Mechanism and function of protein prenylation

描述(申请人提供)：RAS蛋白是法尼化的，抑制法尼化可以防止细胞转化，这一发现引起了人们对这种翻译后修饰的兴趣激增；目前，几种靶向法尼基转移酶的候选药物正在进行第二阶段的试验。然而，尽管取得了这样的成功，我们目前对异丙苯基转移酶的酶学的了解仍然不完整，特别是关于确切的化学机制。此外，关于蛋白质预烯基化的生物学功能的许多方面仍然不清楚。在这个项目的头三年里，我们对戊烯基转移酶的结构和机制有了很大的了解；我们在理解这种修饰的功能方面也取得了进展。这一更新应用的具体目的是：(1)完成对蛋白质法尼基和香叶基香叶基转移酶催化反应的化学机理的研究。将进行立体化学和动力学同位素效应实验。(2)利用我们对类异戊二烯类似物的研究所获得的结构洞察力，设计了一类新的蛋白法尼基转移酶双底物抑制剂。(3)鉴定通过与类异戊二烯相互作用识别预烯基化蛋白质和多肽的额外蛋白质。这些化合物将使用光亲和捕获方法进行识别，该方法使用生物素化的戊烯基半胱氨酸和戊烯基化肽，并结合用于目标识别的质谱分析。这些研究将确定这种现象有多普遍，并评估存在多少不同的结构类别。(4)使用合成方法和噬菌体展示相结合的方法来获得靶向于前烯基化蛋白及其受体之间相互作用的抑制剂。这类抑制剂将有助于阐明蛋白质预烯基化的生物学功能。上述目标的完成应该会为蛋白质预烯基化的机制和功能提供更多的见解。

DESCRIPTION (provided by applicant): The discovery that Ras proteins are farnesylated and that inhibition of farnesylation prevents cellular transformation has generated an explosion of interest in this post-translational modification; currently, several drug candidates that target farnesyl transferase are in Phase 2 trials. However, despite this success, our current understanding of the enzymology of prenyltransferases remains incomplete particularly regarding the precise chemical mechanism. Moreover, many aspects concerning the biological function of protein prenylation remain unclear. In the first three years of this project, we have gained significant insights into the structure and mechanism of prenyltransferases; we have also made progress in understanding functional aspects of this modification. The specific aims of this renewal application are to: (1) Complete our studies of the chemical mechanisms of the reactions catalyzed by protein farnesyl and geranylgeranyl transferase enzymes. Stereochemical and kinetic isotope effect experiments will be performed. (2) Use the structural insights we have gained from our studies with isoprenoid analogues to design a new class of bisubstrate inhibitors of protein farnesyltransferase. (3) Identify additional proteins that recognize prenylated proteins and peptides through interactions with the isoprenoid. These will be identified using a photoaffinity capture method employing biotinylated prenylcysteines and prenylated peptides coupled with mass spectral analysis for target identification. These studies will determine how common this phenomenon is and evaluate how many different structural classes exist. (4) Use a combination of synthetic methods and phage display to obtain inhibitors that target the interaction between prenylated proteins and their receptors. Such inhibitors will be useful for clarifying the biological function of protein prenylation. Completion of the above aims should provide additional insights into the mechanism and function of protein prenylation.