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.

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir (preprint)

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at scale, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~300-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to shed light on the design of next generation protease inhibitors.

A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2 (preprint)

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 g of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants (preprint)

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

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.

Ultrapotent SARS-CoV-2 neutralizing antibodies effectively block new-emerging mutational variants (preprint)

Accumulating mutations on SARS-CoV-2 Spike (S) protein may increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, in a panel of receptor binding domain (S-RBD) specific monoclonal antibodies (mAbs) with high neutralizing potency against authentic SARS-CoV-2, at least 6 of them were found to efficiently block the pseudovirus of 501Y.V2, a highly transmissible SARS-CoV-2 variant with escape mutations. The top 3 neutralizing Abs (13G9, 58G6 and 510A5) exhibited comparative ultrapotency as those being actively pursued for clinical development. Interestingly, the antigenic sites for the majority of our neutralizing Abs overlapped with a single epitope (13G9e) on S-RBD. Further, the 3-dimensional structures of 2 ultrapotent neutralizing Abs 13G9 or 58G6 in complex with SARS-CoV-2 S trimer demonstrated that both Abs bound to a steric region within S472–490. Moreover, a specific linear region (S450–457) was identified as an additional target for 58G6. Importantly, our cryo-electron microscopy (cryo-EM) analysis revealed a unique phenomenon that the S-RBDs interacting with the fragments of antigen binding (Fabs) of 13G9 or 58G6 encoded by the IGHV1-58 and the IGKV3-20 gene segments were universally in the ‘up’ conformation in all observed particles. The potent neutralizing Abs presented in the current study may be promising candidates to fulfill the urgent needs for the current pandemic of SARS-CoV-2, and may of fundamental value for the next-generation vaccine development.

Evaluation on the Diagnostic Efficiency of Different Methods in Detecting COVID-19. (preprint)

Objective: To evaluate the diagnostic efficiency of different methods in detecting COVID-19.Methods: PubMed, Web of Science and Embase databases were searched for identifing eligible articles. All data were calculated utilizing Meta Disc 1.4, Revman 5.3.2 and Stata 12. The diagnostic efficiency was assessed via these indicators including summary sensitivity and specificity, positive likelihood ratio (PLR), negative LR (NLR), diagnostic odds ratio (DOR), summary receiver operating characteristic curve (sROC) and calculate the AUC. Results: 18 articles (3648 cases) were included. EPlex: pooled sensitivity was 0.94; specificity 1.0; PLR 90.91; NLR 0.07; DOR 1409.49; AUC=0.9979, Q*=0.9840. Panther Fusion: pooled sensitivity was 0.99; specificity 0.98; PLR 42.46; NLR 0.02; DOR 2300.38; AUC=0.9970, Q*=0.9799. Simplexa: pooled sensitivity was 1.0; specificity 0.97; PLR 26.67; NLR 0.01; DOR 3100.93; AUC=0.9970, Q*=0.9800. Cobas®: pooled sensitivity was 0.99; specificity 0.96; PLR 37.82; NLR 0.02; DOR 3754.05; AUC=0.9973, Q*=0.9810. RT-LAMP: pooled sensitivity was 0.98; specificity 0.99; PLR 36.22; NLR 0.04; DOR 751.24; AUC=0.9905, Q*=0.9596. Xpert Xpress: pooled sensitivity was 0.99; specificity 0.97; PLR 27.44; NLR 0.01; DOR 3488.15; AUC=0.9977, Q*=0.9829.Conclusions: These methods (ePlex, Panther Fusion, Simplexa, Cobas®, RT-LAMP and Xpert Xpress) bear higher sensitivity and specificity, and might be efficient methods complement to the gold standard.

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.

Evaluation on the diagnostic efficiency of different methods in detecting COVID-19. (preprint)

Objective: To evaluate the diagnostic efficiency of different methods in detecting COVID-19 to provide preliminary evidence on choosing favourable method for COVID-19 detection. Methods: PubMed, Web of Science and Embase databases were searched for identifing eligible articles. All data were calculated utilizing Meta Disc 1.4, Revman 5.3.2 and Stata 12. The diagnostic efficiency was assessed via these indicators including summary sensitivity and specificity, positive likelihood ratio (PLR), negative LR (NLR), diagnostic odds ratio (DOR), summary receiver operating characteristic curve (sROC) and calculate the AUC. Results: 18 articles (3648 cases) were included. The results showed no significant threshold exist. EPlex: pooled sensitivity was 0.94; specificity was 1.0; PLR was 90.91; NLR was 0.07; DOR was 1409.49; AUC=0.9979, Q*=0.9840. Panther Fusion: pooled sensitivity was 0.99; specificity was 0.98; PLR was 42.46; NLR was 0.02; DOR was 2300.38; AUC=0.9970, Q*=0.9799. Simplexa: pooled sensitivity was 1.0; specificity was 0.97; PLR was 26.67; NLR was 0.01; DOR was 3100.93; AUC=0.9970, Q*=0.9800. Cobas: pooled sensitivity was 0.99; specificity was 0.96; PLR was 37.82; NLR was 0.02; DOR was 3754.05; AUC=0.9973, Q*=0.9810. RT-LAMP: pooled sensitivity was 0.98; specificity was 0.99; PLR was 36.22; NLR was 0.04; DOR was 751.24; AUC=0.9905, Q*=0.9596. Xpert Xpress: pooled sensitivity was 0.99; specificity was 0.97; PLR was 27.44; NLR was 0.01; DOR was 3488.15; AUC=0.9977, Q*=0.9829. Conclusions: These methods (ePlex, Panther Fusion, Simplexa, Cobas, RT-LAMP and Xpert Xpress) bear higher sensitivity and specificity, and might be efficient methods complement to the gold standard.

The Chest CT Features of Coronavirus Disease 2019 (COVID-19) in China: A Meta-analysis of 19 Trials (preprint)

ObjectiveThis study aimed to summarize the characteristics of chest CT imaging in Chinese patients with Coronavirus Disease 2019 (COVID-19) to provide reliable evidence for further guiding clinical routine. MethodsPubMed, Embase and Web of Science databases were thoroughly searched to identified relevant articles involving the features of chest CT imaging in Chinese patients with COVID-19. All data were analyzed utilizing R software version i386 4.0.0. Random-effects models were employed to calculate pooled mean differences. Results19 trials incorporating 1332 cases were included in the study. The results demonstrated that the incidence of ground-glass opacities (GGO) was 0.79, consolidation was 0.34; mixed GGO and consolidation was 0.46; air bronchogram sign was 0.41; crazy paving pattern was 0.32; interlobular septal thickening was 0.55; reticulation was 0.30; bronchial wall thickening was 0.24; vascular enlargement was 0.74. subpleural linear opacity was 0.28; intrathoracic lymph node enlargement was 0.03; pleural effusions was 0.03. The distribution in lung: the incidence of central was 0.05; peripheral was 0.74; peripheral involving central was 0.38; diffuse was 0.19; unifocal involvement was 0.09; multifocal involvement was 0.57; unilateral was 0.16; bilateral was 0.83; The incidence of lobes involved (>2) was 0.70; lobes involved ([less double equals]2) was 0.35. ConclusionGGO, vascular enlargement, interlobular septal thickening more frequently occurred in patients with COVID-19. Peripheral, bilateral, involved lobes >2 might be the features of COVID-19 in the distribution aspect. Therefore, based on the aboved features of COVID-19 in chest CT imaging, it might be a promising means for identifying COVID-19.

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.

COVID-19 discharged patients in Hunan, China: correlation between early features and prognosis (preprint)

Purpose      To describe the correlation between the clinical, laboratory and radiological findings with hospitalization days in Coronavirus Infected Disease-19 (COVID-19) discharged patients. Method In this multicenter study, we retrospectively identified 153 discharged patients with COVID-19 pneumonia from Jan 16, 2020 to Feb 26, 2020 in Hunan province. Patients were grouped based on the hospitalization days: Group 1 (hospitalization days≦12 days) and Group 2((hospitalization days> 12days). Demographic, clinical characteristics and laboratory findings on admission and the imaging features of the first Chest CT on admission were analyzed. The differences between groups were analyzed using univariate logistic regression to find the impact factors. Results The cohort included 153 discharged patients (85 males and 68 females, with the mean age of 42.32±14.03 years old). 90(58.8%) patients had hospitalization days≦12 and 63(41.2%) patients had hospitalization days>12. 44(48.9%) patients in Group1 and 28(44.4%) in Group 2 had been to Wuhan. In both Group1 and Group2, most common symptoms at onset were fever (62.2%, 60.3%) and cough (33.3%, 50.8%). Cough was occurred more common in Group 2(50.8%) than Group 1(33.3%) with a significant difference (p=0.03). 6(6.7%) patients in Group1 and 10(15.9%) in Group2 had admitting diagnosis as non-pneumonia (p=0.07), some of them occurred mild pneumonia during hospital stay. White blood cell (2.2%, 9.5%) and neutrophil (9.5%) count above normal were more common on in Group 2 (p=0.04, p=0.04). Patients in Group 2 had higher concentration of aspartate aminotransferase (P=0.04) than Group 1. Most of patients had multiple lesions (75.6%, 69.8%) with bilateral distribution (73.3%, 58.7%) in both groups. Mixed ground-glass opacity (GGO) and consolidation appearance were seen in most patients. GGO components > consolidation appearance were more common in Group 1(31.1%) than in Group 2(8.0%) with a significant difference between groups (P<0.01). Patients had cough at onset disease (OR, 0.47; 95%CI, 0.23 to 0.96, p=0.04) and CT represented as GGO components more than consolidation (OR, 4.84; 95%CI, 1.80 to 13.04, p<0.01) were associated with hospitalization days. Conclusions COVID-19 non-pneumonia patients with longer hospitalization days might have the persistent symptoms or pneumonia occurrence after admission. Chest CT could help prompt diagnosis and monitor disease progression, GGO/consolidation >1 in mixed lesions was associated with shorter hospitalization days. Special attention should be paid to the role of radiological features in monitoring disease prognosis.

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.