One on-DNA ligand against many targets: selectivity profiling for DEL hit validation

The Challenge

Fibroblast activation protein (FAP) is a target for radioligands, CAR-T cells and small-molecule drug conjugates. Its catalytic pocket lies about 30 Å deep, so FAP ligands are sensitive to linkers and attachment points. When a hit from a DNA-encoded library (DEL) is validated on its DNA tag, two questions decide whether the numbers hold: does it bind the species variants used in animal studies, and does the tag interfere with binding?

The Approach

The clinical-stage FAP ligand oncoFAP was coupled to DNA through a C6 or a PEG4-C6 linker, each single- and double-stranded at the surface end. The constructs were immobilized on individual molecular holograms (molograms) by DNA-directed immobilization (DDI). Human, mouse and canine FAP and five control proteins were titrated over the same layout by single-cycle kinetics.

Key Results

• Species profile: the single-stranded constructs bind human, mouse and canine FAP with K_D values from 700 pM to 10 nM.
• Clean selectivity: no response above the 0.5 pg/mm² noise level on five control proteins, at concentrations up to 100 µM.
• A DNA-tag design rule: double-stranded constructs do not bind at all, while on human FAP linker length changes the rates only about two-fold.
• Self-referencing readout: the control traces stay flat without any reference subtraction.

Why It Matters

Focal molography readily supports ‘one-against-many’ profiling, providing rapid insights into target selectivity and cross-reactivity. The selectivity panel gives confidence that the signal is the target and nothing else. The duplex rule applies to any DEL follow-up on a deep binding pocket.

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