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.The novel neuropeptide spexin (SPX), which is encoded by the C12ORF39 gene, was originally discovered using bioinformatics tools 1,2 . The predicted mature SPX peptide sequence of 14 amino acids flanked by dibasic cleavage sites is evolutionarily conserved across vertebrate species [1][2][3][4][5] . SPX expression at the mRNA and/or protein level has been documented in brain regions and peripheral tissues of humans, mice, rats, and goldfish 1-8 , suggesting multiple physiological functions of SPX. Recently, SPX was implicated in regulation of feeding behaviors and related metabolic processes. SPX mRNA levels are markedly decreased in the fat of obese humans, and administration of SPX leads to weight loss in diet-induced obese rodents 9 . SPX also suppresses appetite in goldfish 5 . In addition, SPX is likely involved in reproduction, cardiovascular/renal function, and nociception 4,10 . The precise roles of SPX in these processes, however, are not well understood due to a lack of information on the SPX receptor. Recently, we demonstrated that SPX is an endogenous ligand that acts at GALR2 and GALR3 but not at GALR1, while GAL activates all three receptor subtypes with relatively low potency and affinity for GALR3 11 .The SPX and GAL genes likely emerged through a local duplication from a common ancestor gene, and as a result, their mature peptides share several conserved residues, including Trp2, Thr3, Tyr9, Leu10, and Gly12 11,12 . Like SPX, GAL is widely expressed in the central nervous system and peripheral tissues [13][14][15][16] . The actions of SPX and GAL in appetite behavior and reproduction, however, appear to oppose each other. For instance, levels of circulating GAL, along with neuro...

Synergistic effect of spexin and progesterone on pain sensitivity attenuation in ovariectomized rats

“…Galanin has three receptor subtypes including galanin receptor 1 (GALR1), galanin receptor 2 (GALR2) and galanin receptor 3 (GALR3), GALR1 and GALR3 induce inhibitory Gi coupled signaling. Spexin was discovered to activate GALR2 and GALR3 but not GALR1 . On the other hand GALR2 has both presynaptic and postsynaptic actions, activation of presynaptic GalR2 couples to Gi/o and stops the activation of calcium channels thereby strongly reduces glutamate release and hence nociceptive input to the dorsal horn which leads to a reduction in TRPV1 sensitization in dorsal root ganglion; while postsynaptic GALR2 triggers stimulatory Gq coupled signaling .…”

“…Spexin was discovered to activate GALR2 and GALR3 but not GALR1 . On the other hand GALR2 has both presynaptic and postsynaptic actions, activation of presynaptic GalR2 couples to Gi/o and stops the activation of calcium channels thereby strongly reduces glutamate release and hence nociceptive input to the dorsal horn which leads to a reduction in TRPV1 sensitization in dorsal root ganglion; while postsynaptic GALR2 triggers stimulatory Gq coupled signaling . Therefore, both GALR1 and GALR2 have analgesic effects because GALR2 action is in presynaptic site, so its analgesic effects is similar to GALR1 .…”

Evolutionary and Comparative Genomics to Drive Rational Drug Design, with Particular Focus on Neuropeptide Seven-Transmembrane Receptors

“…(2015), humans often possess fewer, and different, paralogs within a gene family compared to other species. Therefore, by using a variety of paralogs from within a gene family from an array of species, different peptide sequences that are capable of binding human 7TMRs of interest at varying potencies and affinities can be analysed (Kim et al ., 2014a) and the function of amino acids in receptor binding ascertained (Reyes-Alcaraz et al ., 2016). Once the functions of individual amino acids within a peptide have been analysed then mutational experiments can be conducted to specifically alter the binding affinity of the peptide sequences to create novel ligands.…”

“…2). During the divergence of the GAL/SPX and GALR1/2/3 system, GALR2 appears to have become an intermediate form as it responds to both SPX and GAL with high affinity, whereas GALR1 and GALR3 acquired significant preference to GAL and SPX, respectively (Reyes-Alcaraz et al ., 2016). …”

Escitalopram affects spexin expression in the rat hypothalamus, hippocampus and striatum

“…Moreover, SPX exhibits even higher potency toward GALR3 than galanin [13]. Interestingly an artificial SPX-based human GALR2 receptor agonist exerts an anxiolytic effect in mice [14]. On the other hand, SPX does stimulate intestinal peristaltic movement through GALR2-dependent activation of L-type calcium VDCC channels in the murine smooth muscle cells [15].…”