Characterization of antiviral compounds using Bio-Layer Interferometry.

Area of Science:

• Biochemistry and Molecular Biology
• Drug Discovery and Development
• Biophysical Chemistry

Background:

• Small molecule-protein interactions are crucial for biological functions and disease treatment.
• Biolayer interferometry (BLI) offers a sensitive, high-throughput approach for studying these interactions, particularly in antiviral discovery.
• Challenges like nonspecific binding and ligand drift can hinder BLI accuracy.

Purpose of the Study:

• To present optimized BLI protocols for characterizing small molecule-protein interactions.
• To address and provide solutions for common BLI pitfalls.
• To demonstrate BLI's utility in calculating binding affinities for clinically relevant antiviral compounds.

Main Methods:

• Development and application of optimized BLI protocols.
• Characterization of interactions between PF74 and HIV-1 capsid protein (CA).
• Analysis of Lenacapavir (LEN) with HIV-1 CA and Nirmatrelvir (NIR) with SARS-CoV-2 Mpro.

Main Results:

• Demonstrated BLI as a powerful tool for calculating binding affinities (K_D values) from micro to sub-nanomolar ranges.
• Quantified binding affinities for HIV-1 CA interactions with PF74 and LEN.
• Confirmed the covalent interaction between Nirmatrelvir and SARS-CoV-2 Mpro.

Conclusions:

• Optimized BLI protocols facilitate accurate characterization of small molecule-protein binding.
• BLI is effective for determining binding affinities and interaction types for antiviral drug candidates.
• These protocols support high-throughput screening and testing of novel antivirals and derivatives.