Winter Jon J G, Anderson Malcolm, Blades Kevin, Brassington Claire, Breeze Alexander L, Chresta Christine, Embrey Kevin, Fairley Gary, Faulder Paul, Finlay M Raymond V, Kettle Jason G, Nowak Thorsten, Overman Ross, Patel S Joe, Perkins Paula, Spadola Loredana, Tart Jonathan, Tucker Julie A, Wrigley Gail
AstraZeneca , Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom.
J Med Chem. 2015 Mar 12;58(5):2265-74. doi: 10.1021/jm501660t. Epub 2015 Feb 26.
Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.
组成型激活突变体KRas通过内在机制和GTP酶激活蛋白催化机制显示出降低的GTP水解速率，导致Ras通路的持续激活。我们描述了一项使用X射线晶体学的片段筛选活动，该活动导致在Ras:SOS复合物上发现了三个片段结合位点。在这些位点上结合的工具化合物的鉴定使得能够探索两种新的Ras通路抑制方法，即通过稳定或共价修饰Ras:SOS复合物来防止Ras重新加载GTP。最初，我们鉴定了在两个不同位点与Ras:SOS复合物可逆结合的配体，但这些化合物作为抑制剂的效力不足，无法验证我们的稳定化假设。我们通过证明对Ras上的Cys118进行共价修饰导致一种抑制Ras与Raf之间SOS介导相互作用的新机制，并有效抑制突变型（G12C和G12V）和野生型Ras中标记GDP的交换来得出结论。
