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High-throughput atomistic glycan shield model of fully-glycosylated full-length SARS-CoV-2 spike protein in a viral membrane provides insights to design spike protein inactivators

A consortium of three Italian research teams, working at the Institute of Biomedical Technologies of the National Research Council (CNR-ITB), at the University of Brescia (UniBs), and at the University of Genoa (UniGe), has been awarded computational time at the EGI-ACE (through the EGI-ACE call for use cases) advanced computing services to support investigational studies on fully-glycosylated full-length SARS-CoV-2 spike protein. The insights gained from this computational study could provide key atomistic details to design spike protein inactivators for the development of new agents against SARS-CoV-2.

The SARS-CoV-2 Spike protein is a glycoprotein that plays a key role in receptor recognition, viral attachment and entry into host cells. It is composed of two subunits: S1, containing a receptor-binding domain (RBD) that recognizes and binds to the host receptor angiotensin-converting enzyme 2 and S2, responsible for viral fusion and entry. Due to its indispensable functions, spike protein is of great significance for subsequent development of drugs and protection devices against COVID-19. Dense surface glycosylation on the spike protein covers most of the spike surface and the identification of the hotspot regions of protein surface free from glycan is essential to design its inactivators. Several models and molecular dynamics studies have been performed to investigate the dynamic behaviour of spike protein and some of them also provide information on the structural and dynamic details of individual and collective glycans [1]. Our consortium has already performed molecular dynamics studies to evaluate S1 subunit variants located in the RBD and close to insertion 680 SPRRA R↓SV 687 that forms a cleavage motif RxxR for furin-like enzymes [2]. Comparative studies between the wild type protein and its variants highlighted some critical regions for the repositioning of drugs and for the development of polyanionics polymers with virucidal activity.

Aim of the Project

To develop compounds able to inactivate the spike protein we will evaluate the occupancy of the spike protein by glycans and we will identify glycan holes, thus providing opportunities for inactivators to bind [1,3]. In addition, a network analysis will be performed on the glycan shield of spike protein to find the glycans that are most important for an effective shield. Finally, the influence of glycans on the conformational states of the functional loops of the protein will be evaluated. The information collected in this study will be used for the repositioning of putative antivirals drugs and to design anionic polymers, both targeted to the spike glycan holes. The inhibitory activity of the identified compounds derived as spike-ligand complexes will be evaluated by molecular dynamics and experimentally validated by Surface Plasmon Resonance.

The consortium combines the expertise from different areas (Bioinformatics, Biochemistry and Medicinal Chemistry). It is composed by three Italian research teams: the first team is part of the bioinformatics laboratory at the Institute of Biomedical Technologies of the National Research Council (CNR-ITB) (https://www.itb.cnr.it/en/bioinformatics-milan/) whose members (Pasqualina D’Ursi, Andrea Manconi, Alessandro Orro) have matured consolidated skills in the field of molecular modelling and drug discovery as well as in the implementation and maintenance of specialized bioinformatics infrastructures in several National and European projects. The second team is part of the Macromolecular Interaction Analysis Unit (MIAU), Department of Molecular and Translational Medicine of the University of Brescia, and its members (Marco Rusnati and Maria Milanesi) are involved in the study of ligand/receptor and drug/target interactions of physiological, pathological and pharmacological relevance by means of surface plasmon resonance and microscale thermophoresis technologies. The third team is part of the Department of Pharmacy, University of Genoa (https://www.difar.unige.it/) and its members (Paola Fossa and Matteo Uggeri) have solid expertise in the field of drug discovery and drug design mainly concerning the application of different computational strategies for the rational design of ligands targeted to Cystic Fibrosis Transmembrane conductance Regulator (CFTR), Trace Amine Receptors (TAARs1), serotoninergic receptors (5HT1) and PDE4 inhibitors. This activity will involve the training in drug discovery studies and in advanced biosimulations of young researchers. Results from this use case will be shared with the scientific community through public repositories.

Computing resources

This research activity is supported by the EGI-ACE (Advanced Computing for EOSC) project.  EGI-ACE is a project coordinated by the EGI Foundation aimed at empowering researchers from all disciplines to collaborate in data- and compute-intensive research through free-at-point-of-use services. Through this project, EGI offers compute and storage resources, compute platform services, data management services and related user support and training through this call.

References

Articles

  1. Strizzi S, Bernardo L, D’Ursi P, Urbinati C, Bianco A, Limanaqi F, Manconi A, Milanesi M, Macchi A, Di Silvestre D, Cavalleri A, Pareschi G, Rusnati M, Clerici M, Mauri P, Biasin M. An innovative strategy to investigate microbial protein modifications in a reliable fast and sensitive way: A therapy oriented proof of concept based on UV-C irradiation of SARS-CoV-2 spike protein. Pharmacol Res. 2023 Aug;194:106862. doi: 10.1016/j.phrs.2023.106862. Epub 2023 Jul 20. PMID: 37479104.
  2. Milanesi M, Uggeri M, Biasin M, Strizzi S, Bernardo L, DePalma A, Mauri P, Manconi A, Orro A, Urbinati C, Rusnati M, Fossa P and DUrsi P. An antiviral strategy against SARS-CoV2 spike protein based on reducing agents. Manuscript in preparations

Contribution to oral communication

  1. Inactivation mechanism of SARS-CoV2 by UV-C light exposure. Milanesi M, Biasin M, Bernardo L, DePalma A, Mauri P, Urbinati C, Rusnati M and DUrsi P. Bioexcel summer school on biomolecular simulations 2022. June 12-17, 2022, Science and Technology Park of Sardinia, Cagliari, Italy
  2. Effetto dell’irraggiamento UV-C sulla proteina Spike, D’Ursi P.  and  Bernardo L., UV-day, June 21, 2022, Palazzo Brera, Milano, Italy
  3. Radiation exploitment for molecular identikit of pathogens in spaceflight explorations (RAMSES): impiego delle radiazioni come metodo di screening di patogeni in ambiente spaziale. Strizzi S, Bernardo L, D’Ursi P, Urbinati C, Bianco A, Limanaqi F, Manconi A, Milanesi M, Macchi M, Di Silvestre D, Cavalleri A, Pareschi G, Rusnati M, Clerici M, Mauri PL, Biasin M. Simposio di “Biomedicina Spaziale per le Future Missioni di Esplorazione Umana dello Spazio: a Call to Action”, March 15-17, Rome

Posters

  1. An antiviral strategy against SARS-CoV2 spike protein based on reducing agents. Uggeri M, Milanesi M, Biasin M, Strizzi S, Bernardo L, De Palma A, Mauri P, Manconi A, Orro A, Urbinati C, Rusnati M, DUrsi P and Fossa P. XXVII National Meeting in Medicinal Chemistry. September 11-14, 202,2 Bari, Italy
  2. UV-C exposure suggests a SARS-CoV2 spike protein antiviral strategy based on reducing agents. Milanesi M, Biasin M, Strizzi S, Bernardo L, DePalma A, Mauri P, Manconi A, Uggeri M, Fossa P, Urbinati C Rusnati M and DUrsi P. 18th Annual Meeting of the Bioinformatics Italian Society (BITS 2022). June 27-29, 2022, Verona, Italy