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Design, Synthesis and Characterization of Galanin Receptor Selective Ligands
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ulo Langel)ORCID iD: 0000-0001-9671-0354
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Galanin is a 29/30 amino acid long bioactive peptide discovered over 30 years ago when C-terminally amidated peptides were isolated from porcine intestines. The name galanin originates from a combination of the first and last amino acids - G from glycine and the rest from alanine. The first 15 amino acids are highly conserved throughout species, which indicates that the N-terminus is important for receptor recognition and binding. Galanin exerts its effects by binding to three different G protein-coupled receptors, which all differ according to regional distribution, the affinity for shortened galanin fragments, as well as the intracellular G-protein signaling cascade used. When first discovered, galanin was found to cause muscle contraction as well as hyperglycemia.  Over the years, galanin has been reported to be involved in a wide variety of biological functions, for example food intake and neurogenesis, and pathological functions, for example epilepsy and depression.

Determining the specific involvement of the three different galanin receptors in biological and pathological processes is limited by the small amount of galanin receptor selective/specific ligands available as research tools. Furthermore, the fast degradation of peptides limits the administration routes in animal studies.

This thesis aims at developing new galanin receptor-selective ligands to help delineate the involvement of the three different galanin receptors.

Paper 1 presents the shortest galanin fragment with a galanin receptor 2 specific binding preference where only a single amino acid substitution was made, Ala5Ser in galanin (2-11). In addition, G-protein coupled receptor signaling were evaluated through both a classical second messenger assay and a real-time label-free technique in cells overexpressing the receptor as well as low receptor expression.

Paper 2 demonstrates that the neuroprotective effects of galanin in a kainic acid-induced excitotoxic animal model were mediated through galanin receptor 1. Furthermore, a new robust protocol for evaluating G-protein signaling using a label-free real time impedance technique was presented and compared to two different classical second-messenger assays.

Paper 3 presents a series of systemically active galanin receptor 2 selective ligands subsequently evaluated in two different depression-like animal models.

Paper 4 investigates a mutated form of human galanin which was found in epilepsy patients and binding and signaling properties of the mutated associated ligand p.(A39E) was examined.

In conclusion, this thesis presents the discovery of eight new galanin ligands, which can be used to evaluate the galaninergic system as well as to help investigate the possible use of peptides as pharmaceuticals in different diseases.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2017. , p. 96
Keywords [en]
galanin, GAL1R, GAL2R, depression, epilepsy
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-139880ISBN: 978-91-7649-721-0 (print)ISBN: 978-91-7649-722-7 (electronic)OAI: oai:DiVA.org:su-139880DiVA, id: diva2:1075554
Public defence
2017-03-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2017-03-08 Created: 2017-02-20 Last updated: 2017-03-06Bibliographically approved
List of papers
1. Ala(5)-galanin (2-11) is a GAL2R specific galanin analogue
Open this publication in new window or tab >>Ala(5)-galanin (2-11) is a GAL2R specific galanin analogue
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2016 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 60, p. 75-82Article in journal (Refereed) Published
Abstract [en]

It is over 30years since the regulatory peptide galanin was discovered by Professor Mutt and co-workers. Galanin exerts its effects by binding to three galanin G-protein coupled receptors, namely GAL1R, GAL2R and GAL3R. Each galanin receptor has a different distribution in the central nervous system and the peripheral nervous system as well as distinctive signaling pathways, which implicates that the receptors are involved in different biological- and pathological effects. The delineation of the galaninergic system is however difficult due to a lack of stable, specific galanin receptor ligands. Herein, a new short GAL2R specific ligand, Ala(5)-galanin (2-11), is presented. The galanin (2-11) modified analogue Ala(5)-galanin (2-11) was tested in (125)I-galanin competitive binding studies for the three galanin receptors and the G-protein coupled receptor signaling properties was tested by the ability to influence second-messenger molecules like inositol phosphate and cyclic adenosine monophosphate. In addition, two different label-free real-time assays, namely EnSpire® based on an optical biosensor and xCELLigence® based on an electric biosensor, were used for evaluating the signaling properties using cell lines with different levels of receptor expression. Ala(5)-galanin (2-11) was subsequently found to be a full agonist for GAL2R with more than 375-fold preference for GAL2R compared to both GAL1R and GAL3R. The single amino acid substitution of serine to alanine at position 5 in the short ligand galanin (2-11) resulted in a ligand subsequently unable to bind neither GAL3R nor GAL1R, even at concentrations as high as 0.1mM.

Keywords
Agonist, Dynamic mass redistribution, Galanin, GAL2R specific ligand, GPCR, Impedance, Neuropeptide, Label-free real-time assay
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-136177 (URN)10.1016/j.npep.2016.08.008 (DOI)000389398800011 ()27592409 (PubMedID)
Available from: 2016-11-30 Created: 2016-11-30 Last updated: 2017-11-29Bibliographically approved
2. Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus
Open this publication in new window or tab >>Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus
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2016 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 58, p. 83-92Article in journal (Refereed) Published
Abstract [en]

The neuropeptide galanin is widely distributed in the central and peripheral nervous systems and part of a bigger family of bioactive peptides. Galanin exerts its biological activity through three G-protein coupled receptor subtypes, GAL1–3R. Throughout the last 20 years, data has accumulated that galanin can have a neuroprotective effect presumably mediated through the activation of GAL1R and GAL2R. In order to test the pharmaceutical potential of galanin receptor subtype selective ligands to inhibit excitotoxic cell death, the GAL1R selective ligand M617 and the GAL2R selective ligand M1145 were compared to the novel GAL1/2R ligand M1154, in their ability to reduce the excitotoxic effects of intracerebroventricular injected kainate acid in rats.

The peptide ligands were evaluated in vitro for their binding preference in a competitive 125I-galanin receptor subtype binding assay, and G-protein signaling was evaluated using both classical signaling and a label-free real-time technique. Even though there was no significant difference in the time course or severity of the kainic acid induced epileptic behavior in vivo, administration of either M617 or M1154 before kainic acid administration significantly attenuated the neuronal cell death in the hippocampus. Our results indicate the potential therapeutic value of agonists selective for GAL1R in the prevention of neuronal cell death. 

Keywords
Galanin, Galanin receptor subtype selective ligands, GAL1R, GAL2R, Label-free real-time technology, XCELLigence, Excitotoxicity, M1154
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-125756 (URN)10.1016/j.npep.2015.12.009 (DOI)000382414500011 ()
Funder
Swedish Research CouncilStiftelsen Olle Engkvist ByggmästareHelge Ax:son Johnsons stiftelse
Available from: 2016-01-18 Created: 2016-01-18 Last updated: 2017-11-30Bibliographically approved
3. Novel systemically active galanin receptor 2 ligands in depression-like behavior
Open this publication in new window or tab >>Novel systemically active galanin receptor 2 ligands in depression-like behavior
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2013 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 127, no 1, p. 114-123Article in journal (Refereed) Published
Abstract [en]

Neuropeptide galanin and its three G-protein coupled receptors, galanin receptor type 1-galanin receptor type 3 (GalR1-GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression-like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide-based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant-like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (<0.5mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressant-like effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression.

Keywords
animal models of depression, depression, galanin, galanin receptor type 2, neuropeptide, tail suspension test
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-95760 (URN)10.1111/jnc.12274 (DOI)000325007300012 ()
Note

AuthorCount:11;

Available from: 2013-11-05 Created: 2013-11-04 Last updated: 2018-01-11Bibliographically approved
4. Galanin pathogenic mutations in temporal lobe epilepsy
Open this publication in new window or tab >>Galanin pathogenic mutations in temporal lobe epilepsy
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2015 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 24, no 11, p. 3082-3091Article in journal (Refereed) Published
Abstract [en]

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatment.

National Category
Chemical Sciences Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-119176 (URN)10.1093/hmg/ddv060 (DOI)000355674000007 ()
Available from: 2015-08-10 Created: 2015-07-29 Last updated: 2017-12-04Bibliographically approved

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