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

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

Background

The CoV disease 2019 (COVID-19) catastrophe, which resulted in approximately six million fatalities globally in around two years, has highlighted the need for better comprehension and combating the transmission and emergence of respiratory viruses. This information will aid in the development of more effective antiviral techniques to address 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. Hence, a 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 low-cost, stable COVID-19 antivirals that could be easily modified against the 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 constructed on rigid α-helicoidal huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast kinase target of rapamycin 1 (TOR1) (HEAT)-like scaffold termed αReps.

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

Results

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

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

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

F9-C2 introduction in the nasal cavity during or before SARS-CoV-2 infections significantly inhibited the multiplication of the viral strain with the D614G mutation inside the nasal epithelium in hamsters. The viral titers in nasal swabs and the nasal cavity, the primary SARS-CoV-2 replication site, were reduced by this therapy, as were all of the infection's inflammatory indicators. 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 the viral spread in the proximal setting because of their substantial stability and efficacy against SARS-CoV-2 variants.

Conclusions

To summarize, the study findings demonstrated that two biosynthetic protein sequences, namely C2 and F9, had a strong affinity for the SARS-CoV-2 RBD and effectively prevented SARS-CoV-2 entrance in cultured cells (in vitro). The neutralizing EC50 values were reduced to the 10 nM range by assembled αReps through non-covalent and covalent connections. Moreover, in the hamster model of SARS-CoV-2, instilling an αRep dimer into the nasal cavity substantially decreased 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. 

Altogether, the present study depicted that the artificial proteins, αReps, could be developed into SARS-CoV-2 treatments targeting novel viral variants. Stable proteinaceous inhibitors, such as αReps and their derivates, could be a promising option to threaten future pandemics connected with diverse emerging respiratory viruses following initiatives to stabilize them in the nasal cavity and technical improvement in binder selection.

*Important notice

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

Written by

Shanet Susan Alex

Shanet Susan Alex, a medical writer, based in Kerala, India, is a Doctor of Pharmacy graduate from Kerala University of Health Sciences. Her academic background is in clinical pharmacy and research, and she is passionate about medical writing. Shanet has published papers in the International Journal of Medical Science and Current Research (IJMSCR), the International Journal of Pharmacy (IJP), and the International Journal of Medical Science and Applied Research (IJMSAR). Apart from work, she enjoys listening to music and watching movies.