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

摘要： 在过去的二十年里，治疗性激酶抑制剂的开发取得了空前的成功 针对细胞信号级联所必需的蛋白酶。激酶面临的主要挑战 抑制剂药物治疗存在个体间变异性、药物功效受损、不可避免的耐药性， 毒性所有这些性质都强烈依赖于细胞内药物浓度，这可能是深刻的 受异质组织渗透、药物转运和溶酶体药物隔离的影响。然而，在这方面， 目前还没有技术可以定量检测激酶抑制剂的细胞内浓度， 活细胞中的药物与亚细胞空间分辨率。受激拉曼散射（SRS）显微镜是一种 一种新兴的化学成像技术，可以监测分子特定的振动特征， 分子浓度的定量、空间分辨测量。我们建议开发新的SRS- 的方法，以使非扰动，定量测定单细胞药物暴露的第一 时间第一种方法使用pH分配理论来推导基于溶酶体的细胞溶质药物浓度 弱碱性药物的药物隔离。第二种方法使用超灵敏的傅里叶变换SRS 技术和先进化学计量分析直接测定细胞溶质药物浓度。我们将使用 这些创新的方法，以确定EGFR抑制剂渗透和药物螯合在体外使用三维 肿瘤球体和体内使用背皮褶室小鼠模型。此外，我们将系统地 改变药物化合物的物理化学性质并确定它们对药物转运的影响， 隔离和渗透这个提议的第二个目标是阐明细胞反应的异质性 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遗传畸变， 还包括它们的表型状态和微环境。传统的增殖测定测量总体 细胞群体的高度异质性药物反应，并且不能在3D成像中分辨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速率测量方法 技术基于氘脉冲标记。我们将验证使用增长率变化作为准确的 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的影响，细胞内药物暴露，和细胞 微环境对细胞药物反应的影响拟议的工作建立在我们强大的专业知识，在无标签光学 成像和解决药物发现和开发的关键挑战，提供前所未有的 测量能力。所开发的技术和方法可广泛应用于小分子 药物，具有巨大的潜力，以加速早期药物发现和个性化药物筛选。

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.