Abstract
Protein-small molecule hybrids are structures capable of combining the inhibitory properties of small molecules and the specificities of binding proteins. However, discovery of such synergistic conjugates is a substantial engineering challenge. Here, we describe pharmacophore-driven antibody discovery as a high throughput approach to hybrid discovery. In this approach, we use a yeast display antibody library containing reactive noncanonical amino acids (ncAAs) and further diversify it by conjugating the library to four sulfonamide pharmacophores. Yeast display binding screens with each of the resulting billion-member hybrid collections against bovine carbonic anhydrase (bCA) yielded diverse collections of hybrids. Individual hybrids exhibited double digit nanomolar binding affinities, and frequently exhibited inhibitory properties in solution, despite the fact that the screens were based solely on binding phenotypes. Deep sequencing of sorted populations revealed that enrichments were strongly pharmacophore-dependent. In particular, screens with a potent pharmacophore led to collections of hybrids varying substantially in pharmacophore attachment point and antibody sequence features, while screens with moderate or weak pharmacophores led to collections with much narrower sets of enriched attachment points and antibody sequence features. Identification of the most frequently isolated CDR-H3 sequences and clustering CDR-H3 sequences by similarity within enriched populations provided further evidence for pharmacophore-dependent sorting outcomes. Experimental binding assays in which the pharmacophore warhead used during screening was replaced by another warhead indicated that isolated clones can tolerate alternative pharmacophores, but tend to prefer the warhead used during screening. Overall, these efforts demonstrate the utility of introducing pharmacophores into antibody libraries along with several lines of evidence that screening outcomes are pharmacophore-driven. These findings advance our understanding of hybrid discovery and highlight opportunities to pursue hybrids as research tools and potential therapeutic leads.