Engineered protein-small molecule conjugates empower selective enzyme inhibition

S ummary Specific, potent ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. A fibronectin protein library was yeast displayed with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enriched specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibited potent, specific inhibition of carbonic anhydrase isoforms with superior efficacy to acetazolamide or protein alone including an 80-fold specificity increase and 9-fold potency gain. PriSMs were engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveals platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.