Screening with D. melanogaster to Discover Inhibitors G12V Mutated kRAS

描述(由申请人提供)：体细胞KRAS突变，称为致癌KRAS，在许多癌症中被发现的比率很高。例如，在结肠癌中，这一比例为40%。突变的KRAS允许EGF系统绕过其自然控制而持续运行，锁定在促生长模式(野生型KRAS，但不是致癌性KRAS，自我失活)。与这一机制一致，突变的KRAS的存在预示着结直肠癌患者对抗EGF单抗治疗缺乏反应，如Panitumumab(Vectibix)和西妥昔单抗(Erbitux)。这项资助的目标是发现在不影响正常KRAS的情况下，专门抑制致癌KRAS的化合物。当这些分子用于致癌的KRAS患者时，有望恢复抗EGF疗法的抗癌效果，如Panitumumab和西妥昔单抗。不幸的是，之前许多针对致癌KRAS的努力都失败了，KRAS已被许多癌症研究人员宣布为“难对付的”靶点。然而，我们最近开发了一种独特的筛选模型，使用转基因的黑腹果蝇，在它们的翅膀上表达突变的致癌KRAS(G12V)，同时在它们身体的其余部分保持正常的KRAS。我们计划利用这种苍蝇作为筛选平台，寻找在不影响野生型KRAS的情况下抑制突变KRAS的药物。突变果蝇的表型是一只“卷曲的”翅膀，而在检测中的“击中”将是一种防止形成卷曲的翅膀缺陷的化合物。苍蝇的使用允许在整个动物中进行筛选，这有望产生比细胞培养或生化分析中的筛选更可靠的“命中”。此外，整个动物模型允许对安全性进行初步评估，因为只有那些允许正常、健康的苍蝇发育，同时阻止它们翅膀上的突变KRAS的化合物才会被选为“Hits”。对苍蝇有毒性的化合物不会通过筛查。由赠款资助的活动包括进一步确定转基因果蝇和化验的特征，建立区分致癌类型和野生型KRAS抑制剂的分析方法，筛选潜在的抑制剂，“HITS”的结构优化，以及在PK、安全性和KRAS相关的异种移植研究中优化“HITS”的特征。

DESCRIPTION (provided by applicant): Somatic kRAS mutations, termed oncogenic kRas, are found at a high rate in many cancers. For example, in colon cancer, the rate is 40%. Mutated KRAS allows the EGF system to bypass its natural controls and operate continuously, locked in a pro-growth mode (wild-type kRas, but not oncogenic KRAS, is self- inactivating). Consistent with this mechanism, the presence of mutated KRas predicts lack of response to anti- EGF monoclonal antibody therapeutics such as panitumumab (Vectibix) and cetuximab (Erbitux) in colorectal cancer. The goal of this grant is to discover compounds that specifically inhibit oncogenic kRas without affecting normal kRAS. Such molecules would be expected to restore the anti-cancer efficacy of anti-EGF therapeutics such as panitumumab and cetuximab when administered to patients with oncogenic kRas. Unfortunately, many previous efforts to target oncogenic KRas have failed, and kRAS has been declared an "intractable" target by many cancer researchers. However, we recently developed a unique screening model using a genetically modified D. melanogaster that expresses mutant, oncogenic kRAS (G12V) in their wings while maintaining normal kRAS in the rest of their bodies. We plan to use this fly as a screening platform to search for drugs that inhibit mutant kRAS while not affecting wild-type kRAS. The phenotype for the mutated fly is a "crimped" wing, and a "hit" in the assay would be a compound that prevents formation of the crimped wing defect. The use of the fly permits screening in a whole animal, which is expected to produce more reliable "hits" than screening in cell culture or in biochemical assays. In addition, the whole animal model permits a preliminary evaluation of safety, because only compounds that permit development of normal, healthy flies while blocking the mutant kRAS in their wings will be selected as "hits". Compounds toxic in flies will not pass the screen. Activities to be funded by the grant include further characterizing the transgenic fly and the assay, establishing an assay to differentiate inhibitors of oncogenic and wild-type kRAS, screening for potential inhibitors, structural optimization of "hits", and characterization of optimized "hits" in PK, safety and kRAS-relevant xenograft studies.