD. The surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were undertaken to verify the binding affinity of Mpro-ligand. Omicron BA.1.1 and BA.2 variants were used to evaluate the antiviral activity of the focused compounds in non-cytotoxicity concentrations. For introducing the molecular mechanism, computational modeling and NMR spectra were employed to predict the binding mode and binding site of Mpro-ligand. Results From a library of 83 natural compounds, acteoside, licochalcone B, licochalcone D, linoleic acid, and physcion showed the satisfactory inhibition effect on Mpro with IC50 from 1.93 to 42.96 µM, which were further verified by SPR. Showing the excellent binding affinity, acteoside was witnessed to gain valuable insights into the thermodynamic signatures by ITC and presented antiviral activity on Omicron BA.1.1 and BA.2.3 variants in vitro. The results revealed that acteoside inhibited Mpro via forming the hydrogen bond between 7-H of acteoside and Mpro. Conclusion Acteoside is regarded as a representative active natural compound in XFBD to inhibit replication of SARS-CoV-2, which provides the antiviral evidence and some insight into the identifications of SARS-CoV-2 Mpro natural inhibitors.

Repeat-Dose Toxicity of Candidate Omicron COVID-19 Vaccine in Sprague-Dawley Rats

Due to a large number of mutations in the spike protein and immune escape, the Omicron variant (B.1.1.529) has become a predominant variant of concern (VOC) strain. To prevent the disease, we developed a candidate inactivated vaccine (Omicron COVID-19 Vaccine (Vero Cell), Inactivated). To evaluate the safety of the vaccine, we tested the repeat-dose toxicity in Sprague-Dawley (SD) rats. The doses were administered randomly to three groups: physiological saline solution (control), aluminum adjuvant in PBS solution adjuvant (adjuvant group), and low-dose and high-dose omicron vaccines (vaccine group) for 6 weeks. The SD rats were allowed to recover for 4 weeks after withdrawal. We evaluated the physiological condition of the rats, including their ophthalmological condition, body weight, food intake, body temperature, blood biochemistry, urine, neutralizing antibody, inflammation at the injection site, and organs weight. In summary, no dose-dependent adverse toxicological changes were observed, and a recovery trend was obvious, which proved the preclinical safety of the candidate omicron vaccine and provided evidence for clinical trials in humans.

Boundary Guided Semantic Learning for Real-time COVID-19 Lung Infection Segmentation System (preprint)

The coronavirus disease 2019 (COVID-19) continues to have a negative impact on healthcare systems around the world, though the vaccines have been developed and national vaccination coverage rate is steadily increasing. At the current stage, automatically segmenting the lung infection area from CT images is essential for the diagnosis and treatment of COVID-19. Thanks to the development of deep learning technology, some deep learning solutions for lung infection segmentation have been proposed. However, due to the scattered distribution, complex background interference and blurred boundaries, the accuracy and completeness of the existing models are still unsatisfactory. To this end, we propose a boundary guided semantic learning network (BSNet) in this paper. On the one hand, the dual-branch semantic enhancement module that combines the top-level semantic preservation and progressive semantic integration is designed to model the complementary relationship between different high-level features, thereby promoting the generation of more complete segmentation results. On the other hand, the mirror-symmetric boundary guidance module is proposed to accurately detect the boundaries of the lesion regions in a mirror-symmetric way. Experiments on the publicly available dataset demonstrate that our BSNet outperforms the existing state-of-the-art competitors and achieves a real-time inference speed of 44 FPS.

BCS-Net: Boundary, Context and Semantic for Automatic COVID-19 Lung Infection Segmentation from CT Images (preprint)

The spread of COVID-19 has brought a huge disaster to the world, and the automatic segmentation of infection regions can help doctors to make diagnosis quickly and reduce workload. However, there are several challenges for the accurate and complete segmentation, such as the scattered infection area distribution, complex background noises, and blurred segmentation boundaries. To this end, in this paper, we propose a novel network for automatic COVID-19 lung infection segmentation from CT images, named BCS-Net, which considers the boundary, context, and semantic attributes. The BCS-Net follows an encoder-decoder architecture, and more designs focus on the decoder stage that includes three progressively Boundary-Context-Semantic Reconstruction (BCSR) blocks. In each BCSR block, the attention-guided global context (AGGC) module is designed to learn the most valuable encoder features for decoder by highlighting the important spatial and boundary locations and modeling the global context dependence. Besides, a semantic guidance (SG) unit generates the semantic guidance map to refine the decoder features by aggregating multi-scale high-level features at the intermediate resolution. Extensive experiments demonstrate that our proposed framework outperforms the existing competitors both qualitatively and quantitatively.

Close contact management mode and epidemiological characteristics analysis of COVID-19 in Chengdu, China (preprint)

As a potential source of infection, the risk of close contacts has gradually increased. Close contact management mode and epidemiological characteristics for 20,254 close contacts from January 22, 2020 to April 1, 2021 in Chengdu, China were analyzed. The relationship with index cases was mainly co-passengers (73.52%) and relatives (13.64%), and the frequency of contact was mainly occasional contact (68.31%). 277 (1.37%) close contacts who were converted into cases were mainly found in the first and second nucleic acid tests (58.27%), and the contact mode was mainly by sharing transportation (37.55%). In terms of close contact management time, both the supposed ((13.00±3.23) vs. (12.84 ±6.91)) and actual ((14.13±24.02) vs. (14.00±12.98)) isolation time in Chengdu were longer than that of outer cities (P<0.001). Tracing and management of close contacts is a two-way management measure, which requires the cooperation among departments. Enhancing existing monitoring and response capabilities can control the spread of the epidemic to a certain extent.

Improving Lab Culture through Self-Assessment: A Case Study (preprint)

Purpose Motivated by perceived dissatisfaction within our lab’s changed working environment brought about by the COVID-19 pandemic, we performed a self-assessment of our lab culture through anonymous surveys and live sessions. Methods In Survey 1, we asked each lab member to identify and rank up to 10 values that are important for a healthy lab environment. They were then asked to rate how well the lab embodied those values at two time points: before the COVID-19 pandemic while working onsite, and at the time of the survey while working remotely (10 months into the pandemic). In a series of live group sessions, we reviewed relevant literature and the survey results to finalize ten themes. We then reflected on each theme and proposed action items to address any deficiencies. Finally, we conducted Survey 2 after the self-assessment to judge the group’s finalized themes, implemented changes, and overall satisfaction with the assessment process. Results Themes identified were attitude, accountability, teamwork/collaboration, communication, diversity/inclusion, emotional intelligence, integrity, training, well-being, and adaptability in crisis-management. All lab members liked the self-assessment process and felt their voices were heard. On average, there was a 12% increase in satisfaction across all themes from the start to end of the lab assessment. Conclusion We successfully assessed the culture of our lab and subsequently improved lab member satisfaction. The success of this team project suggests that other scientific labs could benefit from similar interactive self-assessments.

Novel inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives (preprint)

The global emergence of SARS-CoV-2 has triggered numerous efforts to develop therapeutic options for COVID-19 pandemic. The main protease of SARS-CoV-2 (Mpro), which is a critical enzyme for transcription and replication of SARS-CoV-2, is a key target for therapeutic development against COVID-19. An organoselenium drug called ebselen has recently been demonstrated to have strong inhibition against Mpro and antiviral activity but its molecular mode of action is unknown preventing further development. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of ebselen derivatives. A free selenium atom bound with cysteine 145 of Mpro catalytic dyad has been revealed by crystallographic studies of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct, formation of a phenolic by-product is confirmed by mass spectrometry. The target engagement of these compounds with an unprecedented mechanism of SARS-CoV-2 Mpro inhibition suggests wider therapeutic applications of organo-selenium compounds in SARS-CoV-2 and other zoonotic beta-corona viruses.

Organoselenium mild electrophiles in the inhibition of Mpro and SARSCoV-2 replication (preprint)

New Ebselen-like derivatives resulted to be very strong in vitro inhibitors of SARS-CoV-2 main protease. We demonstrated that this activity mainly depends on the electrophilicity of the selenium atom that is considerably higher in the N-substituted 1,2- benzoselenazol-3(2H)-ones respect to the corresponding diselenides allowing it to be rapidly attached by free thiols affording sulfur-selenium intermediates that are further subjected to thiol exchange processes. This data paints a very complex scenario that requires us to consider Ebselen and Ebselen-like derivatives as potential electrophilic substrates for the several other free thiols present in the cell beside the target free cysteine. The sulfur selenium intermediates are milder electrophiles that could be theoretically implicated in both the detoxification process as well as in the final enzymatic inhibition. We here demonstrated that the in vitro inhibition activity is not fully reproduced in the prevention of viral replication in the cell-based assay. This indicates that the structure of the substituents introduced in the Ebselen scaffold is a crucial factor to control the reactivity of the selenated molecule in the network of thiol exchanges, as well as for molecular recognition of the targeted enzymatic cysteine. For this reason, an in-depth investigation is strongly desirable to better understand how to increase the activity and the selectivity of Ebselen derivatives overcoming the issues of the apparent PAINS-like role of Ebselen. Furthermore, besides the antiviral activity, thee selected compounds also showed a different ability to reduce the virus-induced cytopathic effect, indicating that other mechanisms could be implicated. One may consider here the well-known cytoprotective antioxidant activity of Ebselen and its derivatives.

A follow-up study of children infected with SARS-CoV-2 from Western China (preprint)

Background: To clarify the characteristic and the duration of positive nucleic acid in children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including asymptomatic children. Methods: A total of 32 children confirmed with SARS-CoV-2 infection between January 24 and February 12, 2020 from four provinces in Western China were enrolled in this study and followed up until discharge and quarantine 14 days later. Results: Eleven children (34%) were asymptomatic, among whom six children had normal computed tomographic (CT) scan images. Age and gender were not associated with clinical symptoms or the results of CT scan in children infected with SARS-CoV-2. The concentrations of white blood cells and neutrophils were higher in children with asymptomatic infection than in children with clinical symptoms or CT abnormalities. Patients who presented with CT abnormalities had lower D-dimer or lower total bilirubin than those who had normal CT scan but clinical symptoms. All children recovered and no one died or was admitted to the pediatric intensive care unit (PICU). The mean duration of positive SARS-CoV-2 nucleic acid was 15.4 (SD=7.2) days and similar for both asymptomatic children and children with symptoms or CT abnormalities. We found a significant negative correlation between the lymphocyte count and the duration of positive nucleic acid test. Conclusions: Children with asymptomatic infection should be quarantined for the same duration as symptomatic patients infected with SARS-CoV-2. The clinical significance and mechanism behind the negative correlation between the number of lymphocytes and the duration of positive SARS-CoV-2 needs further study.

Structural basis for the inhibition of COVID-19 virus main protease by carmofur, an antineoplastic drug (preprint)

The antineoplastic drug Carmofur was shown to inhibit SARS-CoV-2 main protease (Mpro). Here the X-ray crystal structure of Mpro in complex with Carmofur reveals that the carbonyl reactive group of Carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 M) and it is a promising lead compound to develop new antiviral treatment for COVID-19.

Structure-Based Design, Synthesis and Biological Evaluation of Peptidomimetic Aldehydes as a Novel Series of Antiviral Drug Candidates Targeting the SARS-CoV-2 Main Protease (preprint)

SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 M and 0.04 M respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 M and 0.33 M, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 [A] resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.

Structure of RNA-dependent RNA polymerase from 2019-nCoV, a major antiviral drug target (preprint)

A novel coronavirus (2019-nCoV) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12), which catalyzes the synthesis of viral RNA, is a key component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. Here we report the cryo-EM structure of 2019-nCoV full-length nsp12 in complex with cofactors nsp7 and nsp8 at a resolution of 2.9-[A]. Additional to the conserved architecture of the polymerase core of the viral polymerase family and a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain featured in coronaviral RdRp, nsp12 possesses a newly identified {beta}-hairpin domain at its N-terminal. Key residues for viral replication and transcription are observed. A comparative analysis to show how remdesivir binds to this polymerase is also provided. This structure provides insight into the central component of coronaviral replication/transcription machinery and sheds light on the design of new antiviral therapeutics targeting viral RdRp. One Sentence SummaryStructure of 2019-nCov RNA polymerase.

Structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting COVID-19 (preprint)

A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1-4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 M. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.