(164h) Systematic Evaluation of Protein-Small Molecule Conjugates on Yeast Surface

Proteins made of canonical amino acids have limited chemical diversity, which constrains their interactions with biological targets, including in enzyme active sites. Noncanonical amino acids (ncAAs) and efficient conjugation reactions such as “click” chemistry offer powerful strategies to add small molecule functionalities to protein-based therapeutics. Here, we demonstrate our ability to construct and characterize diverse conjugates on the yeast surface during our systematic evaluation of fibronectin-based protein-small molecule “hybrid” conjugates. Starting from 6 recently described fibronectin-maleimide-sulfonamide conjugates, we systematically varied amino acid attachment points and small molecule warheads to generate a combinatorial set of hybrids. Our flow cytometric analyses identified new hybrid structures that show isoform-specific carbonic anhydrase binding with double-digit nanomolar affinities despite drastic linker and small molecule warhead alterations from the original conjugates. We observed that amino acid side chains can greatly change the hybrid affinity even when a strong warhead like acetazolamide is used. In addition, we found new hybrids containing ncAAs and a lower potency small molecule warhead that showed high affinity to the target enzyme. Biochemical characterization of a promising hybrid in solution indicates that observations made on the yeast surface are consistent with observations made with the same hybrid in soluble form. These findings highlight promising opportunities for discovering protein-small molecule hybrids on the yeast surface by integrating a broad chemical toolkit with powerful yeast display screening and analysis platforms.