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Antibody-Inspired Affinity Measurements of Fluorescein-Linked Small Molecule Ligands

preprint
submitted on 28.05.2020 and posted on 29.05.2020 by Marco Catalano, Sebastian Oehler, Luca Prati, Nicholas Favalli, Gabriele Bassi, Jörg Scheuermann, Dario Neri
The availability of reliable methods for the characterization of the binding of small molecule ligands to protein targets is crucially important for Drug Discovery. We have adapted a method, routinely used for the characterization of monoclonal antibodies (Enzyme-linked immunosorbent assay, or “ELISA”), to small molecule ligands, using fluorescein conjugates and anti-fluorescein antibodies as detection reagents. The new small molecule-ELISA methodology was tested using a panel of binders specific to carbonic anhydrase II, with dissociation constants ranging between 6 uM and 14 nM. An excellent agreement was found between ELISA measurements and fluorescence polarization results. The methodology was also extended to BIAcore measurements and implemented for ligands coupled to oligonucleotides. Small molecule-ELISA procedures are particularly useful in the context of DNA-encoded libraries, for which hit validation procedures need to be performed on dozens of candidate molecules and hit compounds can be conveniently resynthesized on DNA.

Funding

Fulfilling Paul Ehrlich’s Dream: therapeutics with activity on demand

European Research Council

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Understanding and Exploiting the Molecular Targeting of Tumor Neo-vasculature

Swiss National Science Foundation

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History

Email Address of Submitting Author

marco.catalano@pharma.ethz.ch

Institution

Swiss Federal Institute of Technology (ETH Zürich)

Country

Switzerland

ORCID For Submitting Author

0000-0001-8257-3977

Declaration of Conflict of Interest

D.N. is a cofounder and shareholder of Philochem AG.

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