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

天然产品含有大量具有基本功能的不同蛋白质。他们靶点的解卷代表着一个独特的机会，可以解开新的抗癌策略。我们在这里应用化学蛋白质组学来确定活的癌细胞中天然产物新卡西林的靶点。新卡西林是一种有效的抗癌化合物，几十年前就被发现，但仍缺乏功能表征。在初步研究中，我们在天然产物上合成了一个乙炔标签，并对蛋白质组进行处理，以确定其细胞靶标(S)。有趣的是，只有一种蛋白质，即在细胞迁移中起关键作用的囊泡胺运输蛋白(VAT-1)，已被确定为显著的HIT。由于新卡西林也表现出独立于VAT-1的强大的抗增殖作用，因此肯定存在其他尚未确定的靶点。为了破译这些靶点，我们的目标是通过加入天然产品中存在但在第一代探头设计中缺失的标志性立体中心来改进我们最初的探头。靶标鉴定将遵循我们建立的化学蛋白质组学程序，选定的蛋白质命中将通过过度表达和结晶学验证化合物结合。这项工作的另一个重点是已经确定的热门增值税-1。我们的初步验证揭示了癌细胞迁移的推定作用，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性不佳的蛋白质的细胞功能，并开发其在未来癌症治疗中的潜力。本文提出的研究将包括在天然产物存在和不存在的情况下进行免疫共沉淀以破译相互作用伙伴，与化合物的共结晶和结构阐明以解开结合模式并使改进的结合剂合理化。这项提案的总体目标是破译新卡西林靶标的全谱，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的衍生物。

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.