Time-resolved FRET-based allostery sensors for any protein kinase drug target

适用于任何蛋白激酶药物靶标的时间分辨 FRET 变构传感器

基本信息

批准号：
9887709
负责人：
Nicholas Mark Levinson
金额：
$ 37.55万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9887709
关键词：
Active Sites Affinity Allosteric Site Antineoplastic Agents Architecture Area Benchmarking Binding Biological Assay CCNE1 gene Cancer Patient Chemicals Chemistry Clinical Complex Crowding Custom Cyclin-Dependent Kinase Inhibitor Cysteine Development Disease Dissociation Drug Screening Drug Targeting Dyes Employment Excision FGFR1 gene Failure Fluorescence Fluorescence Resonance Energy Transfer Fluorescent Probes Funding Growth High Prevalence Human Incidence Kinetics Label Legal patent Malignant Neoplasms Malignant neoplasm of prostate Mediating Mitotic Monitor Movement Neuroblastoma Neurosecretory Systems Phosphotransferases Play Procedures Protein Kinase Proto-Oncogene Proteins B-raf Publishing Reader Readiness Resistance Resolution Series Signal Transduction Site Structure Sulfhydryl Compounds System Technology Therapeutic Time Toxic effect Treatment Failure Work aurora kinase A base cancer therapy combat design drug development drug discovery high throughput screening inhibitor/antagonist kinase inhibitor nanosecond new technology next generation novel novel anticancer drug novel strategies overexpression protein complex scaffold screening screening program sensor sensor technology small molecule src-Family Kinases success tool unnatural amino acids

项目摘要

ABSTRACT The protein kinases are the top class of drug targets for the development of new cancer therapeutics. Existing kinase inhibitors, which target the highly-conserved active sites of kinases, have major limitations including poor selectivity and a high incidence of clinical resistance leading to treatment failure. New allosteric inhibitors, which bind in other pockets outside the kinase active site and trigger structural changes that block kinase activity, are far more selective and are highly effective at overriding clinical resistance to conventional kinase inhibitors. However, allosteric kinase inhibitors have proven extremely challenging to identify with existing drug screening technologies, and are only available for a small handful of kinases. A major reason for this failure of existing drug screening technologies is that they cannot detect the atomic- scale structural changes that define the mode of action of allosteric kinase inhibitors. We have developed a game-changing high-throughput screening technology, based on nanosecond time-resolved fluorescence, that can identify allosteric inhibitors by tracking with atomic resolution the structural changes they trigger in the kinase drug target. Applying this technology to the mitotic protein kinase Aurora A, we have shown that it can simultaneously track inhibitor binding affinity and allosteric effects on the kinase, can classify inhibitors into different allosteric subtypes, and is sufficiently accurate, rapid and scalable to handle high-throughput screening projects. To maximize the impact of the technology on the drug discovery pipeline, several technical barriers need to be surmounted to expand the scope of the technology beyond the current single drug target Aurora A. Our current technology is based on a chemical labeling procedure for incorporating fluorescent probes, cysteine labeling, that is not readily applicable to many important kinase drug targets due to the presence of cysteine residues important for structural integrity and catalytic function. In this proposal, we broaden the scope of the technology to make it applicable to the majority of the ~500 human kinases by developing a series of new tools for site-specific probe incorporation and by expanding the range and type of small molecules that can be identified in screening. Finally, we benchmark the suitability of the technology for real-world drug discovery efforts by performing a high-throughput screening project to identify novel allosteric inhibitors of at least one protein kinase for which no allosteric inhibitors are currently available. The success of this project will bring an entirely-new allosteric drug discovery technology into being, with unique capabilities that no existing technology can provide. Employment of this approach could jumpstart the discovery of allosteric kinase inhibitors for a large number of important cancer drug targets, broadening the range of therapeutic options for cancer patients and providing a much-needed new approach for combating the high prevalence of clinical resistance to first-line kinase inhibitor therapies.

抽象的蛋白激酶是开发新癌症治疗剂的最重要的药物靶标。现存的靶向高度保存的激酶活性位点的激酶抑制剂的主要局限性包括较差选择性和临床抗性的高发病率导致治疗失败。新的变构抑制剂，在激酶活性位点外的其他口袋中结合，并触发阻断激酶活性的结构变化，是更具选择性，并且在对传统激酶抑制剂的临床耐药性上具有非常有效的效果。但是，变构激酶抑制剂已被证明非常具有挑战性，以确定现有药物筛查技术，仅适用于少数激酶。现有药物筛查技术失败的主要原因是它们无法检测到原子尺度结构变化，定义了变构激酶抑制剂的作用方式。我们已经开发了基于纳米秒时间分辨的荧光，改变游戏规则的高通量筛查技术，可以通过使用原子分辨率跟踪它们在激酶中触发的结构变化来识别变构抑制剂药物目标。将该技术应用于有丝分裂蛋白激酶Aurora A，我们已经证明它可以同时跟踪抑制剂结合亲和力和对激酶的变构影响，可以将抑制剂分类为不同的变构亚型，并且足够准确，快速且可扩展以处理高通量筛选项目。为了最大化技术对药物发现管道的影响，几个技术障碍需要克服以扩大技术的范围，而不是当前的单一药物目标极光A。我们当前的技术基于用于结合荧光探针的化学标记程序，半胱氨酸标记，由于存在，这不容易适用于许多重要的激酶药物靶标半胱氨酸残基对结构完整性和催化功能很重要。在此提案中，我们扩大了范围通过开发一系列新的新型人类激酶，使其适用于〜500个人类激酶的技术用于特定地点的探针掺入的工具，并通过扩展可以是的小分子的范围和类型在筛选中确定。最后，我们基准了该技术对现实药物发现工作的适用性通过执行高通量筛选项目以鉴定至少一种蛋白质的新型变构抑制剂目前没有变构抑制剂的激酶。该项目的成功将使完全新的变构毒物发现技术与现有技术无法提供的独特功能。这种方法的使用可能会开始发现大量重要癌症靶标的变构激酶抑制剂，扩大范围癌症患者的治疗选择，并提供了一种急需的新方法来对抗高对一线激酶抑制剂疗法的临床抗性患病率。