Phorbol ester analogs are a promising class of anticancer therapeutics and HIV latency reversing agents that interact with cellular membranes to recruit and activate protein kinase C (PKC) isoforms. However, it is unclear how these esters interact with membranes and how this might correlate with the biological activity of different phorbol ester analogs. Here, we have employed dynamic nuclear polarization (DNP) NMR to characterize phorbol esters in a native cellular context. The enhanced NMR sensitivity afforded by DNP and cryogenic operation reveals topological heterogeneity of ¹³C-21,22-phorbol-myristate-acetate (PMA) within T cells utilizing ¹³C–¹³C correlation and double quantum filtered NMR spectroscopy. We demonstrate the detection of therapeutically relevant amounts of PMA in T cells down to an upper limit of ∼60.0 pmol per million cells and identify PMA to be primarily localized in cellular membranes. Furthermore, we observe distinct ¹³C-21,22-PMA chemical shifts under DNP conditions in cells compared to model membrane samples and homogenized cell membranes, that cannot be accounted for by differences in conformation. We provide evidence for distinct membrane topologies of ¹³C-21,22-PMA in cell membranes that are consistent with shallow binding modes. This is the first of its kind in-cell DNP characterization of small molecules dissolved in the membranes of living cells, establishing in-cell DNP-NMR as an important method for the characterization of drug-membrane interactions within the context of the complex heterogeneous environment of intact cellular membranes. This work sets the stage for the identification of the in-cell structural interactions that govern the biological activity of phorbol esters.

中文翻译：

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用细胞内动态核极化 NMR 波谱解析人 T 细胞中蛋白激酶 C 调节剂的拓扑异质性


佛波酯类似物是一类很有前途的抗癌治疗药物和 HIV 潜伏期逆转剂，可与细胞膜相互作用以募集和激活蛋白激酶 C （PKC） 亚型。然而，目前尚不清楚这些酯如何与膜相互作用，以及这与不同佛波酯类似物的生物活性有何关联。在这里，我们采用动态核极化 （DNP） NMR 在天然细胞环境中表征佛波酯。DNP 和低温操作提供的增强 NMR 灵敏度揭示了 T 细胞内 ¹³C-21,22-佛波醇-肉豆蔻酸酯 （PMA） 的拓扑异质性，利用 ^{13 C-13}C 相关性和双量子滤波 NMR 波谱。我们证明了在 T 细胞中检测到治疗相关量的 PMA，最高可达每百万个细胞 ∼60.0 pmol，并确定 PMA 主要位于细胞膜中。此外，与模型膜样品和匀浆细胞膜相比，我们在 DNP 条件下在细胞中观察到明显的 ¹³C-21,22-PMA 化学变化，这不能用构象的差异来解释。我们为细胞膜中 ¹³C-21,22-PMA 的不同膜拓扑结构提供了证据，这些拓扑结构与浅结合模式一致。这是首个对溶解在活细胞膜中的小分子进行细胞内 DNP 表征的试验，将细胞内 DNP-NMR 确立为在完整细胞膜的复杂异质环境中表征药物-膜相互作用的重要方法。 这项工作为鉴定控制佛波酯生物活性的细胞内结构相互作用奠定了基础。