Abstract
Most drugs work by binding to receptors on the cell surface. Quantification of binding kinetics between drug and membrane protein is an essential step in drug discovery. Current methods for measuring binding kinetics involve extracting the membrane protein and labeling, and both have issues. Surface plasmon resonance (SPR) imaging has been demonstrated for quantification of protein binding to cells with single-cell resolution, but it only senses the bottom of the cell and the signal diminishes with the molecule size. We have discovered that ligand binding to the cell surface is accompanied by a small cell membrane deformation, which can be used to measure the binding kinetics by tracking the cell edge deformation. Here, we report the first integration of SPR imaging and cell edge tracking methods in a single device, and we use lectin interaction as a model system to demonstrate the capability of the device. The integration enables the simultaneous collection of complementary information provided by both methods. Edge tracking provides the advantage of small molecule binding detection capability, while the SPR signal scales with the ligand mass and can quantify membrane protein density. The kinetic constants from the two methods were cross-validated and found to be in agreement at the single-cell level. The variation of observed rate constant between the two methods is about 0.009 s−1, which is about the same level as the cell-to-cell variations. This result confirms that both methods can be used to measure whole-cell binding kinetics, and the integration improves the reliability and capability of the measurement.
Highlights
Receptor–ligand reactions are central to much of the field of pharmacology, and more than 60% of drugs on the market target at cell membrane proteins [1]
We have developed an edge tracking method that detects nanometer-scale changes in cell shape accompanied by the membrane protein binding events for label-free kinetic quantification that is capable of measuring small molecule binding kinetics to membrane receptors on the whole-cell surface [8,9]
We report the integration of the edge tracking method to a dual-mode surface plasmon resonance (SPR) imaging system for simultaneous measurement of the kinetics of ligand–receptor binding on whole cells with both edge tracking and SPR
Summary
Receptor–ligand reactions are central to much of the field of pharmacology, and more than 60% of drugs on the market target at cell membrane proteins [1]. There are several major challenges when studying membrane protein binding kinetics. Extraction is a tedious, laborious task of physically separating the membrane protein from its membrane environment [2]. The conformation of the membrane protein may change due to the detergents [3,4]. These issues plague the study of membrane protein outside the membrane. Fluorescence labeling is commonly used, but it is not feasible for small molecules, which poses a problem, to pharmacology, since nearly 90%
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