Non-perturbative imaging of intracellular drug exposure and drug response of kinase inhibitors

激酶抑制剂细胞内药物暴露和药物反应的非微扰成像

• 批准号: 10391453
• 负责人: Dan Fu
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391453
• 关键词: 3-Dimensional; Address; Biological Assay; Cells; Chemicals; Data; Deuterium; Disease; Dorsal; Drug Compounding; Drug Exposure; Drug Kinetics; Drug Screening; Drug Transport; Drug resistance; Environment; Enzymes; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Fourier Transform; Genetic; Goals; Growth; Heterogeneity; Image; Imaging Techniques; In Vitro; Label; Malignant Neoplasms; Measurement; Measures; Methods; Microscopy; Modeling; Molecular; Monitor; Penetration; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physiologic pulse; Play; Population; Property; Proteins; Resolution; Role; Signal Transduction; Techniques; Technology; Time; Tissues; Toxic effect; Work; base; cancer cell; cell growth; drug development; drug discovery; drug efficacy; drug response prediction; improved; in vivo; inhibitor therapy; innovation; innovative technologies; inter-individual variation; kinase inhibitor; mouse model; novel; optical imaging; precision drugs; public health relevance; rate of change; response; small molecule; success; theories; therapeutic development; tumor; vibration

Abstract: The past two decades have witnessed unparalleled success in the development of therapeutic kinase inhibitors targeting protein enzymes that are essential for cellular signaling cascades. The major challenges in kinase inhibitor drug treatment are interindividual variability, compromised drug efficacy, inevitable drug resistance, and toxicity. All of these properties depend strongly on intracellular drug concentration, which can be profoundly influenced by heterogeneous tissue penetration, drug transport, and lysosomal drug sequestration. However, currently there is no technology that can quantitatively examine intracellular concentration of kinase inhibitor drugs in living cells with subcellular spatial resolution. Stimulated Raman scattering (SRS) microscopy is an emerging chemical imaging technique that monitors molecule-specific vibrational signatures to provide quantitative, spatially resolved measurements of molecular concentration. We propose to develop novel SRS- based methods to enable non-perturbative, quantitative determination of single cell drug exposure for the first time. The first method uses the pH partition theory to derive cytosolic drug concentration based on lysosomal drug sequestration of weakly basic drugs. The second method uses an ultrasensitive Fourier-transform SRS technique and advanced chemometric analysis to directly determine cytosolic drug concentration. We will use these innovative methods to determine EGFR inhibitor penetration and drug sequestration in vitro using 3D tumor spheroids and in vivo using the dorsal skinfold chamber mouse model. In addition, we will systematically vary the physicochemical properties of drug compounds and determine their influence on drug transport, sequestration, and penetration. The second goal of this proposal is to elucidate the heterogeneity of cell response to kinase inhibitor drug treatment. Drug response of cancer cells depend on not only their genetic aberrations, but also their phenotypic states and microenvironments. Traditional proliferation assays measure the ensemble response of a cell population and are unable to resolve the highly heterogeneous drug response of cells in a 3D environment. We propose to develop a quantitative, high sensitivity single-cell growth-rate measurement technique based on deuterium pulse labeling. We will validate the use of growth rate change as an accurate predictor of drug response. By combining single cell drug exposure and drug response measurements in 3D tumor spheroids, we will further dissect the influence of drug penetration, intracellular drug exposure, and cell microenvironment on cell drug response. The proposed work builds on our strong expertise in label-free optical imaging and addresses key challenges in drug discovery and development by providing unprecedented measurement capabilities. The technologies and methods developed can be broadly applied to small molecule drugs, with great potentials to accelerate early stage drug discovery and empower personalized drug screening.

抽象的： 过去二十年见证了治疗性激酶抑制剂的开发取得了无与伦比的成功 靶向对细胞信号级联至关重要的蛋白质酶。激酶的主要挑战 抑制剂药物治疗存在个体差异、药物疗效受损、不可避免的耐药性和 毒性。所有这些特性都强烈依赖于细胞内药物浓度，这可以深刻地影响细胞内药物浓度。 受异质组织渗透、药物转运和溶酶体药物封存的影响。然而， 目前还没有技术可以定量检测细胞内激酶抑制剂的浓度 活细胞中的药物具有亚细胞空间分辨率。受激拉曼散射 (SRS) 显微镜是一种 新兴的化学成像技术，可监测分子特定的振动特征，以提供 分子浓度的定量、空间分辨测量。我们建议开发新颖的SRS- 基于方法首次实现单细胞药物暴露的非扰动定量测定 时间。第一种方法利用 pH 分配理论根据溶酶体浓度推导出胞浆药物浓度。 弱碱性药物的药物隔离。第二种方法使用超灵敏傅立叶变换 SRS 技术和先进的化学计量分析直接测定胞质药物浓度。我们将使用 这些创新方法使用 3D 确定 EGFR 抑制剂体外渗透和药物隔离 肿瘤球体和体内使用背侧皮褶室小鼠模型。此外，我们还将系统地 改变药物化合物的理化性质并确定它们对药物转运的影响， 隔离和渗透。该提案的第二个目标是阐明细胞反应的异质性 以激酶抑制剂药物治疗。癌细胞的药物反应不仅取决于它们的基因畸变， 还有它们的表型状态和微环境。传统的增殖测定测量整体 细胞群的反应，无法解析 3D 细胞的高度异质药物反应 环境。我们建议开发一种定量、高灵敏度的单细胞生长速率测量方法 基于氘脉冲标记的技术。我们将验证增长率变化的使用是否准确 药物反应的预测因子。通过在 3D 中结合单细胞药物暴露和药物反应测量 对于肿瘤球体，我们将进一步剖析药物渗透、细胞内药物暴露和细胞的影响 微环境对细胞药物反应的影响。拟议的工作建立在我们在无标签光学方面强大的专业知识之上 成像并通过提供前所未有的技术解决药物发现和开发中的关键挑战 测量能力。所开发的技术和方法可广泛应用于小分子 药物，具有加速早期药物发现和个性化药物筛选的巨大潜力。