(296d) Study of the Spike (S) Glycoprotein from the Sars-Cov-2 As a Possible Source of Translocating Peptides of Biomedical Interest

Membranes are natural selective barriers necessary for the correct function of human cells as they control the entry of diverse molecules into the intracellular space. Although small and polar molecules such as amino acids and ions can come across the membrane through channels, larger macromolecules such as proteins and nucleic acids generally fail to do so. This represents a significant obstacle for efficient intracellular drug delivery, which is essential to increase the bioavailability of pharmacological agents for the treatment of numerous diseases^1,2. Commonly, diverse methods have been studied to mediate the uptake of large molecules, including mechanical and electrical transfection techniques, but also viral carrier systems².

Some of these methods have been successfully tested in vitro but exhibit biosafety and cytotoxicity issues³. In consequence, there is an increasing interest in the development of novel delivery methods with low cytotoxicity but also high transduction efficiency¹. An attractive alternative is the cell-penetrating peptides (CPPs), which can be obtained from different origins including signal peptides, viral proteins, or antimicrobial peptides⁴. CPPs are generally short peptides with lengths between 5-30 amino acids, positively charged or amphipathic, and rich in arginine and lysine³. Due to their ability to intermingle with the phospholipids of membrane bilayers, some CPPs are able to undergo translocation, and consequently can be considered as promising candidates for the delivery of biologically active molecules to cells.

The novel coronavirus SARS-CoV-2 has attracted significant attention over the past few months as it is responsible for the current global sanitary emergency where more than 1.3 M cases have been confirmed and over 70,000 people have died⁵. The Spike (S) glycoprotein has been thought to be responsible as the major determinant of the viral tropism towards human cells⁶. This protein has a 180 kDa molecular weight and is displayed at the viral surface as a trimer composed of two major domains⁷. The first one is the S1, which contains the receptor-binding domain (RBD) responsible for mediating the receptor binding (Angiotensin-converting enzyme 2). The second one is the S2, which allows the membrane fusion through the exposure of a fusion protein that is activated by proteolytic cleavage in a site upstream (S2’) and proteolytically primed at the interface of the S1 and S2 domains. Transmission of the genetic material into the host cells has been attributed to proteases in priming, receptor binding, and some ionic interactions controlling the stability of the virus⁷.

By recognizing the strong interaction between de spike (S) glycoprotein of SARS-CoV-2 and the angiotensin-converting enzyme 2 from the cellular membrane of the lung cells, here we aimed at finding motifs that could serve as possible sources of peptides capable of intermingling with membranes, and eventually with superior translocating potency. For this purpose, a prediction of the tertiary structure of the S protein from the SARS-CoV-2 was performed by homology, using Phyre Server, with the S protein from the bat coronavirus RaTG13. This was selected due to its closeness to the SARS-CoV-2 virus as it shares more than 93% of the identity in the S gene⁸. Simultaneously, the structure was also predicted de novo using the iTASSER Server to assure that predictions were robust enough for biophysical interaction studies. To determine the motifs with significant membrane activity (and potential translocation ability), a prediction of the possible formed transmembrane helices was carried out using TMHMM Server v. 2.0. Finally, the selected sequences were studied into detail via molecular dynamics (MD), using a mode membrane.

References:

4. Derakhshankhah, and S. Jafari “Cell penetrating peptides: A concise review with emphasis on biomedical applications” Biomedicine & Pharmacotherapy, 108, 1090–1096. (2018)
5. W. Huang, and H.-J. Lee “Cell-penetrating peptides for medical theranostics and targeted drug delivery” Peptide Applications in Biomedicine, Biotechnology and Bioengineering359–370. (2018)
6. W. Huang, H.-J. Lee, L. M. Tolliver and R. S. Aronstam “Delivery of Nucleic Acids and Nanomaterials by Cell-Penetrating Peptides: Opportunities and Challenges” BioMed Research International, 1–16. (2015)
7. A. El-Baky, V. N. Uversky, and E. M. Redwan “Virucidal activity of cell-penetrating peptides of viral origin”Journal of Biomolecular Structure and Dynamics 36, 1739–1746. (2017)
8. H University and Medicine “Coronavirus Resource Center” (2020)
9. J.G. Hulswit, C.A.M. de Haan1 and B.-J. Bosch “Coronavirus Spike Protein and Tropism Changes” Coronaviruses Advances in Virus Research, 29–57. (2016)
10. A. Jaimes, N. M. André, J. K. Millet, and G. R. Whittaker “Structural modeling of 2019-novel coronavirus (nCoV) spike protein reveals a proteolytically-sensitive activation loop as a distinguishing feature compared to SARS-CoV and related SARS-like coronaviruses” (2020)
11. Lan, J. Ge, J. Yu, S. Shan, H. Zhou, S. Fan, Q. Zhang, X. Shi, Q. Wang, L. Zhang and X. Wang “Structure of the SARS-CoV-2 spike receptor – binding domain bound to the ACE2 receptor” Nature. (2020)