Potent, specific 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. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

有效的特异性配体推动精准医学和基础生物学发展。蛋白质、肽和小分子构成有效的配体类别。然而，更大的分子多样性将有助于寻找配体，以针对具有挑战性的目标引发精确的生物活性。我们展示了一个发现蛋白质-小分子 (PriSM) 杂交体的平台，将独特的药效团活性和形状与受限的、可高效工程化的蛋白质结合起来。在该平台中，纤连蛋白文库显示在酵母上，其中单个半胱氨酸通过马来酰亚胺-聚（乙二醇）接头与乙酰唑胺偶联。磁性和流式细胞仪分类丰富了碳酸酐酶亚型的特异性结合剂。孤立的 PriSM 表现出强大的，特异性抑制碳酸酐酶亚型，功效优于单独使用乙酰唑胺或蛋白质，包括 80 倍的特异性增加和 9 倍的效力增加。PriSM 设计有多个接头长度、蛋白质偶联位点和针对两种不同亚型的序列，这揭示了平台灵活性和分子设计的影响。PriSM 促进了高效工程配体的分子多样性。