Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors

Abstract

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.

Keywords: Binding affinity determination; Binding thermodynamics; Chemical denaturation; GPCR.

Figure 1

Left Panel: Fluorescence emission spectra of A2a-8M using an excitation wavelength of 280 nm. A2a-8M spectra were obtained in the absence (squares) and in the presence (circles) of 5.5 M GuHCl. There is an almost five-fold decrease in intensity at the highest GuHCl concentration. Right Panel: Fluorescence emission of A2a-8M at 338 nm using an excitation wavelength of 280 nm from 0 to 5.5M GuHCl increased as a linear gradient (squares). The red and green solid lines represent the native and denatured states baselines. The solid line is the calculated curve with the best parameters from non-linear least squares fit for a two state transition model (ΔG = 3.96 kcal/mol and m = 1.37 kcal/mol×M).

Figure 2

GuHCl induced denaturation of A2A-8M in the absence (squares) and presence of 2 μM ZM241385 (circles). In all cases the protein concentration was 0.167 μM. Denaturation curves were obtained by measuring the fluorescence intensity at 338 nm. In this figure, the normalized curves have been plotted as a function of the concentration of denaturant. A significant increase in protein stability is observed in the presence of the ligand as evidenced by the shift in C_1/2 to higher denaturant concentrations. Analysis of the data yielded a K_d of 2.0nM in perfect agreement with the one obtained by SPR. All the thermodynamic data is summarized in Table 1.

Figure 3

Representative results from surface plasmon resonance experiments. A2a-8M was captured on a NTA chip via its His tag and subsequently amine coupled to the chip surface.Ligand binding to A2a-8M is shown for ZM241385 at concentrations of 111, 37 12.3, 4.1, 1.4 nM, respectively. On and off rates were used to calculate the K_d (1.8 nM). All the SPR data is summarized in Table 1.

Figure 4

Correlation between K_d values (nM units) determined by chemical denaturation and surface plasmon resonance. The coefficient for the logarithmic plot was obtained by linear least squares forcing the intercept to zero. If the intercept is allowed to float it is equal to 0.25 and the slope 0.87. Ligands in the graph are SCH442416, TC-G1004, ZM241385, SCH58261, PSB1115, Istradefyllin, LUF5834.