PO.CH01.02 · 化学
调整SAR-MAP化学生物学平台以靶向肿瘤免疫检查点
Adapting the SAR-MAP chemical biology platform to target tumor immune checkpoints
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
临床前药物发现中的一个主要挑战是将复杂原代人类细胞的深度细胞分析适配到可扩展的小分子筛选及后续的构效关系(SAR)研究中。当目标是靶向细胞间相互作用(如肿瘤免疫检查点)时，情况尤其如此。我们此前开发了通过多重活性分析进行构效关系研究(SAR-MAP；PMC12313981)，这是一个单细胞化学生物学平台，可定量绘制化合物中微小的结构变化如何在人类细胞内驱动不同的功能反应。SAR-MAP将磷酸化特异性流式细胞术与荧光细胞条形码相结合，可并行高效分析数十个关键的细胞内信号节点、表面标志物和细胞状态。该平台揭示了rocaglate天然产物家族此前未被认识的机制异质性，包括一个赋予选择性抗白血病活性的结构特征。在此，我们将SAR-MAP适配至原代人类巨噬细胞，并建立了一个可扩展的筛选检测方法，能够在单次实验中剖析IFN-gamma响应通路、免疫检查点调控和基本的免疫相关信号节点。从外周血中分离单核细胞，进行分化，并使用IFN-gamma以荧光条形码的96孔板格式将其极化为M1样促炎状态，该格式适合筛选和迭代SAR。多重活性分析定量了包括PD-L1、CD14、CD206和CD32在内的关键功能和表面标志物，证实相对于基线巨噬细胞，IFN-gamma极化能稳健地诱导PD-L1。这一适配能够同时评估调控免疫检查点表达的多种机制，包括直接的PD-L1阻断、IFN-gamma调节以及JAK-STAT1或mTOR通路干扰。选择性调节这些通路的小分子不仅具有转化相关性，还可能阐明此前未被认识的调控免疫的靶点和机制。总之，这些研究进一步确立了SAR-MAP作为一种可扩展、高内涵的方法，用于将化学结构与原代人类细胞中的免疫调控联系起来。
查看英文原文 English abstract
A major challenge in preclinical drug discovery is adapting deep cellular profiling of complex primary human cells to scalable small-molecule screening and subsequent structure-activity relationship (SAR) studies. This is especially the case when the goal is to target an intercellular interaction, such as a tumor immune checkpoint. We previously developed Structure-Activity Relationships by Multiplexed Activity Profiling (SAR-MAP; PMC12313981), a single cell chemical biology platform that quantitatively maps how small structural changes in compounds can drive distinct functional responses within human cells. SAR-MAP integrates phospho-specific flow cytometry with fluorescent cell barcoding to efficiently profile dozens of key intracellular signaling nodes, surface markers, and cell states in parallel. This platform revealed previously unrecognized mechanistic heterogeneity for the rocaglate natural product family, including a structural feature that confers selective anti-leukemia activity. Here we adapted SAR-MAP to primary human macrophages and established a scalable screening assay capable of dissecting IFN-gamma responsive pathways, immune checkpoint regulation, and fundamental immune-relevant signaling nodes within a single experiment. Monocytes were isolated from peripheral blood, differentiated, and polarized to an M1-like proinflammatory state using IFN-gamma in a fluorescently barcoded 96-well format suited for screening and iterative SAR. Multiplexed activity profiling quantified key functional and surface markers including PD-L1, CD14, CD206, and CD32, confirming that IFN-gamma polarization robustly induces PD-L1 relative to baseline macrophages. This adaptation enables simultaneous evaluation of multiple mechanisms regulating immune checkpoint expression, including direct PD-L1 blockade, modulation of IFN-gamma, and JAK-STAT1 or mTOR pathway interference. Small molecules that selectively modulate these pathways not only have translational relevance but may also illuminate previously unrecognized targets and mechanisms governing immune regulation. Together, these studies further establish SAR-MAP as a scalable, high-content approach for linking chemical structure to immune regulation in primary human cells.
利益披露 Disclosure
H. L. Thirman, None..
S. Medina, None..
J. M. Irish, None.