The ability of biologically active molecules to access intracellular targets remains a critical barrier in drug development. While assays for measuring cellular uptake exist, they often fail to distinguish between membrane-associated or endosomal trapped compounds and those that successfully reach the cytosol. Here, we present the Chloroalkane HaloTag Azide-based Membrane Penetration (CHAMP) Assay, a novel high-throughput method that employs a minimally disruptive azide tag to report the cytosolic accumulation of diverse molecules in mammalian cells. The CHAMP assay utilizes HaloTag-expressing cells and strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry to quantify the presence of azide-tagged test compounds in the cytosol. We demonstrate the versatility of this approach by evaluating the accumulation profiles of small molecules, peptides, and proteins, revealing how structural variations and stereochemical differences influence cytosolic penetration. Our findings with cell-penetrating peptides confirm established structure-activity relationships, with longer polyarginine sequences showing enhanced accumulation. Additionally, we observed that C -terminal amidation and D-amino acid substitutions significantly impact cellular penetration. When applied to supercharged proteins and antibiotics, CHAMP successfully discriminates between compounds with varying accumulation capabilities. This method provides a robust platform for screening cytosolic accumulation while minimizing the confounding effects of large tags on molecular permeability, potentially accelerating the development of therapeutics targeting intracellular pathways.

生物活性分子进入细胞内靶点的能力仍然是药物开发中的关键障碍。虽然存在测量细胞摄取的测定方法，但它们往往无法区分膜结合或溶酶体捕获的化合物和成功到达细胞质的化合物。在这里，我们提出了氯代烷烃 HaloTag 咪唑𬭩（CHAMP）渗透测定法，这是一种新型高通量方法，采用最小干扰的咪唑𬭩标签来报告多种分子在哺乳动物细胞中的细胞质积累情况。CHAMP 测定法利用表达 HaloTag 的细胞和应变促进的叠氮-炔烃环加成 (SPAAC) 化学反应来量化测试化合物中细胞质内的叠氮标记的存在。我们通过评估小分子、肽类和蛋白质的累积谱图，展示了这种方法的灵活性，并揭示了结构变化和立体化学差异如何影响细胞质渗透。我们的细胞穿透肽结果证实了已建立的构效关系，具有较长多精氨酸序列的化合物显示出增强的积累。此外，我们观察到C端酰胺化和D氨基酸替代物显著影响细胞穿透能力。当应用于超荷电蛋白质和抗生素时，CHAMP 成功区分了累积能力不同的化合物。该方法为筛选细胞质累积提供了稳健平台，并最大限度地减少了大标签对分子渗透性造成的干扰效应，有可能加速针对细胞内途径的治疗药物开发。