Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals

Abstract

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8–18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

Inhibition of influenza virus replication by artificial proteins (αReps) targeting its RNA-polymerase

“…A main advantage of αReps is their solubility and stability even in reducing conditions such as in the intracellular environment where they could block different steps of the virus cycle. They can either target viral glycoproteins, such as the spike of SARS-CoV-2 to block infection (Thébault et al, 2022) or bind to intracellular viral proteins like the HIV-1 nucleocapsid and to negatively interfere with virus maturation (Hadpech et al, 2017).…”

“…A large αRep library has been assembled and was demonstrated on a wide range of unrelated protein targets to be a generic source of tight and specific binders (Valerio-Lepiniec et al, 2015). Thus, αReps were previously selected as interactors of the receptor binding domain of the spike of SARS-CoV-2 and shown to block infection of cultured cells and in hamsters (Thébault et al, 2022), or of HIV-1 nucleocapsid and to negatively interfere with virus maturation (Hadpech et al, 2017).…”

Customizably designed multibodies neutralize SARS-CoV-2 in a variant-insensitive manner

“…Given the critical relevance of the S-ACE2 specific recognition for SARS-CoV-2 entry into the host cell, several inhibitors for this protein-protein interaction (hereafter called binders) have been developed as candidates for therapeutic intervention or as the base of detection devices ( 10 – 12 ). However, the continuous emergence of SARS-CoV-2 variants of concern (VOCs), characterized by mutations located in crucial positions of S, almost invariably reduced the neutralizing capacity of natural antibodies ( 3 ), nanobodies ( 13 ), and designed proteins ( 14 – 16 ). Therefore, there is a continuous demand for developing adaptable molecular scaffolds capable of efficiently blocking the entry of SARS-CoV-2 variants.…”