Investigations into Linker Effects of DNA-VHL Ligand Conjugates by Multiplexed Affinity Measurements Using Focal Molography

The determination of kinetic binding properties of DNA-tagged compounds binding to a target protein is an important step in the development of nucleic acid-based proteolysis targeting chimeras (PROTACs). Focal molography (FM) is a recent addition to the toolbox of instruments that allow for measuring kinetic binding data of drugs binding to a target protein with high experimental throughput. Here, we applied focal molography to characterize the binding of DNA-VHL ligand conjugates-these are "DNA-PROTAC" compounds-to VHL. We synthesized two libraries of 20 such compounds with diverse amino acid linkers by solid-phase amide coupling and used FM to measure their affinity to VHL in both singleplex and 20-plex multiplexed measurement formats. Systematic comparison of equilibrium and kinetic fitting approaches reveals strong within-format correlations that preserve compound rankings despite systematic offsets in absolute K D values. The multiplexed format achieves 20fold higher throughput while yielding stronger structureactivity correlations with lipophilicity compared to singleplex measurements. Our analysis identifies hydrophobicity as the primary driver of the contribution of the linker part to overall linker-VHL ligand affinity for VHL in the case of DNA-PROTACs. These findings validate multiplexed FM as a robust platform for PROTAC structureactivity relationship studies and provide design principles for optimizing linker-E3 ligase interactions.