Application of an Innovative Technology to Develop Low Toxicity Kinase Inhibitors

描述（由申请人提供）：激酶抑制是多种疾病的可行治疗方法，包括癌症、糖尿病和阿尔茨海默病。尽管小分子抑制剂在激酶抑制领域占据了中心地位，但毒性和耐药性出现的挑战其有效性和影响有限。人们越来越认识到需要结合多种药物来靶向多种信号通路来治疗癌症，这使得这些已经很严峻的挑战变得更加复杂。必须迅速探索开发有效的新型激酶抑制剂的每一种可能途径。干扰激酶-底物相互作用的肽有可能作为低毒性、高特异性的试剂来满足该领域未满足的关键需求。我们建议结合多种创新技术来开发和测试体内新型致癌激酶肽抑制剂。使用反向凝胶内激酶测定与新型高分辨率色谱聚焦蛋白质分离相结合，将鉴定具有最高观察到的催化效率的天然存在的蛋白激酶CK2底物。磷酸受体位点将通过电子转移解离辅助质谱法测定，蛋白质-蛋白质相互作用位点将通过化学位移微扰介导的核磁共振波谱法探测。将设计和分析底物竞争性伪底物类肽抑制剂和对接位点类肽抑制剂的体外和体内激酶抑制活性。对于体内分析，将使用一种新方法监测激酶抑制活性，以确定小鼠肠胃外给药后外周血单核细胞中 CK2 和底物的磷酸化状态。这些目标的成功完成将提供原理证明，证明现有 IMAT 资助的技术的创新应用可以促进新型肽激酶抑制剂的开发，以治疗癌症和其他以异常激酶活性为基础的疾病发生或进展的疾病。

DESCRIPTION (provided by applicant): Kinase inhibition is a viable treatment approach for multiple diseases including cancer, diabetes, and Alzheimer's. Although small molecule inhibitors have occupied center stage in the kinase inhibition arena, challenges with toxicity and emergence of resistance have limited effectiveness and impact. The growing recognition of the need to combine agents to target multiple signaling pathways to treat cancers compounds these already significant challenges. It is imperative that every possible route to developing effective new kinase inhibitors be explored with alacrity. Peptides that interfere with kinase-substrate interactions have the potential to serve as low toxicity, high specificity agents to fill a critical unmet need in this area. We propose to combine multiple innovative technologies to develop and test, in vivo, novel peptide inhibitors of an oncogenic kinase. Using the reverse in-gel kinase assay in combination with novel high-resolution chromatofocusing-based protein separation, naturally occurring Protein Kinase CK2 substrates with the highest observed catalytic efficiency will be identified. Phosphoacceptor sites will be determined by Electron Transfer Dissociation-assisted mass spectrometry, and sites of protein-protein interaction will be probed by Chemical Shift Perturbation-mediated Nuclear Magnetic Resonance spectroscopy. Substrate- competitive pseudosubstrate-class peptide inhibitors, and docking site-class peptide inhibitors will be designed and analyzed for kinase inhibitory activity in vitro and in vivo. For in vivo analyses, kinase inhibitory activity will be monitored using a novel approach to determine the phophorylation state of CK2 and substrates in peripheral blood mononuclear cells after parenteral administration in mice. The successful completion of these aims will provide proof-of-principle that the innovative application of an existing IMAT-funded technology can facilitate the development of new peptide kinase inhibitors to treat cancers and other diseases where aberrant kinase activity underlies disease initiation or progression.