Department of Chemistry and Chemical Biology , Harvard University , 12 Oxford St. , Cambridge , Massachusetts 02138 , United States.
J Am Chem Soc. 2018 Mar 28;140(12):4259-4268. doi: 10.1021/jacs.7b11639. Epub 2018 Mar 15.
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.
许多治疗方法会引起细胞类型特异性的多药理学，这是由小分子与整个蛋白质组之间的分子识别事件网络执行的。然而，测量构成蛋白质组甚至常见治疗药物（如非甾体抗炎药 (NSAIDs)）与小分子之间分子关联的结构受到无法绘制小分子互作组的限制。为了解决这一差距，我们开发了一种称为通过光亲和标记进行小分子互作组映射的平台 (SIM-PAL)，并将其应用于 NSAID 结合位点的细胞内直接特征描述。SIM-PAL 使用 (1) 全蛋白质组中 NSAID 衍生物的光化学偶联，以及 (2) 共轭肽的富集和同位素重编码，以及 (3) 基于靶向质谱的分配。使用 SIM-PAL，我们确定了 NSAID 互作组，其中包含超过 1000 种显着富集的蛋白质，并直接描述了近 200 种代表光 NSAID 与 Jurkat 和 K562 细胞中蛋白质直接结合的共轭肽。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）。共轭肽揭示了从细胞膜到细胞核的 NSAID 直接结合位点，以及三种光 NSAID 在组蛋白 H2A 和 H2B 上的特定结合热点。细胞热转移测定法表明 NSAID 结合稳定了 COX-2 和组蛋白 H2A。由于小分子稳定蛋白质复合物是一种功能获得性调节机制，因此可以想象 NSAIDs 通过这些更广泛的蛋白质组相互作用影响生物过程。SIM-PAL 能够描述 NSAID 结合位点热点，并且易于绘制几乎任何感兴趣分子的全局结合位点。
