Biosynthetic proteins targeting the SARS-CoV-2 spike as antivirals

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 -Rep 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 and pathogenicity. 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.

Identification of potent inhibitors of SARS-CoV-2 exoribonuclease by fluorescence polarization assay

The emergence of SARS-CoV-2 has triggered a pandemic with devastating consequences to the world. One of the proteins essential to the virus life cycle is nsp14, which is a bifunctional protein that encodes a 3'to 5' exoribonuclease activity in its N-terminus, and a methyl transferase activity in its C-terminus. Nsp14 in complex with the accessory protein nsp10 is involved in a proofreading mechanism that ensures the genetic stability of its massive viral genome, and is associated to the resistance against nucleotide analogs targeting the polymerase nsp12. Because of its key role, nsp14-nsp10 complex constitutes an attractive target for antiviral development. Here we present a fluorescence polarization (FP) assay development to measure the exoribonuclease activity and its inhibition in vitro. The FP method is sensitive, robust, amenable to miniaturization and offers confirmation by visualizing the degradation of the fluorescent RNA in acrylamide gels. We performed a screening of a focused library of 113 metal chelators at 20 and 5 μM compound concentration and IC50 measurement of 9 hits showing efficiency at micromolar level. We also tested the focused library in SARS-CoV-2 infected Vero cells and we confirmed 3 hits previously detected in the in vitro screening out of 6 promising inhibitors. In conclusion the FP method proposed is a reliable tool to discover inhibitors of the SARS-CoV-2 exoribonuclease activity and will help to find new antivirals to be used in combination with nucleoside analogs.

Evaluation of formulations to improve SARS-CoV-2 viability and thermostability after lyophilisation.

In the context of the COVID-19 pandemic, virus collections such as EVA-GLOBAL play a key role in the supply of viruses and related products for research. Freeze-drying techniques for viruses represent a method of choice for the preservation of strains and their distribution without the need for a demanding cold chain. Here, we describe an optimised lyophilisation protocol usable for SARS-CoV-2 strains that improves preservation and thermostability. We show that sucrose used as an adjuvant represents a simple and efficient stabilizer providing increased protection for long-term preservation and shipment of the virus under different climatic conditions.

Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis

The ongoing Corona Virus Disease 2019 (Covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and higherror rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of Covid-19.