Frontal affinity chromatography-mass spectrometry useful for characterization of new ligands for GPR17 receptor

Abstract

The application of frontal affinity chromatography-mass spectrometry (FAC-MS), along with molecular modeling studies, to the screening of potential drug candidates toward the recently deorphanized G-protein-coupled receptor (GPCR) GPR17 is shown. GPR17 is dually activated by uracil nucleotides and cysteinyl-leukotrienes, and is expressed in organs typically undergoing ischemic damage (i.e., brain, heart and kidney), thus representing a new pharmacological target for acute and chronic neurodegeneration. GPR17 was entrapped on an immobilized artificial membrane (IAM), and this stationary phase was used to screen a library of nucleotide derivatives by FAC-MS to select high affinity ligands. The chromatographic results have been validated with a reference functional assay ([(35)S]GTPgammaS binding assay). The receptor nucleotide-binding site was studied by setting up a column where a mutated GPR17 receptor (Arg255Ile) has been immobilized. The chromatographic behavior of the tested nucleotide derivatives together with in silico studies have been used to gain insights into the structure requirement of GPR17 ligands.

Scheme 1a:

^a Reaction conditions: (a) (i) POCI_3, (CH₃O)₃ PO;(ii) NH₄HCO₃; b) (i) n-Bu₃N, DMF; (ii) CDI, DMF;(iii) CH₃OH,DMF;(iv) (n-Bu₃NH)₂(P₂O₇H₂), DMF; (v) NH₄HCO₃; (c) (i) n-Bu₃N, DMF; (ii) CDI, DMF; (iii) CH₃OH, DMF; (iv) (n-Bu₃NH)(HPO₄), DMF; (v) NH₄HCO₃.

Figure 1.

Frontal affinity chromatography—mass spectrometry of library A (a) and library B (b). Shown are extracted breakthrough curves for each analyte of library A and library B. Mixtures of ligands in the presence of the three reference compounds each at 1 fiM were infused through the GPR17-IAM-I column using the mass spectrometer in negative mode.

Figure 2.

[³⁵S]GTPyS experiments: (a) dose—response curves of some compounds of library A in 1321N1 cells expressing wild-type GPR17; (b) antagonistic effect of 12 on UDP-glucose stimulation of wild-type GPR17 in the [³⁵S]GTPyS binding, where each point is the mean ± SEM of three independent experiments run in triplicate; (c) dose—response curves of UDP (3), 4, and 13 in 1321N1 cells expressing mutated GPR17; (d) antagonistic effect of cangrelor (1) and MRS 2179 (2) on UDP-glucose stimulation of mutated GPR17 in the [³⁵S]GTPyS binding, where each point is the mean ± SEM of three independent experiments run in triplicate.

Figure 3.

Forced unbinding profile of UDP. To elucidate the effect of a single point mutation of the arginine residue (Arg255) of GPR17, potentially involved in nucleotide binding, simulations of the forced unbinding of UDP from the wild type (WT) and the mutant (R255I) receptor models are compared. Panel a shows the pulling energy developed to unbind UDP from WT (in magenta) and from R255I (in black). The detected significant difference in energy peak intensities suggests that the mutation actually affects the binding of UDP. The distances between the couples of atoms involved in the formation of hydrogen/electrostatic bonds occurring between the ligand and either WT or R255I receptors are reported in panels b and c. In the case of WT, for almost the whole duration of the simulation, residue Arg255 holds the ligand near the pocket, thus allowing UDP to maintain interactions with other residues, among which are Tyrl85, Tyr251, and Tyr262 (b). The same interactions are loosen up at the early stages of simulation in R255I receptor (c). In panel d, some representative frames of the unbinding pathway of UDP (red sticks) from GPR17 (blue cartoon) are reported.

Figure 4.

Binding pose hypothesis of nucleotide derivatives on GPR17. The picture shows the two different possible binding modalities hypothesized for bisphosphate and triphosphate compounds on GPR17. Panels a and b show a macroscopic view of a typical diphosphate compound (MRS 2179, panel a, spheres representation) and of a triphosphate one (4, panel b, spheres representation) docked to the receptor model (cartoon representation). Panels c and d show the molecular surface of the pocket identified for MRS 2179 and 4, respectively. Two possible target arginine residues of ligand phosphate moieties are highlighted (yellow, stick representation): Arg255 for MRS 2179 phosphates and Arg87 for 4 phosphates. Regions with hydrophobic, hydrophilic, and mild polar features are reported in green, purple, and gray, respectively. Phosphates of MRS 2179 and 4 involved in arginine binding are reported in magenta and highlighted with arrows. It is evident that different arginine residues (Arg255 and Arg87) participate in ligand binding in the cases of MRS 2179 and 4, respectively (see text for more detail).