Structural remodeling of SARS-CoV-2 spike protein glycans reveals the regulatory roles in receptor binding affinity

Glycans of the SARS-CoV-2 spike protein are speculated to play functional roles in the infection processes as they extensively cover the protein surface and are highly conserved across the variants. To date, the spike protein has become the principal target for vaccine and therapeutic development while the exact effects of its glycosylation remain elusive. Experimental reports have described the heterogeneity of the spike protein glycosylation profile. Subsequent molecular simulation studies provided a knowledge basis of the glycan functions. However, there are no studies to date on the role of discrete glycoforms on the spike protein pathobiology. Building an understanding of its role in SARS-CoV-2 is important as we continue to develop effective medicines and vaccines to combat the disease. Herein, we used designed combinations of glycoengineering enzymes to simplify and control the glycosylation profile of the spike protein receptor-binding domain (RBD). Measurements of the receptor binding affinity revealed the regulatory effects of the RBD glycans. Remarkably, opposite effects were observed from differently remodeled glycans, which presents a potential strategy for modulating the spike protein behaviors through glycoengineering. Moreover, we found that the reported anti-SARS-CoV-(2) antibody, S309, neutralizes the impact of different RBD glycoforms on the receptor binding affinity. Overall, this work reports the regulatory roles that glycosylation plays in the interaction between the viral spike protein and host receptor, providing new insights into the nature of SARS-CoV-2. Beyond this study, enzymatic remodeling of glycosylation offers the opportunity to understand the fundamental role of specific glycoforms on glycoconjugates across molecular biology.Covert art LegendsThe glycosylation of the SARS-CoV-2 spike protein receptor-binding domain has regulatory effects on the receptor binding affinity. Sialylation or not determines the “stabilizing” or “destabilizing” effect of the glycans. (Protein structure model is adapted from Protein Data Bank: 6moj. The original model does not contain the glycan structure.)SignificanceGlycans extensively cover the surface of SARS-CoV-2 spike (S) protein but the relationships between the glycan structures and the protein pathological behaviors remain elusive. Herein, we simplified and harmonized the glycan structures in the S protein receptor-binding domain and reported their regulatory roles in human receptor interaction. Opposite regulatory effects were observed and were determined by discrete glycan structures, which can be neutralized by the reported S309 antibody binding to the S protein. This report provides new insight into the mechanism of SARS-CoV-2 S protein infection as well as S309 neutralization.

V367F mutation in SARS-CoV-2 spike RBD emerging during the early transmission phase enhances viral infectivity through increased human ACE2 receptor binding affinity

The current pandemic of COVID-19 is caused by a novel coronavirus SARS-CoV-2. The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring RBD mutations during the early transmission phase have altered the receptor binding affinity and infectivity, firstly we analyzed in silico the binding dynamics between SARS-CoV-2 RBD mutants and the human ACE2 receptor. Among 32,123 genomes of SARS-CoV-2 isolates (January through March, 2020), 302 non-synonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations (MDS). The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The MDS also indicated that it would be difficult for bat SARS-like CoV to infect humans. However, the pangolin CoV is potentially infectious to humans. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding ELISA, surface plasmon resonance, and pseudotyped virus assays. Phylogenetic analysis of the genomes of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F + D614G) which may derivate from recombination. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines. Importance A novel coronavirus SARS-CoV-2 has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether the mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and made them more infectious, has been the research hotspot. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase and increasing human ACE2 receptor binding affinity and infectivity. Our study provides insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin. The continuing surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is critical to the development of new COVID-19 drugs and vaccines against these variants during the sustained COVID-19 pandemic.

Emergence of SARS-CoV-2 spike RBD mutants that enhance viral infectivity through increased human ACE2 receptor binding affinity

ABSTRACTThe current global pandemic of COVID-19 is caused by a novel coronavirus SARS-CoV-2. The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring mutations in the RBD have altered the receptor binding affinity and infectivity, firstly we analyzed in silico the binding dynamics between mutated SARS-CoV-2 RBDs and the human ACE2 receptor. Among 1609 genomes of SARS-CoV-2 strains isolated during the early transmission phase, 32 non-synonymous RBD mutants were identified and found clustered into nine mutant types under high positive selection pressure. Applying molecular dynamics simulations, three mutant types (V367F, W436R, N354D/D364Y) displayed higher binding affinity to human ACE2, likely due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The increased infectivity of one mutant (V367F) circulating worldwide was further validated by performing receptor-ligand binding ELISA, surface plasmon resonance, and pseudotyped virus assays. Genome phylogenetic analysis of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F + D614G), which emerged later and formed a distinct sub-cluster. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of SARS-CoV-2 under high selection pressure and supports the continuing surveillance of spike mutations to aid in the development of COVID-19 drugs and vaccines.

Association of sleep among 30 antidepressants: a population-wide adverse drug reaction study, 2004–2019

Background Sleep is one of the most essential processes required to maintain a healthy human life, and patients experiencing psychiatric illness often experience an inability to sleep. The aim of this study is to test the hypothesis that antidepressant compounds with strong binding affinities for the serotonin 5-HT2C receptor, histamine H1 receptors, or norepinephrine transporter (NET) will be associated with the highest odds of somnolence. Methods Post-marketing cases of patient adverse drug reactions were obtained from the United States Food and Drug Administration Adverse Events Reporting System (FAERS) during the reporting window of January 2004 to September 2019. Disproportionality analyses of antidepressants reporting somnolence were calculated using the case/non-case method. The reporting odds-ratios (ROR) and corresponding 95% confidence interval (95% CI) were computed and all computations and graphing conducted in R. Results There were a total of 69,196 reported cases of somnolence out of a total of 7,366,864 cases reported from January 2004 to September 2019. Among the 30 antidepressants assessed, amoxapine (n = 16) reporting odds-ratio (ROR) = 7.1 (95% confidence interval [CI] [4.3–11.7]), atomoxetine (n = 1,079) ROR = 6.6 (95% CI [6.2–7.1]), a compound generally approved for attention deficit hyperactivity disorder (ADHD), and maprotiline (n = 18) ROR = 6.3 (95% CI, 3.9–10.1) were the top three compounds ranked with the highest reporting odds of somnolence. In contrast, vortioxetine (n = 52) ROR = 1.3 (95% CI [1.0–1.8]), milnacipran (n = 58) ROR = 2.1 (95% CI [1.7–2.8]), and bupropion (n = 1,048) ROR = 2.2 (95% CI [2.1–2.4]) are least significantly associated with somnolence. Moreover, levomilnacipran (n = 1) ROR = 0.4 (95% CI [0.1–2.9]) was not associated with somnolence. Conclusion Among the thirty tested antidepressants, consistent with the original hypothesis, amoxepine has strongest 5-HT2C receptor binding affinity and has the highest reporting odds of somnolence. Atomoxetine, ranked second in reporting odds of somnolence overall, binds to the NET with with the strongest binding affinity among the thirty compounds. Mirtazapine, a tetracyclic antidepressant, was ranked 11th in reporting odds of somnolence and had the strongest H1 receptor binding affinity. This study provides an informative ranking of somnolence among thirty antidepressant compounds with an already wide array of clinical indications as well as provides insight into potential drug repurposing in psychopharmacology.