Applications of the Microscale Thermophoresis Binding Assay in COVID-19 Research

Abstract

As the COVID-19 pandemic progresses, new variants of SARS-CoV-2 continue to emerge. This underscores the need to develop optimized tools to study such variants, along with new coronaviruses that may arise in the future. Such tools will also be instrumental in the development of new antiviral drugs. Here, we introduce microscale thermophoresis (MST) as a reliable and versatile tool for coronavirus research, which we demonstrate through three different applications described in this report: (1) binding of the SARS-CoV-2 spike receptor binding domain (RBD) to peptides as a strategy to prevent virus entry, (2) binding of the RBD to the viral receptor ACE2, and (3) binding of the RBD to ACE2 in complex with the amino acid transporter SLC6A20/SIT1 or its allelic variant rs61731475 (p.Ile529Val). Our results demonstrate that MST is a highly precise approach to studying protein–protein and/or protein–ligand interactions in coronavirus research, making it an ideal tool for studying viral variants and developing antiviral agents. Moreover, as shown in our results, a unique advantage of the MST assay over other available binding assays is the ability to measure interactions with membrane proteins in their near-native plasma membrane environment.

Functional Characterization of the Lysosomal Peptide/Histidine Transporter PHT1 (SLC15A4) by Solid Supported Membrane Electrophysiology (SSME)

“…MST measurements were performed with the Monolith NT.115 instrument (NanoTemper Technologies GmbH). Membrane preparation of HEK293 cells overexpressing PHT1-GFP were obtained as described elsewhere [28]. Equal amounts of the membrane preparation were loaded into Monolith capillaries (NanoTemper; MO-K022) together with the indicated concentrations of Lhistidine; both dissolved in 10mM citric acid buffer at pH 5.0.…”

Novel Antiviral Agents: Synthesis, Molecular Modelling Studies and Biological Investigation

“…Furthermore, the MST approach can be used to examine whether or not membrane proteins are expressed in intracellular membranes. Notably, the strategy outlined in this study could be applied to different coronaviruses, including novel viral variants [ 6 ].…”