Artificial proteins as specific and versatile neutralizing binders targeting the spike of SARS-CoV-2

In a recent article published on bioRxiv*preprint server, researchers demonstrated that biosynthetic proteins called αReps dealing with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) could be novel SARS-CoV-2 antivirals

Study: Biosynthetic proteins targeting the SARS-CoV-2 spike as antivirals. Image Credit: Naeblys/Shutterstock

Background

The 2019 CoV disease (COVID-19) disaster, which has resulted in an estimated six million deaths worldwide in about two years, has highlighted the need to better understand and address virus transmission and emergence respiratory. This information will help in the development of more effective antiviral techniques to deal with future pandemics and epidemics.

SARS-CoV-2 S binds to angiotensin-converting enzyme 2 (ACE2) receptors in hosts, allowing the virus to enter the cell. Therefore, one possible technique for developing COVID-19 antivirals is to target this interaction.

About the study

In the present work, the researchers aimed to identify ligands that block the SARS-CoV-2-ACE2 interaction. They wanted to develop stable, inexpensive COVID-19 antivirals that could be easily modified against emerging SARS-CoV-2 variants.

The team identified candidates recognizing the SARS-CoV-2 S receptor binding domain (RBD). For this, they screened a phage-display collection of biosynthetic protein sequences built on rigid α-helical huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and yeast target kinase of the rapamycin 1 (TOR1) (HEAT)-like scaffold called αReps.

Competitive binding assays were conducted among αReps to analyze their mechanism of neutralization of SARS-CoV-2. Additionally, the researchers showed how bioengineering αRep could stimulate the neutralizing action of SARS-CoV-2 using a multivalent form. In addition, they assessed the SARS-CoV-2 neutralization capacity of these αReps in vitro and live.

Results

The results of the study indicated that among the artificial proteins analyzed, two, namely C2 and F9, bind to SARS-CoV-2 RBD with nanoscale affinities, exhibiting neutralizing action. in vitro and identify different sites, with F9 spanning the ACE2 binding motif. The authors found that C2 and F9 significantly inhibited SARS-CoV-2 entry into cultured cells. These two compounds neutralized the virus through different pathways, with C2 binding at a location distant from the receptor binding motif of ACE2 while F9 competes with ACE2 for RBD binding.

For SARS-CoV-2 neutralization, a trivalent αRep form called C2-foldon and the F9-C2 fusion protein had affinities of 0.1 nM and a half-maximal effective concentration (EC50) of 8–18 nM. Both the C2-foldon homotrimer and the F9-C2 heterodimer showed more robust SARS-CoV-2 neutralizing capacity than the two parental αReps, with a half-maximal inhibitory concentration (IC50) ranging from 3 to 12 nM. Additionally, virus entry was prevented at lower concentrations by αReps assembled via non-covalent or covalent connections, with a 20-fold increase in activity for a trimeric αRep.

These αReps derivatives effectively neutralized SARS-CoV-2 Omicron, δ, γ and β variants. Notably, with EC50 values ​​ranging from 13 to 32 nM, F9-C2 or C2-foldon successfully neutralized SARS-CoV-2 mutants, such as the Omicron and Delta variants.

Introduction of F9-C2 into the nasal cavity during or before SARS-CoV-2 infections significantly inhibited the multiplication of the virus strain with the D614G mutation inside the nasal epithelium in hamsters . Viral titers in nasal swabs and the nasal cavity, the primary site of SARS-CoV-2 replication, were reduced by this therapy, as were all inflammatory indicators of infection. However, the treatment did not completely block SARS-CoV-2 infection in the nasal cavity.

Overall, the scientists mentioned that αReps represent a viable approach for COVID-19 therapies to target the nasal cavity and reduce viral spread in the proximal setting due to their substantial stability and efficacy against variants of the SARS-CoV-2.

conclusion

In summary, the study results demonstrated that two biosynthetic protein sequences, namely C2 and F9, had high affinity for SARS-CoV-2 RBD and effectively prevented SARS-CoV-2 from entering cells. cells in culture (in vitro). Neutralizing EC50 values ​​were reduced to the 10 nM range by αReps assembled via non-covalent and covalent connections. Additionally, in the hamster model of SARS-CoV-2, instillation of an αRep dimer into the nasal cavity significantly reduced viral pathogenicity and replication. A C2 homotrimer and the F9-C2 fusion protein potently inhibited SARS-CoV-2 mutants, even the antigenically foreign Omicron variant.

Overall, the present study showed that the engineered proteins, αReps, could be developed into SARS-CoV-2 treatments targeting novel viral variants. Stable protein inhibitors, such as αReps and their derivatives, could be a promising option to threaten future pandemics related to various emerging respiratory viruses following initiatives to stabilize them in the nasal cavity and technical improvement in the selection of binders.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

Journal reference:

  • Biosynthetic proteins targeting the SARS-CoV-2 spike as antivirals; Stéphanie Thébault, Nathalie Lejal, Alexis Dogliani, Amélie Donchet, Agathe Urvoas, Marie Valerio-Lepiniec, Muriel Lavie, Cécile Baronti, Franck Touret, Bruno da Costa, Clara Bourgon, Audrey Fraysse, Audrey Saint-Albin-Deliot, Jessica Morel, Bernard Klonjkowski, Xavier de Lamballerie, Jean Dubuisson, Alain Roussel, Philippe Minard, Sophie Le Poder, Nicolas Meunier, Bernard Delmas. bioRxiv. do I: https://doi.org/10.1101/2022.05.10.491295 https://www.biorxiv.org/content/10.1101/2022.05.10.491295v1

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