Characterization of antiviral compounds using Bio-Layer Interferometry

Small molecule-protein interactions underpin many biological functions and play an integral role in the treatment and prevention of several human diseases. These interactions can be key to understanding the mechanism of action of these compounds. Previous methods of determining protein-protein or protein-antibody interactions have been well established; however, the use of BLI in antiviral discovery is a promising and relatively new avenue. The high-throughput nature of this method in tandem with its pM sensitivity allows for quick and seamless identification of hit compounds. Here we discuss ways to overcome common pitfalls that can occur while using BLI such as nonspecific binding (NSB) and ligand drift while offering possible solutions. Characterizing small molecule-protein interactions is not trivial and optimizing the experimental conditions is imperative. To address this gap in knowledge, we present optimized BLI protocols for the study of three cases of protein-small molecule interactions: PF74 or Lenacapavir (LEN) with HIV-1 capsid protein (CA), and Nirmatrelvir (NIR) with SARS-CoV-2 Mpro. LEN and NIR are of particular interest because they are clinically relevant, and PF74, a well-studied control, was the first compound reported to target the LEN binding site. We demonstrate that BLI can be a powerful and effective tool in calculating the binding affinities between a protein and small molecule. These newly designed methods enabled calculation of K _D values, the affinity between ligand and analyte, ranging from the micro to the sub-nanomolar range for CA binding events and confirmed the covalent interaction between NIR and Mpro. These protocols will facilitate efficient testing of new antivirals or derivatives in a high- throughput format.