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

摘要： 在过去的二十年里，我们见证了治疗用的激酶抑制剂的发展取得了前所未有的成功。 靶向对细胞信号级联至关重要的蛋白质酶。蛋白水解酶的主要挑战 抑制药物治疗是个体间的差异、药物疗效受损、不可避免的耐药性，以及 毒性。所有这些特性都强烈地依赖于细胞内药物浓度，而细胞内药物浓度对细胞内药物浓度有很大影响。 受异质组织渗透、药物转运和溶酶体药物隔离的影响。然而， 目前还没有一种技术可以定量检测细胞内的激酶抑制物浓度 活细胞中具有亚细胞空间分辨率的药物。受激拉曼散射(SRS)显微镜是一种 新兴的化学成像技术，监测分子特定的振动特征，以提供 分子浓度的定量、空间分辨率测量。我们建议开发新型的受激拉曼散射- 首次实现单细胞药物暴露的非微扰、定量测定的方法 时间到了。第一种方法基于溶酶体，利用pH分配理论推导出细胞内药物浓度 弱碱性药物的药物封存。第二种方法使用超灵敏的傅里叶变换SRS 技术和先进的化学计量学分析，直接测定胞浆药物浓度。我们将使用 这些创新的方法使用3D确定EGFR抑制剂的体外渗透和药物封存 肿瘤球体和活体采用背侧皮皱室小鼠模型。此外，我们还将系统地 改变药物化合物的物理化学性质并确定其对药物传输的影响， 隔离和渗透。这项建议的第二个目标是阐明细胞反应的异质性 到激酶抑制剂的药物治疗。癌细胞的药物反应不仅取决于它们的遗传变异， 还包括它们的表型状态和微环境。传统的细胞增殖检测方法衡量的是整体 细胞群体的反应，并且不能在3D中解析细胞的高度异质性药物反应 环境。我们建议开发一种定量的、高灵敏度的单细胞生长率测量方法。 基于氚脉冲标记的技术。我们将验证使用增长率变化作为一种准确的 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 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 微环境对细胞药物反应的影响。建议的工作建立在我们在无标签光学方面的强大专业知识基础上 成像和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实现个性化药物筛选的巨大潜力。

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.