Cell–cell interactions and communication are crucial to the proper function of complex mammalian physiology including neurocognitive and immune system functions. While many tools are available for observing and perturbing intracellular processes, relatively few exist to probe intercellular processes. Current techniques for studying interactions often rely on direct protein contact, and few can manipulate diverse, functional outputs with tunable protein expression. To address these limitations, we have developed a small-molecule approach based on a trimethoprim prodrug–enzyme pair capable of reporting the presence of two different engineered cell populations with programmable protein outputs. The approach relies on bacterial nitroreductase enzyme catalysis, which is orthogonal to normal mammalian biology, and diffusion of trimethoprim from “activator” cells to “receiver” cells. We test this strategy, which can theoretically regulate many different types of proteins, using biochemical and in vitro culture assays with optical and cytokine protein readouts. This describes the first small-molecule approach capable of detecting and controlling engineered cell–cell outputs, and we anticipate future applications that are especially relevant to the field of immuno-oncology.

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基于工程细胞相互作用的可编程蛋白质输出的化学方法

细胞间相互作用和通讯对于复杂哺乳动物生理机能的正常功能至关重要，包括神经认知和免疫系统功能。虽然有许多工具可用于观察和干扰细胞内过程，但用于探测细胞间过程的工具相对较少。当前用于研究相互作用的技术通常依赖于直接的蛋白质接触，很少有人能够通过可调的蛋白质表达来操纵多样化的功能性输出。为了解决这些限制，我们开发了一种基于甲氧苄啶前药-酶对的小分子方法，能够报告两种不同工程细胞群的存在，并具有可编程的蛋白质输出。该方法依赖于细菌硝基还原酶的催化作用，与正常的哺乳动物生物学正交，甲氧苄啶从“激活”细胞扩散到“受体”细胞。我们测试了这个策略，它理论上可以调节许多不同类型的蛋白质，使用生化和具有光学和细胞因子蛋白读数的体外培养测定。这描述了第一种能够检测和控制工程细胞-细胞输出的小分子方法，我们预计未来与免疫肿瘤学领域特别相关的应用。