Parallel kinetic validation of DNA-encoded library hits

The Challenge

DEL selections return hundreds of enriched compounds, yet each hit still has to be made again and characterized one by one. Fluorescence polarization, surface plasmon resonance (SPR) and isothermal titration calorimetry validate dozens of compounds per campaign, with high protein use and often without kinetics. Hit validation, not screening, is the bottleneck of DEL-based ligand discovery.

The Approach

Focal molography turns the DNA tag of each hit into its immobilization handle. Every hit is ligated to a chip-specific code and hybridizes to its own molecular hologram (mologram) by DNA-directed immobilization (DDI). A whole pool is loaded in one injection and titrated in one single-cycle kinetics run. Twenty-four on-DNA hits, stereoisomers included, were measured against carbonic anhydrase IX (CAIX) and the antitarget carbonic anhydrase II (CAII), in two runs of about one hour per protein.

Key Results

• Sub-nanomolar binders: the 2R,4R compounds of HIT1 to HIT4 bind CAIX with K_D values of 0.3 to 0.8 nM.
• Selectivity: the same compounds are approximately 10,000- to 20,000-fold selective over CAII.
• Stereochemistry from the same runs: the other stereoisomers and the acetylated controls bind two to four decades more weakly, or not at all.
• Enrichment is not affinity: HIT5, despite its higher enrichment, binds CAIX at 230 ± 70 nM with a fast off-rate.
• Orthogonal agreement: fluorescence polarization reproduced the rank order and the selectivity trend.

Why It Matters

Because the DNA tag of a DEL member is all the MACS Matchmaker needs, the workflow scales to the full output of a selection, with kinetics for every hit and its stereoisomers on one chip.

Data from Favalli N et al., Journal of Medicinal Chemistry (2026), in collaboration with Philochem AG. https://doi.org/10.1021/acs.jmedchem.6c01097