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2026 Favalli et al. - High-Throughput DEL Hit Validation by Focal Molography

Focal molography enables high-throughput, label-free kinetic validation of DNA-encoded library (DEL) hits, measuring 24 compounds in parallel with subnanomolar accuracy.

The Challenge

DNA-encoded libraries (DELs) let medicinal chemists screen millions to billions of compounds against a target in a single experiment. The bottleneck has shifted downstream: established hit-validation methods such as surface plasmon resonance (SPR), fluorescence polarization (FP), and isothermal titration calorimetry (ITC) can only characterize a small fraction of the enriched compounds, so many potentially relevant binders remain uncharacterized.

The Approach

The authors applied focal molography on the MACS® Matchmaker to measure binding kinetics for 24 DEL-derived hits against carbonic anhydrase IX (CAIX) and carbonic anhydrase II (CAII). Each hit was conjugated to a unique 20-mer DNA code and immobilized in parallel via DNA-directed immobilization (DDI) onto spatially encoded molograms, enabling 18-plex single-cycle kinetic measurement on a single regenerable chip.

Key Results

  • Multiplexed throughput: 24 DEL hits plus an SABA reference were characterized in a single run, with the chip format supporting up to 500 kinetic measurements per day.
  • Subnanomolar accuracy: Four top hits (compounds 4, 8, 12, 16, all 2R,4R stereoisomers) reached KD values of 0.3 to 0.8 nM against CAIX.
  • Selectivity profiling: Focal molography resolved roughly 20,000-fold selectivity for CAIX over CAII for the strongest binders.
  • Cross-platform consistency: Kinetic rank order matched SPR and FP across three reference sulfonamides (AAZ, SABA, mSABA).
  • Crude compatibility: Ligation crudes were measured directly without HPLC purification, removing a time-consuming workflow step.

Why It Matters

Hit validation is the central bottleneck in DEL campaigns: screening can produce thousands of enriched compounds, yet downstream characterization is slow and material-intensive. Focal molography closes this gap by combining label-free quantitative kinetics with multiplexed throughput, allowing medicinal chemists to triage and rank DEL hits at the scale modern library outputs demand.

Favalli, N.; Velti, C.; Campari, L.; Heuberger, L.; Fabbri, M.; Müller, M.; Puglioli, S.; Cazzamalli, S.; Neri, D.; Frutiger, A.; Oehler, S. High-Throughput Binding Kinetic Measurements of DNA-Encoded Library-Derived Hits by Focal Molography. Journal of Medicinal Chemistry 2026. https://doi.org/10.1021/acs.jmedchem.6c01097

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