Anticonvulsant activity of a nonpeptide galanin receptor agonist Ku¨ lliki Saar*†, Andrey M. Mazarati†‡, Riina Mahlapuu§, Gerd Hallnemo¶, Ursel Soomets*§, Kalle Kilk*, Sven Hellberg¶, Margus Pooga*ʈ, Bo-Ragnar Tolf¶, Tiejun S. Shi**, Tomas Ho¨ kfelt**, Claude Wasterlain‡, Tamas Bartfai*††, and Ulo¨ Langel*‡‡ *Department of Neurochemistry and Neurotoxicology, Stockholm University, SE-10691 Stockholm, Sweden; §Department of Biochemistry, University of Tartu, EE 50411 Tartu, Estonia; ¶AstraZeneca, SE-15185 So¨derta¨lje, Sweden; ʈEstonian Biocenter, EE-51010 Tartu, Estonia; **Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden; ‡Department of Neurology, University of California School of Medicine, Veterans Administration Medical Center, West Los Angeles, CA 90073; and ††Department of Neuropharmacology, The Harrold L. Dorris Neurological Research Center, La Jolla, CA 92037 Communicated by Philip Siekevitz, The Rockefeller University, New York, NY, March 20, 2002 (received for review November 25, 2001) Galanin is a neuropeptide with a wide variety of biological func- Materials and Methods tions, including that of a strong endogenous anticonvulsant. No Protected amino acids and diamines were from Neosystem nonpeptide ligands, capable of activating galanin receptors, are Laboratoire (Strasbourg, France) or Senn Chemicals (Gentilly, available today. Based on known pharmacophores of galanin, a France). 4-Methylbenzhydrylamine-polystyrene resin and acti- combinatorial library was designed, synthesized, and screened at vators were from Bachem. 7-Amino-4-methylcoumarin (AMC) the rat hippocampal galanin receptor. A low molecular weight was from Senn Chemicals. Trifluoroacetic acid, diisopropyleth- galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cy- ylamine, and dioxane were from Lancaster Synthesis, More- clohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found cambe, U.K. Dichloromethane, N,N-dimethylformamide to displace 125I-galanin with micromolar affinity at Bowes cellular (DMF), and acetonitrile were from Labscan, Dublin. All other and rat hippocampal membranes. Autoradiographic binding assay chemicals were from Sigma-Aldrich. on rat spinal cord sections confirmed the ability of galnon to displace 125I-galanin from its binding sites. Galnon inhibited ade- Synthesis of Combinatorial Library. Synthesis of the combinatorial nylate cyclase activity, suggesting an agonist action at galanin library was started from the initial lead compound, Cbz-Phe- receptors. When injected i.p. galnon reduced the severity and Arg-aromatic amine (A–B–C–D). The initial lead compound increased the latency of pentylenetetrazole-induced seizures in was found by screening a library of the analogues of Trp-Asn-Tyr (a tripeptide combining major pharmacophores in galanin, Trp2, mice and reversed the proconvulsant effects of the galanin recep- 5 9 tor antagonist M35, injected into a lateral ventricle. Intrahippocam- Asn , and Tyr ). We included modifications into all positions in pal injection of galnon also shortened the duration of self-sustain- the initial lead compound, resulting in the following lead struc- ture: A–B–C–D, where A denoted a bulky hydrophobic group, B ing status epilepticus in rats, confirming its agonist properties in a hydrophobic amino acid, C an amino acid with protonated side vivo. Pretreatment of rats with antisense peptide nucleic acid chain (in physiological conditions), and D an aromatic amine targeted to galanin receptor type 1 mRNA abolished the effect of (Table 1). We used a split synthesis approach to create a galnon, suggesting mediation of its anticonvulsant properties combinatorial library of compounds. All coupling steps were through this receptor subtype. These findings introduce a system- carried out separately for each compound; for deprotection the ically active nonpeptide galanin agonist anticonvulsant. separate resins were pooled again. Fragment A–B–C was syn- thesized on 4-methylbenzhydrylamine-polystyrene resin and alanin, a neuropeptide of 29 aa (30 in humans), is widely cleaved and, finally, the component D was coupled in the Gdistributed in the central and peripheral nervous systems solution. Synthesized compounds were purified on Sep-Pak and is involved in regulation of learning and memory (1), feeding cartridges (Waters) by eluting with a stepwise gradient of (2), sexual behavior (3), pain threshold (4), and the release of acetonitrile in water. In total, 20 fractions were obtained, 125 insulin (5) and growth hormone (6). freeze-dried, and screened for activity in I-galanin displace- Recent evidence suggests that galanin is also a potent endog- ment assay. The structure of galnon was deduced reiteratively by enous anticonvulsant (7–9, 28). The importance of galanin using the resin samples saved in each step (for details see receptor agonists for seizure control may arise from their ability Results). to act at both presynaptic and postsynaptic sites to reduce Synthesis of Galnon. Galnon was synthesized following the scheme excitability, to inhibit glutamate but not ␥-aminobutyric acid outlined in Fig. 1. The first step of the synthesis was the coupling release (10), and to hyperpolarize CA3 pyramidal cells by ϩ of 9-fluorenylmethoxycarbonyl (Fmoc)-Lys(tert-butyloxycar- opening K channels, thus dampening seizure activity (11). bonyl, Boc)-OH to AMC. One millimole of Fmoc-Lys(Boc)-OH Nonpeptide ligands are available for receptors of cholecysto- and 0.5 equivalents of dicyclohexylcarbodiimide were separately kinin, angiotensin, bradykinin, and many other neuropeptides dissolved in dioxane, cooled on ice, and then pooled. Reaction (12, 13). Most galanin receptor ligands available today, however, mixture was stirred for 30 min at room temperature, then 0.5 are peptides (14, 15), vulnerable to enzymatic degradation and unable to cross the blood–brain barrier. Despite extensive random screening efforts, only two reports Abbreviations: AMC, 7-amino-4-methylcoumarin; Boc, tert-butyloxycarbonyl; DMF, N,N- on nonpeptide antagonists for galanin receptors have been dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; PTZ, pentylenetetrazole; GalR1, galanin receptor type 1; PNA, peptide nucleic acid; SSSE, self-sustaining status epilepticus; published: spirocoumaranon (16) (Sch 202596) and 2,3-dihydro- PPS, perforant path stimulation. 2-(4-methylphenyl)-1,4-dithiepin-1,1,4,4-tetroxide (17). †K.S. and A.M.M. contributed equally to this work. In this study, we report a nonpeptide galanin receptor ligand, ‡‡To whom reprint requests should be addressed. E-mail: [email protected]. galnon, discovered by application of a combinatorial library The publication costs of this article were defrayed in part by page charge payment. This approach. We demonstrate its agonistic activity in adenylate article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. cyclase assays and on seizure models. §1734 solely to indicate this fact. 7136–7141 ͉ PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.102163499 Downloaded by guest on October 5, 2021 Table 1. The building blocks for the combinatorial library quantified and then exposed to Hyperfilm-Max x-ray film (Am- A-B-C-D, where A is a bulky hydrophobic group, B a ersham Biosciences). hydrophobic amino acid, C an amino acid with protonated side chain (in physiological conditions) and D an aromatic amine Seizure Induction and Quantification. All animal studies were coupled to the C terminus of amino acid C through an approved by and carried out in accordance with the guidelines amide bond of the animal research committees of the University of Califor- ABCDnia, Los Angeles and the West Los Angeles Veterans Admin- istration Medical Center. Fmoc- Phe Lys 7-Amino-4-(methoxymethyl)- coumarin Effect of Galnon in Mice. C57BL͞6J male mice weighing 20–30 g Acetyl- Trp His AMC (Simonsen Laboratories, Gilroy, CA) were anaesthetized with Benzyloxycarbonyl- hPhe* Orn m-Anisidin ketamine (100 mg͞kg i.p.) and xylazine (15 mg͞kg i.p.) and Adamantane-1-carbonyl- Cha† Dab‡ Cyclohexylamine stereotaxically chronically implanted into a lateral ventricle *Homophenylalanine. (ICV) with guide cannulae. Galanin or M35 were injected ICV †Cyclohexylalanine. in freely moving mice (0.5 nmol, in 0.5 ␮l, over 5 min). Control ‡Diaminobutyric acid. animals were treated with saline. Galnon was freshly dissolved in 50% DMSO in saline and administered i.p. in a dose of 2 mg͞kg 15 min before pentyle- equivalent of AMC dissolved in DMF was added to the sym- netetrazole (PTZ, Research Biochemicals, Natick, MA), when metric anhydride solution and the mixture was stirred overnight. the effect of galnon alone was studied, or 5 min after M35 The solvents were evaporated, and Fmoc-Lys(Boc)-AMC was injection, in the coadministration studies. Control animals were precipitated with petrol ether͞ethyl acetate mixture (5͞95 vol͞ treated with 50% DMSO in saline. vol) and dried under vacuum. The Boc group was removed with Seizures were induced by i.p. injection of PTZ in a dose of 40 H O͞trifluoroacetic acid mixture (3͞97 vol͞vol) in ice bath for 2 mg͞kg (when studying the effects of galanin only or galnon only) 5 min, followed by the evaporation of the solvents. The obtained or 30 mg͞kg (when studying the effect of M35 and galnon ϩ Fmoc-Lys-AMC was coupled to chlorotrityl resin by incubation M35). Seizure latency and the highest behavioral seizure score of 2–3 equivalents of Fmoc-Lys-AMC, 4–9 equivalents of diiso- were recorded (9). For statistical purposes, if