Huang Tao, Hosseinibarkooie Seyyedmohsen, Borne Adam L, Granade Mitchell E, Brulet Jeffrey W, Harris Thurl E, Ferris Heather A, Hsu Ku-Lung
Department of Chemistry, University of Virginia McCormick Road, P.O. Box 400319 Charlottesville Virginia 22904 USA
Department of Medicine, University of Virginia School of Medicine Charlottesville Virginia 22903 USA.
Chem Sci. 2021 Jan 21;12(9):3295-3307. doi: 10.1039/d0sc06623k.
Sulfonyl-triazoles are a new class of electrophiles that mediate covalent reaction with tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. Recent studies demonstrate the broad utility and tunability of SuTEx chemistry for chemical proteomics and protein ligand discovery. Here, we present a strategy for mapping protein interaction networks of structurally complex binding elements using functionalized SuTEx probes. We show that the triazole leaving group (LG) can serve as a releasable linker for embedding hydrophobic fragments to direct molecular recognition while permitting efficient proteome-wide identification of binding sites in live cells. We synthesized a series of SuTEx probes functionalized with a lipid kinase fragment binder for discovery of ligandable tyrosines residing in catalytic and regulatory domains of protein and metabolic kinases in live cells. We performed competition studies with kinase inhibitors and substrates to demonstrate that probe binding is occurring in an activity-dependent manner. Our functional studies led to discovery of probe-modified sites within the C2 domain that were important for downregulation of protein kinase C-alpha in response to phorbol ester activation. Our proof of concept studies highlight the triazole LG of SuTEx probes as a traceless linker for locating protein binding sites targeted by complex recognition elements in live cells.
磺酰基 - 三唑是一类新型亲电试剂，可通过硫 - 三唑交换（SuTEx）化学与蛋白质上的酪氨酸残基介导共价反应。最近的研究证明了SuTEx化学在化学蛋白质组学和蛋白质配体发现方面具有广泛的实用性和可调节性。在此，我们提出了一种使用功能化SuTEx探针绘制结构复杂结合元件的蛋白质相互作用网络的策略。我们表明，三唑离去基团（LG）可作为可释放的连接子，用于嵌入疏水片段以指导分子识别，同时允许在活细胞中对结合位点进行全蛋白质组范围的有效鉴定。我们合成了一系列用脂质激酶片段结合剂功能化的SuTEx探针，用于发现活细胞中蛋白质和代谢激酶的催化和调节域中可配体化的酪氨酸。我们用激酶抑制剂和底物进行了竞争研究，以证明探针结合是以活性依赖的方式发生的。我们的功能研究导致在C2结构域内发现了探针修饰位点，这些位点对于佛波酯激活后蛋白激酶C - α的下调很重要。我们的概念验证研究强调了SuTEx探针的三唑LG作为一种无痕连接子，用于在活细胞中定位由复杂识别元件靶向的蛋白质结合位点。
