Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface.

Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hß2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.

Theoretical investigation of selective ligand binding mode of galanin receptors.

The Galaninergic system consist of Galanin and its receptors, involved in neuromodulation and neurotransmission. Galanin regulate its physiologic and pathologic functions by interacting with three G-protein coupled receptors; GalR1, GalR2 and GalR3. The widespread distribution of Galanin and its receptor subtypes in central and peripheral nervous system makes them an attractive drug target for the treatment of neurological diseases. However, subtypes selective ligands paucity and little structural information related to either Galanin receptors and Galanin receptor-ligand complexes hampered the structure-based drug design. Thus computational modeling characterization strategy was utilized for Galanin receptor 3D structure prediction and subtypes ligands binding selectivity. Reported ligands with experimental activity were docked against the homology model of Galanin receptors. Further, the MD simulation and binding free energy calculation were carried out to determine the binding interactions pattern consistency and selectivity towards receptor subtype. Results of binding free energy of per residue indicate key contribution of GalR1 Phe115 and His267 in the selective binding of ligands while Tyr103, Tyr270 and His277 play major role in the selective binding of GalR3 ligands. Our study provide rationale for further in silico virtual screening of small molecules for the development of selective ligands against Galanin receptor subtypes.Communicated by Ramaswamy H. Sarma.

Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors.

Galanin receptors (GALRs) belong to the superfamily of G-protein coupled receptors. The three GALR subtypes (GALR1, GALR2, and GALR3) are activated by their endogenous ligands: spexin (SPX) and galanin (GAL). The synthetic SPX-based GALR2-specific agonist, SG2A, plays a dual role in the regulation of appetite and depression-like behaviors. Little is known, however, about the molecular interaction between GALR2 and SG2A. Using site-directed mutagenesis and domain swapping between GALR2 and GALR3, we identified residues in GALR2 that promote interaction with SG2A and residues in GALR3 that inhibit interaction with SG2A. In particular, Phe103, Phe106, and His110 in the transmembrane helix 3 (TM3) domain; Val193, Phe194, and Ser195 in the TM5 domain; and Leu273 in the extracellular loop 3 (ECL3) domain of GALR2 provide favorable interactions with the Asn5, Ala7, Phe11, and Pro13 residues of SG2A. Our results explain how SG2A achieves selective interaction with GALR2 and inhibits interaction with GALR3. The results described here can be used broadly for in silico virtual screening of small molecules for the development of GALR subtype-specific agonists and/or antagonists.

Preclinical evaluation of intravenous NAX 810-2, a novel GalR2-preferring analog, for anticonvulsant efficacy and pharmacokinetics.

OBJECTIVE: Potential clinical utility of galanin or peptidic analogs has been hindered by poor metabolic stability, lack of brain penetration, and hyperglycemia due to galanin receptor subtype 1 (GalR1) activation. NAX 810-2, a galanin receptor subtype 2 (GalR2)-preferring galanin analog, possesses 15-fold greater affinity for GalR2 over GalR1 and protects against seizures in the mouse 6 Hz, corneal kindling, and Frings audiogenic seizure models. The purpose of these studies was to further evaluate the preclinical efficacy and pharmacokinetics of NAX 810-2 in mice. METHODS: NAX 810-2 was administered by intravenous (i.v.; tail vein, bolus) injection to fully kindled (corneal kindling assay) or naive CF-1 mice (6 Hz assay and pharmacokinetic studies). Plasma NAX 810-2 levels were determined from trunk blood samples. NAX 810-2 was also added to human plasma at various concentrations for determination of plasma protein binding. RESULTS: In the mouse corneal kindling model, NAX 810-2 dose-dependently blocked seizures following intravenous administration (median effective dose [ED50 ], 0.5 mg/kg). In the mouse 6 Hz (32 mA) seizure model, it was demonstrated that NAX 810-2 dose-dependently blocked seizures following bolus administration (0.375-1.5 mg/kg, i.v.; ED50 , 0.7 mg/kg), with a time-to-peak effect of 0.5 h posttreatment. Motor impairment was observed at 1.5 mg/kg, i.v., whereas one-half of this dose, 0.75 mg/kg, i.v., was maximally effective in the 6 Hz test. Plasma levels of NAX 810-2 show linear pharmacokinetics following intravenous administration and a half-life of 1.2 h. Functional agonist activity studies demonstrate that NAX 810-2 effectively activates GalR2 at therapeutic concentrations. SIGNIFICANCE: These studies further suggest the potential utility of NAX 810-2 as a novel therapy for epilepsy.

Homology modeling, docking, and molecular dynamics simulation of the receptor GALR2 and its interactions with galanin and a positive allosteric modulator.

Galanin receptor type 2 (GALR2) is a class A G-protein-coupled receptor (GPCR), and it has been reported that orthosteric ligands and positive allosteric modulators (PAMs) of GALR2 could potentially be used to treat epilepsy. So far, the X-ray structure of this receptor has not been resolved, and knowledge of the 3D structure of GALR2 may prove informative in attempts to design novel ligands and to explore the mechanism for the allosteric modulation of this receptor. In this study, homology modeling was used to obtain several GALR2 models using known templates. ProSA-web Z-scores and Ramachandran plots as well as pre-screening against a test dataset of known compounds were all utilized to select the best model of GALR2. Molecular dockings of galanin (a peptide) and a nonpeptide ligand were carried out to choose the (GALR2 model)-galanin complex that showed the closest agreement with the corresponding experimental data. Finally, a 50-ns MD simulation was performed to study the interactions between the GALR2 model and the synthetic and endogenous ligands. The results from docking and MD simulation showed that, besides the reported residues, Tyr160(4.60), Ile105(3.32), Ala274(7.35), and Tyr163(ECL2) also appear to play important roles in the binding of galanin. The potential allosteric binding pockets in the GALR2 model were then investigated via MD simulation. The results indicated that the mechanism for the allosteric modulation caused by PAMs is the binding of the PAM at pocket III, which is formed by galanin, ECL2, TM2, TM3, and ECL1; this results in the disruption of the Na(+)-binding site and/or the Na(+) ion pathway, leading to GALR2 agonism.

Preferential activation by galanin 1-15 fragment of the GalR1 protomer of a GalR1-GalR2 heteroreceptor complex.

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1-15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1-GalR2 heteromers which may play a significant role in mediating galanin fragment (1-15) signaling. In HEK293T cells evidence for the existence of GalR1-GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET(2) assays. PLA positive blobs representing GalR1-GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1-15) was more potent than galanin (1-29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1-GalR2 transfected cells. The inhibition of CREB by 50nM of galanin (1-15) and of galanin (1-29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1-15) vs galanin (1-29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1-29). In contrast, in NFAT reporter gene assays galanin (1-29) shows a higher efficacy than galanin (1-15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1-GalR2 heteroreceptor complexes induced by galanin (1-15) may contribute to depression-like actions since GalR1 agonists produce such effects.

Coevolution of the spexin/galanin/kisspeptin family: Spexin activates galanin receptor type II and III.

The novel neuropeptide spexin (SPX) was discovered using bioinformatics. The function of this peptide is currently under investigation. Here, we identified SPX along with a second SPX gene (SPX2) in vertebrate genomes. Syntenic analysis and relocating SPXs and their neighbor genes on reconstructed vertebrate ancestral chromosomes revealed that SPXs reside in the near vicinity of the kisspeptin (KISS) and galanin (GAL) family genes on the chromosomes. Alignment of mature peptide sequences showed some extent of sequence similarity among the 3 peptide groups. Gene structure analysis indicated that SPX is more closely related to GAL than KISS. These results suggest that the SPX, GAL, and KISS genes arose through local duplications before 2 rounds (2R) of whole-genome duplication. Receptors of KISS and GAL (GAL receptor [GALR]) are phylogenetically closest among rhodopsin-like G protein-coupled receptors, and synteny revealed the presence of 3 distinct receptor families KISS receptor, GALR1, and GALR2/3 before 2R. A ligand-receptor interaction study showed that SPXs activate human, Xenopus, and zebrafish GALR2/3 family receptors but not GALR1, suggesting that SPXs are natural ligands for GALR2/3. Particularly, SPXs exhibited much higher potency toward GALR3 than GAL. Together, these results identify the coevolution of SPX/GAL/KISS ligand genes with their receptor genes. This study demonstrates the advantage of evolutionary genomics to explore the evolutionary relationship of a peptide gene family that arose before 2R by local duplications.

Positive allosteric modulators to peptide GPCRs: a promising class of drugs.

The task of finding selective and stable peptide receptor agonists with low molecular weight, desirable pharmacokinetic properties and penetrable to the blood-brain barrier has proven too difficult for many highly coveted drug targets, including receptors for endothelin, vasoactive intestinal peptide and galanin. These receptors and ligand-gated ion channels activated by structurally simple agonists such as glutamate, glycine and GABA present such a narrow chemical space that the design of subtype-selective molecules capable of distinguishing a dozen of glutamate and GABA receptor subtypes and possessing desirable pharmacokinetic properties has also been problematic. In contrast, the pharmaceutical industry demonstrates a remarkable success in developing 1,4-benzodiazepines, positive allosteric modulators (PMAs) of the GABAA receptor. They were synthesized over 50 years ago and discovered to have anxiolytic potential through an in vivo assay. As exemplified by Librium, Valium and Dormicum, these allosteric ligands of the receptor became the world's first blockbuster drugs. Through molecular manipulation over the past 2 decades, including mutations and knockouts of the endogenous ligands or their receptors, and by in-depth physiological and pharmacological studies, more peptide and glutamate receptors have become well-validated drug targets for which an agonist is sought. In such cases, the pursuit for PAMs has also intensified, and a working paradigm to identify drug candidates that are designed as PAMs has emerged. This review, which focuses on the general principles of finding PAMs of peptide receptors in the 21st century, describes the workflow and some of its resulting compounds such as PAMs of galanin receptor 2 that act as potent anticonvulsant agents.

Synthesis and biological evaluation of novel pyrimidine derivatives as sub-micromolar affinity ligands of GalR2.

GalR1 and GalR2 represent unique pharmacological targets for treatment of seizures and epilepsy. A novel series of 2,4,6-triaminopyrimidine derivatives were synthesized and found to have sub-micromolar affinity for GalR2. Optimization of a series of 2,4,6-triaminopyrimidines led to the discovery of several analogs with IC50 values ranging from 0.3 to 1 µM.