Analyzing kinetic signaling data for G-protein-coupled receptors
Analyzing kinetic signaling data for G-protein-coupled receptors
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DOI：
10.1038/s41598-020-67844-3
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发表时间：
2020-07-23
影响因子：
4.6
通讯作者：
Bridge, Lloyd J.
中科院分区：
文献类型：
--
作者：
Hoare, Sam R. J.;Tewson, Paul H.;Bridge, Lloyd J.
在经典药理学中，生物测定数据符合一般方程(如剂量反应方程)来确定经验药物参数(如EC50和E-max)，然后用于计算亲和力和疗效等化学参数。在这里，我们对动态、时间进程信号数据使用了类似的方法，以允许以动力学术语对G蛋白偶联受体的信号进行经验和化学定义。在常用的曲线拟合程序GraphPad Prism中，使用通用的时间历程方程(无模型方法)和力学模型方程(机械方法)对实验数据进行分析。文献调查表明，信号时程数据通常符合四种曲线形式之一：直线、联想指数曲线、起伏至零曲线和起伏至稳态曲线。在无模型方法中，信号的初始速率被量化，这是通过对整个时间过程进行曲线拟合来完成的，从而避免了选择曲线的线性部分的需要。结果表明，这四种形状符合以酶动力学为基础的信号机制模型，其形状由信号机制的调节(如受体脱敏、信号降解)决定。信号效率是由激动剂占据的受体(k(Tau))发出信号的初始速率，简单地说就是信号在受到调节机制影响之前的生成速率，可以使用无模型分析来测量。如果机制已知，可以估计信号参数的调节，如受体脱敏速率常数。这项研究将经典药理学中使用的经验和机制方法扩展到动力学信号数据，促进了新疗法在动力学方面的优化。
In classical pharmacology, bioassay data are fit to general equations (e.g. the dose response equation) to determine empirical drug parameters (e.g. EC50 and E-max), which are then used to calculate chemical parameters such as affinity and efficacy. Here we used a similar approach for kinetic, time course signaling data, to allow empirical and chemical definition of signaling by G-protein-coupled receptors in kinetic terms. Experimental data are analyzed using general time course equations (model-free approach) and mechanistic model equations (mechanistic approach) in the commonly-used curve-fitting program, GraphPad Prism. A literature survey indicated signaling time course data usually conform to one of four curve shapes: the straight line, association exponential curve, rise-and-fall to zero curve, and rise-and-fall to steady-state curve. In the model-free approach, the initial rate of signaling is quantified and this is done by curve-fitting to the whole time course, avoiding the need to select the linear part of the curve. It is shown that the four shapes are consistent with a mechanistic model of signaling, based on enzyme kinetics, with the shape defined by the regulation of signaling mechanisms (e.g. receptor desensitization, signal degradation). Signaling efficacy is the initial rate of signaling by agonist-occupied receptor (k(tau)), simply the rate of signal generation before it becomes affected by regulation mechanisms, measurable using the model-free analysis. Regulation of signaling parameters such as the receptor desensitization rate constant can be estimated if the mechanism is known. This study extends the empirical and mechanistic approach used in classical pharmacology to kinetic signaling data, facilitating optimization of new therapeutics in kinetic terms.