Chemical proteomic strategies for deciphering neocarzilin´s mode of action in cancer cells

天然产品涉及大量具有基本功能的不同蛋白质。他们的目标的解卷积代表了解开新抗癌策略的独特机会。我们在这里应用化学蛋白质组学来确定活癌细胞内天然产物neocarzilin的靶点。Neocarzilin是一种有效的抗癌化合物，几十年前就被发现，但仍然缺乏功能特征。在初步研究中，我们合成了带有炔标签的天然产物，并处理了蛋白质组以鉴定其细胞靶点。有趣的是，只有一种蛋白质，囊泡胺转运蛋白（VAT-1）与细胞迁移的重要作用，已被确定为突出的命中。由于新卡西林也表现出不依赖于VAT-1的强抗增殖作用，因此必须存在其他尚未鉴定的靶标。为了破译这些目标，我们的目标是通过掺入天然产物中存在但在第一代探针设计中不存在的签名立体中心来改进我们的初始探针。靶标鉴定将遵循我们建立的化学蛋白质组学程序，并将通过过表达和晶体学验证所选蛋白质命中的化合物结合。这项工作的另一个重点是已经确定的打击增值税-1。我们的初步验证揭示了在癌细胞迁移中的假定作用，这是恶性肿瘤的一个重要特征。需要进一步的化学蛋白质组学研究来了解这种特征不明显的蛋白质的细胞功能，并利用其在未来癌症治疗中的潜力。本文提出的研究将包括在存在和不存在天然产物的情况下的共免疫沉淀，以破译相互作用伴侣，共结晶和化合物的结构解析，以解开结合模式并合理化改进的结合剂。该提案的总体目标是破译新卡西林靶点的全谱，了解其作用模式，研究VAT-1的作用，并找到具有治疗潜力的改进衍生物。

Natural products address a plethora of different proteins with essential functions. The deconvolution of their targets represents a unique opportunity for unravelling new anti-cancer strategies. We here apply chemical proteomics to identify the targets of the natural product neocarzilin within living cancer cells. Neocarzilin is a potent anti-cancer compound which was identified decades ago but still lacks functional characterization. In preliminary studies we synthetically equipped the natural product with an alkyne tag and treated proteomes to identify its cellular target(s). Interestingly, only one protein, the vesicle amine transport protein (VAT-1) with an essential role in cell migration, has been identified as prominent hit. As neocarzilin also exhibits strong anti-proliferative effects which are independent of VAT-1, other yet unidentified targets must exist. To decipher these targets we aim to improve our initial probe by incorporation of a signature stereocenter present in the natural product but absent in the first generation probe design. Target identification will follow our established chemical proteomic procedures and selected protein hits will be validated for compound binding by overexpression and crystallography. Another strong focus of this work is the already identified hit VAT-1. Our initial validation revealed a putative role in cancer cell migration, an important feature of malignant tumors. Further chemical proteomic studies are required to understand the cellular function of this poorly characterized protein and to exploit its potential for future cancer treatment. The studies proposed here will include co-immunoprecipitation in presence and absence of the natural product to decipher interaction partners, co-crystallization and structure elucidation with the compound to unravel the binding mode and rationalize improved binders. The overall aims of this proposal are to decipher the full spectrum of neocarzilin targets, understand its mode of action, investigate the role of VAT-1 and find improved derivatives with therapeutic potential.