High-throughput thermodynamic fingerprinting of protein–ligand interactions by DNA-directed focal molography

The Challenge

Knowing whether a ligand binds through enthalpy or entropy guides compound design beyond what affinity alone reveals. Getting these values for many related ligands is slow. Surface plasmon resonance (SPR) reacts to every temperature-induced change in refractive index and needs 30 to 60 minutes of equilibration per temperature step. Isothermal titration calorimetry (ITC) needs large amounts of protein and titrates one ligand at a time.

The Approach

The authors measured how the regulatory subunit of protein kinase A (PKA-R) binds five cyclic AMP (cAMP) derivatives on the MACS® Matchmaker. They first tested the diffractometric readout under a 25 to 45 to 25 °C temperature ramp and in 50% human serum. They then presented all five derivatives in parallel on one chip by DNA-directed immobilization (DDI) and ran single-cycle kinetics at five temperatures from 15 to 35 °C, followed by van 't Hoff and Eyring analysis.

Key Results

• Temperature stability: The diffractometric signal changed by less than 1 pg/mm² over the full temperature ramp, while the referenced refractometric signal still drifted by up to 1,200 pg/mm².
• Serum tolerance: In 50% human serum, association rates were unchanged and K_D values were at most about 20% higher than in buffer (6-AH-cAMP: 16.5 nM in buffer, 19.7 nM in serum).
• Multiplexed temperature series: Five derivatives at five temperatures were measured on the same chip format, with 64 individual kinetic fits per compound.
• Distinct fingerprints: Four derivatives bound with almost the same free energy (ΔG of -10.5 to -10.9 kcal/mol, K_D of 10 to 19 nM). Two of them balanced enthalpy and entropy equally, while two were more enthalpy-driven.
• Linker position effect: The C2-modified 2-AHA-cAMP bound about 1.5 kcal/mol more weakly (K_D of 177 nM). For the two derivatives with published reference data, focal molography, SPR and ITC agreed in ΔG within 1 to 1.5 kcal/mol.

Why It Matters

Lead optimization often produces series of close analogs with similar affinity. Thermodynamic fingerprints show which of them reach that affinity through different binding forces, information that affinity ranking hides. Because the diffractometric readout tolerates temperature changes without long equilibration, and DDI places many ligands on one chip under matched conditions, comparative thermodynamic profiling moves from a one-ligand-at-a-time experiment toward a multiplexed workflow. The values are apparent and specific to the surface format, which makes them best suited for ranking related ligands.

Oehninger J, Notova S, Frutiger A. High-throughput thermodynamic fingerprinting of protein–ligand interactions by DNA-directed focal molography. PLOS One 2026;21(10):e0354008. Open access (CC BY 4.0). https://doi.org/10.1371/journal.pone.0354008