Institute of Biomedicine, University of Turku , Kiinamyllynkatu 10 C, FI-20520 Turku, Finland.
Institute for Molecular Medicine Finland, University of Helsinki , Tukholmankatu 8, FI-00290 Helsinki, Finland.
Anal Chem. 2017 Apr 18;89(8):4508-4516. doi: 10.1021/acs.analchem.6b04904. Epub 2017 Mar 29.
Ras GTPases act as "molecular switches", alternating between inactive GDP-bound and active GTP-bound conformation. Ras-oncogenes were discovered over three decades ago, but there are still no effective therapies for Ras-driven cancers. So far, drug discovery strategies have been unsuccessful, because of a lack of suitable screening methodologies and well-defined binding pockets on the Ras proteins. Here, we addressed the former by introducing a homogeneous quenching resonance energy transfer (QRET) technique-based screening strategy for Ras interfacial and competitive inhibitors. We demonstrate that using a unique GTP-specific antibody fragment to monitor GTPase cycling in the presence of a guanine nucleotide exchange factor (GEF) and a GTPase activating protein (GAP) is an efficient method for Ras inhibitor high-throughput screening. When compared to a conventional GEF-stimulated nucleotide exchange assay in a proof-of-concept screen, we identified an overlapping set of potential inhibitor compounds but also compounds found exclusively with the new GTP hydrolysis monitoring-based GTPase cycling assay.
Ras GTPases 充当“分子开关”，在非活性 GDP 结合构象和活性 GTP 结合构象之间交替。Ras 癌基因在三十多年前被发现，但目前仍然没有针对 Ras 驱动的癌症的有效治疗方法。到目前为止，由于缺乏合适的筛选方法和 Ras 蛋白上定义明确的结合口袋，药物发现策略一直没有成功。在这里，我们通过引入一种基于均相荧光猝灭共振能量转移 (QRET) 技术的 Ras 界面和竞争性抑制剂筛选策略来解决前者。我们证明，在鸟嘌呤核苷酸交换因子 (GEF) 和 GTP 酶激活蛋白 (GAP) 的存在下，使用独特的 GTP 特异性抗体片段来监测 GTPase 循环，是一种有效的 Ras 抑制剂高通量筛选方法。与传统的 GEF 刺激核苷酸交换测定法在概念验证筛选中的应用相比，我们确定了一组潜在的抑制剂化合物的重叠，但也发现了仅用新的基于 GTP 水解监测的 GTPase 循环测定法发现的化合物。
