A Cell Based Assay Using Virus-like Particles to Screen AM Type Mimics for SARS-CoV-2 Neutralisation

A number of small molecule and protein therapeutic candidates have been developed in the last four years against SARS-CoV-2 spike. However, there are hardly a few molecules that have advanced through the subsequent discovery steps to eventually work as a therapeutic agent. This is majorly because of the hurdles in determining the affinity of potential therapeutics with live SARS-CoV-2 virus. Furthermore, affinity determined for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, at times fails to mimic physiological conditions of the host-virus interaction. To bridge this gap between in vitro and in vivo methods of therapeutic agent screening we report an improved screening protocol for therapeutic candidates using SARS-CoV-2 virus like particles (VLPs). To minimise the interference in affinity studies by bulkier reporters like GFP, a smaller hemagglutinin (HA) tag has been fused to one of the proteins of VLP. This HA tag serves as readout, when probed with fluorescent anti-HA antibodies. Outcome of this study sheds light on the lesser known virus neutralisation capabilities of AM type miniprotein mimics. Further, to assess the stability of SARS-CoV-2 spike - miniprotein complex, we have performed molecular dynamic simulations on the membrane embedded protein complex. Simulation results reveal extremely stable intermolecular interactions between RBD and one of the AM type miniproteins, AM1. Furthermore, we discovered a robust network of intramolecular interactions that helps stabilise AM1. Findings from our in vitro and in silico experiments concurrently highlight advantages and capabilities of mimic based miniprotein therapeutics.