Development of Spexin-based Human Galanin Receptor Type II-Specific Agonists with Increased Stability in Serum and Anxiolytic Effect in Mice

Abstract

The novel neuropeptide spexin (SPX) was discovered to activate galanin receptor 2 (GALR2) and 3 (GALR3) but not galanin receptor 1 (GALR1). Although GALR2 is known to display a function, particularly in anxiety, depression, and appetite regulation, the further determination of its function would benefit from a more stable and selective agonist that acts only at GALR2. In the present study, we developed a GALR2-specific agonist with increased stability in serum. As galanin (GAL) showed a low affinity to GALR3, the residues in SPX were replaced with those in GAL, revealing that particular mutations such as Gln5 → Asn, Met7 → Ala, Lys11 → Phe, and Ala13 → Pro significantly decreased potencies toward GALR3 but not toward GALR2. Quadruple (Qu) mutation of these residues still retained potency to GALR2 but totally abolished the potency to both GALR3 and GALR1. The first amino acid modifications or D-Asn1 substitution significantly increased the stability when they are incubated in 100% fetal bovine serum. Intracerebroventricular administration of the mutant peptide with D-Asn1 and quadruple substitution (dN1-Qu) exhibited an anxiolytic effect in mice. Taken together, the GALR2-specific agonist with increased stability can greatly help delineation of GALR2-mediated functions and be very useful for treatments of anxiety disorder.

Figure 1. Sequence alignment of human (Hu) GAL, GALP, SPX1, and coelacanth (Co) SPX2.

Conserved residues in the peptides are indicated in yellow. SPX-specific residues are in green, while GAL-specific residues are highlighted in violet. The residues in human SPX1 were changed as indicated.

Figure 2. Differential effects of GAL-specific residue substitution in SPX on potencies toward GALR1, GALR2, and GALR3.

The SRE-luc reporter vector and plasmid containing human GALR1, GALR2, or GALR3 were transfected into HEK293 cells that stably expressed Gq_i protein. Cells were then treated with increasing concentrations of mutant peptides, and the receptor activities were determined using the SRE-luc reporter system. (A) Potencies of mutant peptides toward GALR2 (red bars) and GALR3 (blue bars) are expressed as −log[EC₅₀] values. The horizontal dashed lines represent potency of WT SPX toward receptors. (B–D) Dose-response activity fold induction by peptides with multiple substitutions on GALR1 (B), GALR2 (C), and GALR3 (D).WT SPX and GAL were used as control peptides. a, P < 0.05 vs. WT SPX; b, P < 0.05 vs. activity with GALR2.

Figure 3

Effect of single D-amino acid substitution (A) and Asn1 replacement/modification (B) on the potency toward GALR2 and GALR3. The SRE-luc reporter vector and plasmid containing human GALR2 or GALR3 were transfected into HEK293 cells that stably expressed Gq_i protein. Cells were then treated with increasing concentrations of mutant peptides, and the receptor activities were determined using the SRE-luc reporter system. Potencies of mutant peptides toward the GALR2 (red bars) and GALR3 (blue bars) by the mutant peptides are expressed as −log[EC₅₀] values. The horizontal dashed lines represent potency of WT SPX toward receptors. a, P < 0.05 vs. WT SPX

Figure 4

Stability of SPX mutants in the presence of FBS (A) Qu analogs in FBS (B) and human serum (C). Potency of Qu mutants toward GALR2 (D) and GALR3 (E). For stability of SPX mutants, peptides (1 μM) were incubated in serum for 0, 3, 6, 12, 24, 48, and 72 hrs. The remaining activities of the peptides were determined by measuring IP3 production by HEK293 cells that express GALR2 after the indicated times. IP3 production was expressed as percent change, where 100% indicates IP3 production by WT SPX incubated in FBS or human serum for 0 h. For potency of Qu mutants toward GALR2 and GALR3, the SRE-luc reporter vector and plasmid containing human GALR2 or GALR3 were transfected into HEK293 cells that stably expressed Gq_i protein. Cells were then treated with increasing concentrations of mutant peptides, and the receptor activities were determined using the SRE-luc reporter system.

Figure 5. Administration of the GALR2 agonist dN1-Qu displays anxiolytic activity in animal models.

Graphs show the % time in open arm (A), number of open arm entry (B) and center crossing (C). Data are presented as mean ± SEM (n = 9 for VEH and n = 8 for dN1, *p < 0.05 and **p < 0.01 versus VEH and dN1).