The potential of small molecules to localize within subcellular compartments is rarely explored. To probe this question, we measured the localization of Hsp70 inhibitors using fluorescence microscopy. We found that even closely related analogs had dramatically different distributions, with some residing predominantly in the mitochondria and others in the ER. CRISPRi screens supported this idea, showing that different compounds had distinct chemogenetic interactions with Hsp70s of the ER (HSPA5/BiP) and mitochondria (HSPA9/mortalin) and their co-chaperones. Moreover, localization seemed to determine function, even for molecules with conserved binding sites. Compounds with distinct partitioning have distinct anti-proliferative activity in breast cancer cells compared with anti-viral activity in cellular models of Dengue virus replication, likely because different sets of Hsp70s are required in these processes. These findings highlight the contributions of subcellular partitioning and chemogenetic interactions to small molecule activity, features that are rarely explored during medicinal chemistry campaigns.

小分子在亚细胞区室中定位的潜力很少被探索。为了探究这个问题，我们使用荧光显微镜测量了 Hsp70 抑制剂的定位。我们发现，即使是密切相关的类似物也具有显着不同的分布，其中一些主要存在于线粒体中，而另一些则存在于内质网中。 CRISPRi 筛选支持了这一观点，表明不同的化合物与 ER (HSPA5/BiP) 和线粒体 (HSPA9/mortalin) 的 Hsp70 及其共伴侣具有不同的化学遗传学相互作用。此外，定位似乎决定了功能，即使对于具有保守结合位点的分子也是如此。与登革热病毒复制细胞模型中的抗病毒活性相比，具有不同分配的化合物在乳腺癌细胞中具有不同的抗增殖活性，这可能是因为这些过程中需要不同组的 Hsp70。这些发现强调了亚细胞分配和化学遗传学相互作用对小分子活性的贡献，这些特征在药物化学活动中很少被探索。