Guanosine triphosphatase stimulation of oncogenic Ras mutants
Guanosine triphosphatase stimulation of oncogenic Ras mutants
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DOI：
10.1073/pnas.96.12.7065
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发表时间：
1999-06-08
影响因子：
11.1
通讯作者：
Scheffzek, K
中科院分区：
文献类型：
--
作者：
Ahmadian, MR;Zor, T;Scheffzek, K
对Ras作为分子药物靶标的鸟苷三磷酸酶（GTdR）反应的兴趣源于以下观察：在大量人类肿瘤中，Ras在密码子12或61处特征性地突变，更罕见地在密码子13处突变。已发现即使在GTdR活化蛋白存在下，GTdR活性受损是大多数Gly 12/Gln 61突变的致癌性背后的生化原因，从而防止Ras被关断。因此，这些致癌Ras突变体保持组成性激活，并有助于肿瘤细胞的肿瘤表型。在这里，我们表明，鸟苷5 '-三磷酸（CTP）类似物二氨基二苯甲酮-氨基磷酸酯-GTP（DABP-GTP）被野生型Ras水解，但更有效地通过频繁发生的致癌Ras突变体，产生鸟苷5'-二磷酸结合的失活Ras和DABP-Pi+。该反应是独立的Gln 61的存在下，是最显着的Gly 12突变体增强。因此，通过使用DABP-GTP代替GTP，可以挽救致癌Ras突变体的缺陷性GTdR反应，认为Ras的GTdR开关没有不可逆地受损。DABP-GTP的环外芳族氨基对于反应是关键的，并且绕过了固有Ras GTP酶反应的假定限速步骤。Ras结合的DABP-β，γ-亚氨基-GTP的晶体结构显示了一个无序的开关I，并确定Gly 12/Gly 13区域作为容纳DABP-部分的疏水补丁。生化和结构研究有助于定义针对阻断GTP酶反应的抗Ras药物设计的要求。
Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13, Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (CTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wildtype Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi+ The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety, The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.