Close David A, Journigan V Blair, Johnston Paul A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA; Computational Chemical Genomics Screening Center, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15260, USA.
SLAS Discov. 2025 Jan;30:100207. doi: 10.1016/j.slasd.2024.100207. Epub 2024 Dec 31.
Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca mobilization kinetic data to plot curves and calculate EC and IC values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC and IC ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC or IC values required to distinguish compound potencies. EC and IC values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC and IC values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.
本文描述了针对瞬时受体电位（TRP）通道TRPM8、TRPV1和TRPA1的人源化钙动员检测方法的开发、优化和校准。抗TRPM8抗体染色和TRPM8激动剂诱导的钙动员信号均需要在HEK293T细胞中进行hTRPM8的异源表达，二者均可用于优化转染效率。优化了FLIPR钙6染料浓度、加载时间和TRPM8转染细胞的接种密度，并设定了≤0.2%的二甲基亚砜耐受性。尽管相对于最大激动剂诱导的反应，这些差异较小，但TRPM8钙动员检测的静息基线相对荧光单位（RFU）信号在孔间仍表现出显著差异。从钙动员动力学数据中提取最大RFU、累积RFU总和或曲线下面积值，以绘制曲线并计算EC和IC值。基线倍数（FOB）比值数据处理消除了静息基线信号的孔间差异，减小了误差条，改善了曲线拟合，并缩小了95%置信区间的EC和IC范围。FOB比值数据处理降低了变异性，提高了单次实验中重复测量的精度，从而降低了区分化合物效力所需的EC或IC值的最小阈值差异。单次实验中测定的TRPM8激动剂和拮抗剂的EC和IC值与多个独立实验的结果高度一致。TRPM8、TRPV1和TRPA1的基准EC和IC值在先前报道的激动剂和拮抗剂标准范围内。FOB比值数据处理提高了TRP钙动员检测的精度和准确性，增强了其在研究构效关系方面的实用性。
