Investigations into linker effects of DNA-VHL ligand conjugates by multiplexed affinity measurements using focal...

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 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 20-fold higher throughput while yielding stronger structure-activity 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 structure-activity relationship studies and provide design principles for optimizing linker-E3 ligase interactions.