Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
Faculty of Chemistry and Chemical Biology, Dortmund University of Technology, Otto-Hahn-Straße 4a, 44227, Dortmund, Germany.
Sci Rep. 2017 Jun 16;7(1):3687. doi: 10.1038/s41598-017-03973-6.
Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRas using GDP equipped with an electrophilic group at the β-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such β-modified derivatives for Ras of at least 10 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.
长期以来，人们一直认为简单的、可逆的竞争性核苷酸结合抑制 Ras 家族 GTP 酶的 GTP 结合，不太可能用于实验或治疗目的来操纵这些蛋白质的活性。这是由于 GTP 与 GTP 酶的高亲和力以及细胞内 GTP 浓度高，但也存在针对 GTP 酶中高度保守的结合位点的特异性问题。最近的一种方法表明，这些问题可以通过使用 GDP 衍生物来克服，这些衍生物可以与疾病特异性突变体发生共价反应，特别是使用在β-磷酸上带有亲电基团的 GDP 来抑制 KRas。我们在这里表明，这种方法的一个主要缺点是，与 GTP 和 GDP 相比，这种β修饰的衍生物对 Ras 的可逆亲和力至少降低了 10 倍。通过彻底的动力学特性分析，我们表明这导致了共价反应时间太慢，使得这些化合物不适合细胞内使用，但如果能够生成一种假设的具有高可逆亲和力的 GDP/GTP 类似物的反应性 GDP 衍生物，可能是一种可行的替代方法。
