# Investigations into linker effects of DNA–VHL ligand conjugates by multiplexed affinity measurements using focal molography

## TL;DR

This study uses a high-throughput method to measure how DNA-PROTACs bind to a protein called VHL, finding that linker hydrophobicity strongly affects binding affinity.

## Contribution

The study validates multiplexed focal molography for measuring DNA–PROTAC affinity and identifies linker hydrophobicity as a key factor in binding.

## Key findings

- Multiplexed focal molography achieves 20-fold higher throughput than singleplex measurements.
- Linker hydrophobicity is the primary driver of DNA–PROTAC affinity to VHL.
- Structure–activity correlations with lipophilicity are stronger in multiplexed measurements.

## Abstract

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 KD 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.

We measured the affinity of linker-diversified DNA–PROTACs to VHL by focal molography. Our analysis validates multiplexed affinity measurements and identifies hydrophobicity of the linker as the primary driver to linker-VHL ligand affinity for VHL.