Nicholas Favalli,Carola Velti,Lorenzo Campari,Lukas Heuberger,Mosè Fabbri,Marco Müller,Sara Puglioli,Samuele Cazzamalli,Dario Neri,Andreas Frutiger,Sebastian Oehler

Journal of Medicinal Chemistry

DOI: 10.1021/acs.jmedchem.6c01097

Abstract

DNA-encoded libraries (DELs) enable rapid discovery of large pools of small organic ligands against target proteins. Currently available hit validation methodologies are limited in their ability to characterize binding kinetics for a large number of molecules. Only a subset of hits identified by DEL screening are followed up on, while many potentially relevant binders remain uncharacterized. Here, we propose focal molography as a high-throughput, label-free optical methodology for parallel kinetic measurements of DEL-derived hits. The methodology was applied to measure binding kinetics (kon and koff) and dissociation constants (Kd) of DEL-derived ligands against carbonic anhydrase IX and of known ligands to fibroblast activation protein. The binding parameters obtained were consistent with fluorescence polarization, surface plasmon resonance, and inhibition measurements. The data reported in this manuscript support the use of focal molography as a robust DEL-compatible technology for quantitative, high-throughput hit validation.