Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Current methods rely on the catalytic properties of the target protein, and the use of a ligand or downstream reporting systems. Here, we report a novel method using nanobodies that allows the efficient and sensitive screening of potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen <i>Pseudomonas aeruginosa</i>. A nanobody inhibiting the catalytic activity of Cif was selected and used in a displacement sandwich ELISA (dsELISA), where the inhibitory nanobody also interferes with the binding of a small-molecule inhibitor to the active site of Cif. The dsELISA assay correlates strongly with a conventional fluorogenic assay in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic assay method. These data were supported with surface plasmon resonance and Octet biolayer measurement techniques. The novel method described herein relies solely on the binding properties of the specific neutralizing nanobody, and thus is applicable to any pharmacological target for which such a nanobody can be found, independent of any requirement for catalytic activity.

Nanobody-based binding assay for the discovery of potent inhibitors in drug development

2019 Fall National Meeting

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