单细胞成像:临床前药物筛选

        表面等离激元共振显微镜能够获取免标记单细胞的实时原位质量分布图像,从而可以对细胞迁移、收缩和细胞表面的分子相互作用进行研究以及对病毒颗粒,细菌,细胞器等对象的观察研究 。单细胞成像对药物研究、细胞生命活动的研究以及疾病监测具有重要意义。

        测定单抗药物分子与受体的结合动力学常数是临床前药物预筛选,以及生物仿制药评价的重要参考数据之一。目前主流的结合动力学常数方法(Binding Kinetics Assay)利用表面等离子共振技术,测定单抗药物与纯化受体蛋白的结合和解离动力学常数,进行相互作用评价。这种基于纯化蛋白的离体分析方法忽略了细胞表面真实生物化学环境对药物—受体结合行为的巨大影响,容易引入假阳性误差。

        我们近年来发展了一种基于完整细胞测定单抗药物与细胞表面受体分子的原位结合动力学常数的新技术。与传统SPR技术相比,新技术无需分离和纯化药物受体蛋白,直接在完整细胞表面获取药物—受体结合动力学常数,所得数据更能反映药物作用的实际情况,有助于更准确的药物筛选和仿制药评价。与基于荧光的各种结合实验相比,新技术无需分子标记,而且能够获取动力学数据,尤其是解离速率常数这一药效评价的重要参数。这种基于完整细胞的结合动力学常数分析方法兼具Binding assay和Cell-based assay两类技术的优点,可以通过细胞表面的原位结合动力学数据快速、准确地评价药物分子与受体的结合行为。所获取的数据具有明确生物学意义,稳定可靠,易于解读。另外,此方法也可用于纳米药物的作用机制研究和药效评价。

     

        图1. 看似静止的单细胞在SPR显微镜下呈现出丰富的微观运动特征               图2. 外部渗透压改变时单细胞收缩行为具有显著的各向异性
                                       (unpublished data)                                                                               (Wang et al., Langmuir, 2012, 28, 13373)

                           图3. 单个神经元中两个互相追逐的线粒体                               图4. 单细胞电穿孔过程中伴随着电化学阻抗分布的时空演化
                              (Yang et al., Small, 2015, 11, 2878)                                                         (Wang et al., Nature Chemistry, 2011, 3, 249)

代表性文章:

[1] Yu-Wen Su, Wei Wang, Surface plasmon resonance sensing: from purified biomolecules to intact cells, Anal. Bioanal. Chem., 2018, in press.

[2] Wei Wang, Linliang Yin, Laura Gonzalez, Shaopeng Wang, Xiaobo Yu, Seron Eaton, Shengtao Zhang, Hong-Yuan Chen*, Joshua LaBaer*, Nongjian Tao*, In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance. Scientific Reports, 2014, 4, 6609.

[3] Wei Wang, Yunze Yang, Shaopeng Wang, Vinay J Nagaraj, Qiang Liu, Jie Wu and Nongjian Tao*, Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells, Nature Chemistry, 2012, 4, 846-853.

[4] Wei Wang, Kyle Foley, Xiaonan Shan, Shaopeng Wang, Seron Eaton, Vinay J. Nagaraj, Peter Wiktor, Urmez Patel, Nongjian Tao*, Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy, Nature Chemistry, 2011, 3, 249-255.

 

Measuring drug-receptor binding kinetics in intact cells:
A promising cell-based binding assay for drug screening and evaluation

 

    People in molecular medicine generally believe in a maxim proposed by Paul Ehrlich in 1913: corpora non agunt nisi fixata (“drugs will not work unless they are bound”). The drug effects always start from the initial binding of drugs with receptors.Since then, the binding kinetics and affinity between drug candidates and their targets (receptors) have become one of the most important criteria during the preclinical drug screening.Up to now, purified protein-based methods remain the main stream in the drug screening, which usually require the extraction and purification of drug receptors, following by the immobilization of them on a sensing surface, on which the drugs would be introduced to measure the interactionsin vitro. However, it has been well understood that chemical environments play critical roles in the molecular interactions.The elimination of native biological and chemical environments in purified protein-based binding kinetics assay thus likely introduced false-positive and false-negative results in the pre-clinical drug screening.

    We recently developed an intact-cell based binding kinetics assay that is capable of measuring in situ binding kinetics between monoclonal antibody drugs and its receptors in intact native cells. By taking Trastuzumab (Herceptin) and its target, Her2 receptor, as an example, we observed great variations in the Trastuzumab-Her2 binding kinetics in different cell lines. For example, the dissociation rate constant (koff) of Trastuzumab on BT-474 cells is three times faster than that on SK-BR3 cells. Furthermore, we discovered that the binding kinetics of Trastuzumab on Herceptin-resistant breast cancer cells was greatly weakened by comparing tothat on Herceptin-sensitive cells, suggesting new molecular insights into Herceptin-resistance mechanism.

    The proposed intact-cell based binding kinetics assay is highly compatible with the present binding kinetics assay using surface plasmon resonance (SPR), with significant improvements in the sample preparation, experimental procedures and data analysis. We believe the intact-cell based binding kineticsassay would exhibit better performance than the present assayin pre-clinical drug screening, evaluation on bio-similar monoclonal antibody drugs, and studying drug resistance mechanism.

References:

[1] Yu-Wen Su, Wei Wang, Surface plasmon resonance sensing: from purified biomolecules to intact cells, Anal. Bioanal. Chem., 2018, in press.

[2] Wei Wang, Linliang Yin, Laura Gonzalez, Shaopeng Wang, Xiaobo Yu, Seron Eaton, Shengtao Zhang, Hong-Yuan Chen*, Joshua LaBaer*, Nongjian Tao*, In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance. Scientific Reports, 2014, 4, 6609.

[3] Wei Wang, Yunze Yang, Shaopeng Wang, Vinay J Nagaraj, Qiang Liu, Jie Wu and Nongjian Tao*, Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells, Nature Chemistry, 2012, 4, 846-853.

[4] Wei Wang, Kyle Foley, Xiaonan Shan, Shaopeng Wang, Seron Eaton, Vinay J. Nagaraj, Peter Wiktor, Urmez Patel, Nongjian Tao*, Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy, Nature Chemistry, 2011, 3, 249-255.

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