Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC₅₀) = 10 nM) that bind the active site of CypD and also make novel interactions with non-conserved residues in the S2 pocket, an adjacent exo-site. The resulting macrocycles inhibit CypD activity with 21- to >10,000-fold selectivity over other cyclophilins and inhibit mitochondrial permeability transition pore opening in isolated mitochondria. We further exploited S2 pocket interactions to develop the first cyclophilin E (CypE)-selective inhibitor, which forms a reversible covalent bond with a CypE S2 pocket lysine, and exhibits 30- to >4,000-fold selectivity over other cyclophilins. These findings reveal a strategy to generate isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic development.

尽管亲环蛋白是生物学探索和治疗干预的有吸引力的靶标，但尚未描述亚型选择性亲环蛋白抑制剂。我们通过对 256,000 个药物样大环化合物的 DNA 模板库进行体外筛选，发现了新型亲环蛋白抑制剂，用于亲环蛋白 D (CypD) 亲和力。由十个 X 射线共晶结构引导的迭代大环工程产生了有效的选择性抑制剂（半数最大抑制浓度 (IC ₅₀ ) = 10 nM），其结合 CypD 的活性位点，并与 CypD 中的非保守残基产生新的相互作用。 S2 口袋，相邻的外部站点。由此产生的大环化合物抑制 CypD 活性的选择性比其他亲环蛋白高 21 至 >10,000 倍，并抑制分离线粒体中线粒体通透性转换孔的开放。我们进一步利用 S2 口袋相互作用开发了第一个亲环蛋白 E (CypE) 选择性抑制剂，它与 CypE S2 口袋赖氨酸形成可逆共价键，并且比其他亲环蛋白表现出 30 至 > 4,000 倍的选择性。这些发现揭示了一种生成异构体选择性小分子亲环蛋白调节剂的策略，提高了它们作为生物学研究和治疗开发靶标的适用性。