Multiplexed focal molography identifies hydrophobicity as the primary driver of linker-mediated binding affinity in DNA-encoded PROTAC building blocks targeting the E3 ligase VHL.
The Challenge
Proteolysis-targeting chimeras (PROTACs) are a class of therapeutic molecules that work by hijacking the cell's own protein disposal system. A PROTAC consists of two binding modules connected by a molecular linker: one end binds a disease-relevant target protein, the other recruits an E3 ubiquitin ligase, an enzyme that tags proteins for destruction. Von Hippel-Lindau (VHL) is the most widely used E3 ligase in PROTAC design. Understanding how the linker connecting these two modules affects binding to VHL is critical for designing effective degraders, but conventional biosensing methods measure compounds one at a time, making systematic optimization slow.
The Approach
Using the MACS Matchmaker instrument and focal molography, the authors synthesized 20 DNA-tagged VHL ligand conjugates, each bearing a different amino acid linker that mimics the connector used in PROTAC molecules. Each compound was immobilized on a single sensor chip via DNA-directed immobilization (DDI), enabling simultaneous real-time measurement of how strongly all 20 linker variants bind to VHL protein in a single experiment. Both singleplex and 20-plex multiplexed formats were compared.
Key Results
- Affinity range: Dissociation constants (KD) spanned 20 to 550 nM across 20 compounds, confirming that all tested linker structures are tolerated by VHL
- Compound ranking preserved: The three strongest binders (compounds 3, 15, and 19) were consistently identified across all four measurement methods (r = 0.88 singleplex, r = 0.98 multiplexed)
- 20-fold throughput gain: The multiplexed format measured all 20 compounds simultaneously on one chip, eliminating run-to-run variability inherent to sequential singleplex processing
- Lipophilicity drives affinity: Higher linker hydrophobicity correlated with stronger VHL binding across all methods (r = −0.83 in multiplexed equilibrium), identifying c log P as the dominant structure-activity predictor
- Optimal design window: Compounds with c log P values between 0 and +2 offer the best balance between binding affinity and drug-like physicochemical properties
Why It Matters
These findings establish multiplexed focal molography as a validated platform for PROTAC linker structure-activity relationship studies. The ability to screen 20 linker variants simultaneously reduces the experimental cycle from sequential week-long campaigns to a single measurement, directly accelerating medicinal chemistry optimization in targeted protein degradation programs.
Raschke, P., Notova, S., Gatterdam, V., Frutiger, A., & Brunschweiger, A. (2026). Investigations into linker effects of DNA-VHL ligand conjugates by multiplexed affinity measurements using focal molography. RSC Chemical Biology, Advance Article. https://doi.org/10.1039/D6CB00011H
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