A toolbox of promiscuous immobilisation chemistries to identify the targets of natural compounds by chemoproteomics (PromisChemProt)

天然产物已经进化到有利于它们的生产生物体：与人造合成化合物相反，它们已经被进化选择和优化，以与生物目标相互作用，例如作为攻击或防御机制。因此，天然产物构成了一个有希望的创新化学生物学工具和潜在药物池，将受益于系统的探索。靶蛋白可以参与其他生物体的基本细胞通路，并且很多时候尚未被证实为人类医学的药物靶点。表型筛选已经发现了许多具有抗肿瘤活性的天然化合物，但通常不清楚哪些细胞靶点参与并导致观察到的表型结果。然而，获取这些信息，即目标反卷积，对于将可观察到的表型转化为可操作的新化学型和/或新的生物靶点至关重要。在复杂细胞系统的背景下，由于缺乏通用的实验方法，这些筛选命中的目标反褶积常常受到阻碍。特别地，一个特别的可连接类似物的化学合成经常被用来询问一个分子的目标空间。然而，天然产物通常难以合成，这使得基于活性或基于亲和力的目标反卷积方法变得复杂。有了这项研究计划，我们的目标是开发一个强大的化学蛋白质组学管道，允许对任何选择的未经修饰的天然产物进行目标反卷积：更准确地说，我们将建立一个化学工具箱，能够将少量天然化合物固定在珠子上。我们将研究重氮嘧啶在碳插入、光信反应、金催化的环加成和烷氧基化以及酶的复分解等方面的作用，以使天然产物与载试剂珠混合反应。所获得的亲和基质将能够从细胞裂解物中亲和纯化天然化合物的目标，并通过定量质谱法对其进行表征，建立在我们的化学蛋白质组学专业知识基础上。由于天然产物中存在丰富的烯烃和醇功能，我们预计至少有一种利用的化学物质将产生与分子相容的连锁：目标结合事件。由于这种有利的连锁位点无法预测未知的目标，我们的概念依赖于一组化学物质的平行不可知论使用，这将应用于40种确认对癌细胞系起作用的天然化合物。天然产物参与新发现的靶标与表型之间的任何确认的因果关系都将构成药物研究的一个可行的起点，因为缺乏经过验证的靶标正迫切需要替代药物发现范式。

Natural products have evolved to benefit their producing organisms: contrary to synthetic man-made compounds, they have been selected and optimized by evolution to interact with biological targets for example as attack or defense mechanisms. Natural products thus constitute a promising pool of innovative chemical biology tools and potential drugs that would benefit from systematic exploration. Target proteins can be involved in essential cellular pathways of other organisms, and are many times not yet validated drug targets for human medicine. Phenotypic screenings have uncovered many natural compounds with anti-tumor activity but it is often unclear which cellular targets are engaged and lead to the observed phenotypic outcome. Yet, acquiring this information, i.e. target deconvolution, is of utmost importance to convert observable phenotypes into actionable novel chemotypes and/or novel biological targets. Target deconvolution of those screening hits is often deterred by the lack of generic experimental approaches in the context of complex cellular systems. In particular, chemical synthesis of an ad-hoc linkable analogue is often used to interrogate the target space of a molecule. However natural products are often difficult to synthesize which complicates e.g. activity-based or affinity-based target deconvolution approaches. With this research proposal, we ambition to develop a robust chemoproteomics pipeline that allows to perform target deconvolution of any chosen unmodified natural product: More precisely, we will build a toolbox of chemistries able to immobilize small amounts of natural compounds on beads. We will investigate diazirine for carbene insertion, Mitsunobu reaction, gold catalyzed cycloaddition and alkoxylation as well as enyne metathesis as means to promiscuously react natural products with reagent-loaded beads. The obtained affinity matrices will enable the affinity purification of targets of natural compounds from cellular lysates and their characterization by quantitative mass spectrometry, building on our chemical proteomics expertise. Because of the wealth of alkenes and alcohol functionalities present in natural products, we anticipate that at least one of the utilized chemistries will produce a linkage compatible with the molecule:target binding event. Since such propitious linkage site cannot be predicted for unknown targets, our concept relies on the parallel agnostic use of a set of chemistries, which will be applied to forty natural compounds confirmed to act on cancer cell lines. Any confirmed causality link between the engagement of the newly identified target by the natural product and he phenotype will constitute an actionable starting point for pharmaceutical research, where the lack of validated targets is pressing for alternative drug discovery paradigms.