Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

中文翻译：

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光亲和标记的小分子相互作用组图谱揭示了 NSAID 的结合位点热点

许多疗法引发细胞类型特异性多药理学，由小分子和整个蛋白质组之间的分子识别事件网络执行。然而，由于无法绘制小分子相互作用组，因此无法测量蛋白质组和甚至常见疗法（例如非甾体抗炎药 (NSAID)）之间的分子关联的结构。为了解决这一差距，我们开发了一个称为通过光亲和标记 (SIM-PAL) 进行小分子相互作用组映射的平台，并将其应用于特定 NSAID 结合位点的纤维素内直接表征。SIM-PAL 使用 (1) NSAID 衍生物在整个蛋白质组中的光化学偶联和 (2) 偶联肽的富集和同位素记录，用于 (3) 基于质谱的靶向分配。使用 SIM-PAL，我们鉴定了由 1000 多种显着富集的蛋白质组成的 NSAID 相互作用组，并直接表征了近 200 种偶联肽，这些偶联肽代表了来自 Jurkat 和 K562 细胞的蛋白质的 photo-NSAID 的直接结合位点。富集的蛋白质通常被鉴定为复合物的一部分，包括 NSAID 活性的已知靶标（例如，NF-κB）和新的相互作用（例如，AP-2、蛋白酶体）。缀合的肽揭示了从细胞表面到细胞核的直接 NSAID 结合位点，以及组蛋白 H2A 和 H2B 上三种光-NSAID 的特异性结合位点热点。通过细胞热位移测定，NSAID 结合稳定了 COX-2 和组蛋白 H2A。由于蛋白质复合物的小分子稳定是功能调节机制的获得，可以想象，NSAIDs 通过这些更广泛的蛋白质组学相互作用影响生物过程。SIM-PAL 能够表征 NSAID 结合位点热点，并且能够绘制几乎任何感兴趣分子的全局结合位点。