Anticonvulsant Activity of a Nonpeptide Galanin Receptor Agonist

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 structure: 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 synthesized 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 the animal failed propylethylamine, and 1 equivalent of chlorotrityl resin for 2 h. Resin was washed and the Fmoc group was removed with to show seizure of any particular score, a latency of 900 s was piperidine͞DMF (20͞80 vol͞vol). Coupling of Fmoc-cyclohexy- assigned to this score. No behavioral side effects of galnon were lalanine was performed by using 2 equivalents of amino acid, observed. Statistical methods used were t test (latency) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetraflu- Mann–Whitney (seizure score). oroborate, 1-hydroxybenzotriazole, and 4 equivalents of diiso- Effect of Galnon in Rats. propylethylamine. Galnon was cleaved from the resin by apply- Self-sustaining status epilepticus (SSSE) ing trifluoroacetic acid͞dichloromethane mixture (50͞50 vol͞ was induced as described (8). Briefly, male Wistar rats (Simon- sen Laboratories), 10–12 weeks old, were anaesthetized with vol) in four aliquots. The filtrate was evaporated, and the ͞ ͞ obtained product was purified on Sep-Pak cartridges and ana- ketamine (60 mg kg i.p.) and xylazine (10 mg kg i.p.) and lyzed by using a plasma desorption mass spectrometer (model implanted with a bipolar stimulating electrode into the angular Bioion 20, Bioion, Uppsala, Sweden). bundle of the perforant path. A recording electrode-cannula (Plastics One, Roanoke, VA) was implanted ipsilaterally into the Synthesis of Peptides and Peptide Nucleic Acid (PNA) Oligomers. granule cell layer of ventral dentate gyrus. After 7–10 days of Human and rat galanin and M35 were synthesized on a model recovery, SSSE was induced in free-running animals by 30 min 431A peptide synthesizer (Perkin–Elmer͞Applied Biosystems) of the perforant path stimulation (PPS, 20-Hz 10-s trains at 20 as described (18). PNA oligomers with a general sequence V delivered every min together with continuous 2-Hz stimula- biotin-Cys-PNA-Lys-amide were synthesized by using a Perkin– tion). An electroencephalogram was recorded for 24 h and Elmer͞Applied Biosystems stepwise peptide synthesizer model analyzed off-line with HARMONIE software (Stellate Systems, A433 as described (19). The antisense PNA sequence comple- Montreal). Total time spent in seizures and the time of occur- mentary to the position 18–38 of the coding region of galanin rence of the last seizure were calculated. Galanin (American receptor type 1 (GalR1) mRNA was (PNA1): CCA-TTC-CCT- Peptide, Sunnyvale, CA) and M35 were dissolved in saline, and TCA-CTG-AGG-TTC and the scrambled sequence of it galnon was dissolved in 50% DMSO. The compounds were ␮ (PNAscr): GGC-ATG-GCT-GCT-CTC-CGT-CTG (20). injected into the dentate gyrus in a volume of 1 l at a rate of 0.5 ␮l͞min, 10 min after the end of PPS. Control animals were Ligand Binding to Galanin Receptors, Adenylate Cyclase Assay. The treated with vehicle. mixtures of ligands and individual compounds were screened to displace 125I-galanin in equilibrium binding assay with mem- Administration of PNA. In addition to the electrode implantation brane preparations from rat hippocampi and Bowes cells, rich in described above, animals were implanted with the guide cannula galanin binding sites as described in detail (21). Assays of the ICV. Starting 4–6 h after surgery, rats received repeated bilat- effect of galnon on basal and forskolin-stimulated cAMP pro- eral ICV injections of PNA1. Control animals were injected with duction were performed essentially as described by Valkna et al. PNAscr. The PNA oligomers were dissolved in saline, at a (22) in membranes from rat ventral hippocampi. concentration of 4 nmol͞␮l. Injections were delivered to free- moving rats through the injection cannula, placed inside the Autoradiography. Rats were decapitated, and the spinal cord guide cannula, and connected to the 1 ␮l-Hamilton microsyringe lumbar segments L4 and L5 were dissected out, rapidly frozen, placed into the SP 310i microsyringe pump (World Precision cut in a cryostat, and processed for the autoradiographic ligand Instruments, Sarasota, FL). The injection volume was 1 ␮l͞side binding assay of Young and Kuhar (23), as described (20). and the duration was 5 min. PNA were delivered for 5 days, ␮

Galnon was added at 1, 3, and 5 M concentrations. The sections 10–14 h apart. Each rat received a total of 80 nmol of PNA. PPS PHARMACOLOGY were processed in a BAS3000 Bio-Imagine Analyzer (Fuji) and was initiated 12–16 h after the last PNA injection.

Saar et al. PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 ͉ 7137 Downloaded by guest on October 5, 2021 Fig. 1. Synthesis route of galnon. DCC, dicyclohexylcarbodiimide; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetraﬂuoroborate; HOBt, 1-hydroxybenzotriazole; DIEA, diisopropylethylamine; Cha, cyclohexylalanine; TFA, triﬂuoroacetic acid; DCM, dichloromethane.

Results resins (A-B-Lys, A-B-His, A-B-Orn, and A-B-Dab) and tested Reiterative Evaluation of the Combinatorial Library. After the com- them again in binding assay. We found that the mixtures 125 pletion of synthesis of the combinatorial library, we had four containing Lys at position C were most active in I-galanin resins bearing (theoretically) 64 compounds each. The synthe- displacement assay. In the same way, the most active components of A and B were identified, resulting in galnon, Fmoc- sized compounds in these four mixtures were separated and the 125 cyclohexylalanine-Lys-amidomethyl coumarin (according to In- fractions obtained were tested for the ability to displace I- ternational Union of Pure and Applied Chemistry systematic galanin from galanin receptors in rat hypothalamic membranes. nomenclature: 1-[5-amino-1-(4-methyl-2-oxo-2H-chromen-7- The structure of galnon was deduced reiteratively in the synthe- ylcarbamoyl)-pentylcarbamoyl]-2-cyclohexyl-ethyl-carbamic sis, step by step. First, we were able to define that AMC- acid 9H-fluoren-9-ylmethyl ester). The synthesis of galnon is containing compounds were most active in 125I-galanin displace- shown in Fig. 1. The iterative deduction indicated that two more ment assay. Therefore, we coupled AMC to four types of A-B-C compounds from our library could serve as candidates for

7138 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.102163499 Saar et al. Downloaded by guest on October 5, 2021 Table 2. Comparison of galanin and galnon in inhibition of adenylate cyclase activity (basal and forskolin stimulated) in membranes from rat ventral hippocampi and displacement of 125I-galanin from its speciﬁc binding sites

EC50, M of inhibition of adenylate cyclase activity in rat hippocampal

membranes KD,M

Forskolin Rat Bowes cells Basal stimulated hippocampus (GalR1)

Galanin 1.1 ϫ 10Ϫ9 1.1 ϫ 10Ϫ9 0.8 ϫ 10Ϫ9 0.4 ϫ 10Ϫ9 Galnon 8 ϫ 10Ϫ6 10 ϫ 10Ϫ6 4.8 ϫ 10Ϫ6 2.9 ϫ 10Ϫ6

peptidomimetic galanin receptor ligands, however, with lower affinities than galnon at galanin receptors.

Binding Studies and Inhibition of Adenylate Cyclase Activity. 125I- galanin was displaced by galnon in membranes from Bowes cells ␮ (Ki value 2.9 M) and rat ventral hippocampus with a Ki value of 4.8 ␮M (Table 2). The ability of galnon to displace 125I-galanin from its binding sites in a concentration-dependent manner was confirmed in an autoradiographic binding assay on rat spinal cord sections, where the autoradiographic signal of 125I-galanin and its dose-dependent displacement by galnon could be studied best. Attenuation of 125I-galanin binding was detected at 1, 3, and 5 ␮M concentration of galnon by 23 Ϯ 6%, 21 Ϯ 10%, and 42 Ϯ 11%, respectively. The activity of galnon on adenylate cyclase activity was studied. Like galanin, galnon inhibited both basal and forskolin- stimulated adenylate cyclase activity (Table 2). Galnon (10 ␮M) Fig. 2. Effects of galnon (2 mg͞kg i.p.) and͞or M35 (0.5 nmol ICV) on inhibited basal cAMP production by 25%, whereas the inhibitory PTZ-induced seizures on mice. Data are presented as mean Ϯ SEM. (Insets) effect of 10 ␮M galanin was 36%. Inhibition of adenylate cyclase Maximal seizure scores (mean Ϯ SEM). (a) Effect of galnon on PTZ-induced activity suggested that galnon exhibited agonist-like properties at seizures. Galanin (0.5 nmol ICV, E), galnon (2 mg͞kg i.p., Œ) treated animals, galanin receptors. and controls (DMSO i.p., }). *, P Ͻ 0.01 vs. control. (b) Effects of coadministration of M35 and galnon on PTZ-induced seizures. Animals treated with M35 ■ Œ } Ͻ Anticonvulsant Properties of Galnon. Effects of systemic galnon in ( ), with M35 and galnon ( ); controls ( ). *, P 0.05 versus control. mice. In the first set of experiments, we induced seizures with 40 ͞ ͞ ␮ ͞ mg kg of PTZ (i.p.), and injected 2 mg kg (3 mol kg) of galnon receptor antagonist M35, when the latter was administered in a (i.p.) 15 min before PTZ injection. Galnon lowered the maximal dose that alone, as it has been previously reported (8), did not seizure score from 4.5 (control mice) to 1.45 (galnon-treated alter the course of SSSE (0.5 nmol, Fig. 3b). When galnon was mice), and increased seizure latency 3-fold compared with Ͻ injected immediately after M35, both time spent in seizures and controls (P 0.01, Fig. 2a). The anticonvulsant effect of galnon the duration of SSSE were significantly higher than in galnon- was comparable to that of galanin administered ICV in a dose treated rats without M35, and these parameters did not differ 0.5 nmol (9). from those in control animals (Fig. 3b). Next, we examined the pattern of PTZ-induced convulsions In a separate set of experiments, equimolar galnon blocked the ͞ (30 mg kg i.p.) in mice treated ICV with the galanin receptor seizure-facilitating effect of intrahippocampal injections of M35 antagonist M35 and galnon (i.p.). In accordance with our in a amount of 5 nmol, a dose that was previously shown to previous finding (9) M35 increased the severity of PTZ-induced potentiate seizures (9): under these conditions PPS as short as 7 convulsions. Galnon (Fig. 2b) completely abolished the seizure- min is sufficient to induce self-sustaining seizures (9). When an facilitating effect of M35. equimolar amount of galnon was administered into the hip- Effects of intrahippocampal galnon in rats. SSSE consisted of pocampus before M35, total seizure time and duration of SSSE recurrent limbic seizures of varying intensity (stages 1–5), which were reduced from 45 Ϯ 5to13Ϯ 4 min and from 99 Ϯ 11 to lasted for 12–18 h after PPS was discontinued. Behavioral 37 Ϯ 8 min, respectively (Fig. 3c). seizures were accompanied by high-frequency (13ϩ Hz) and Anticonvulsant effect of galnon in antisense PNA-pretreated rats. amplitude (1ϩ mV) discharges with the duration of individual We examined the anticonvulsant effects of galnon in SSSE. As events between 30 s and 3 min. Between the seizures, ictal spikes shown previously, down-regulation of GalR1 by PNA1 did not with the amplitude of 0.8 mV and more and frequency of 3 Hz affect the parameters of SSSE (7). However, in PNA1-pretreated and less were continuously generated (spike frequency of 3 Hz animals, galnon (1 nmol) had no effect on self-sustaining and more was recognized as a seizure) (8). seizures, whereas in control rats injected with scrambled PNA, Intrahippocampal administration of galnon shortened the galnon reduced total seizure time 9-fold and duration of SSSE duration of SSSE and decreased the time spent in seizures in a 7-fold (Fig. 4). dose-dependent manner (Fig. 3a). In the maximal dose used (5 nmol), galnon shortened SSSE duration to 28 Ϯ 8 min, from Discussion Ϯ Ͻ 760 77 min in controls (P 0.05). The anticonvulsant effects Recent massive screening efforts, often in combination with PHARMACOLOGY of galnon were abolished by pretreatment with the galanin classical medicinal chemistry optimization, have led to the

Saar et al. PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 ͉ 7139 Downloaded by guest on October 5, 2021 Fig. 4. Effect of galnon in the rats pretreated with antisense PNA targeted to the GalR1 mRNA (PNA1) or with scrambled PNA (PNAscr). *, P Ͻ 0.05 vs. PNAscr ϩ saline; #, vs. PNAscr ϩ galnon. Open bars: time in seizures; ﬁlled bars: time of the last seizure.

The first nonpeptide galanin receptor ligand, Sch202596 (spirocoumaranon), discovered by random screening of fungal metabo- 125 lites, displaces I-galanin in Bowes cell membranes with IC50 value of 1.7 ␮M (16) but does not appear ideal for optimization because of its complex chemical structure. For dithiepin-1,1,4,4-tetroxide, the second low molecular weight galanin receptor ligand described, ␮ an IC50 value of 0.17 M in Bowes cell membranes was reported (17). This compound behaves as a galanin receptor antagonist in several functional assays, but its chemically reactive nature pre- cludes any therapeutic use. We report in this study on the design and synthesis of galnon, Fmoc-cyclohexylalanine-Lys-amidomethylcoumarin, a low molecular weight, blood–brain barrier-penetrating galanin receptor ligand with agonist properties, both in vitro and in vivo. We synthesized a combinatorial library of 256 compounds Fig. 3. Effects of galnon on SSSE induced by 30 min of PPS. (a) Intrahip- composed of analogues of galanin pharmacophores. Reitera- pocampal injection of galnon 10 min after the end of PPS attenuated SSSE tive screening of binding activities of these compounds re- in a dose-dependent manner. P Ͻ 0.05: * vs. control (50% DMSO). (b) sulted in galnon, with binding affinities to galanin receptors Anticonvulsant effects of galnon in animal model of SSSE were M35 comparable to those of spirocoumaranon and dithiepin- sensitive. M35 was injected 10 min after PPS, followed 5 min later by 1,1,4,4-tetroxide in Bowes cellular membranes. Galnon was galanin or galnon. The amount of galnon, which attenuated SSSE, failed to also tested in an autoradiographic binding assay on rat spinal do so in M35-pretreated rats. P Ͻ 0.05: *, vs. DMSO (control for galnon); #, cord sections, to confirm the binding results obtained in vs. both galnon and saline (control for M35). There were no significant membranes from Bowes cells and rat hippocampus, using a differences between the two control groups (in DMSO group time in Ϯ Ϯ different source of galanin receptors as well as different seizures 395 38 min and the time of the last seizure was 760 77 min; in 125 the saline-treated group these parameters were 510 Ϯ 65 and 1,000 Ϯ 87 methodology. Attenuation of I-galanin binding was detected min, respectively). (c) Galnon inhibited M35-induced facilitation of SSSE. at different concentrations of galnon, confirming its ability to 125 Intrahippocampal injection of M35 (5 nmol) enabled the establishment of displace I-galanin from its binding sites in yet another SSSE after 7 min of PPS, which is not sufficient to induce seizures in naı¨ve system. We cannot rule out the possibility that our library animals (control). When galnon (5 nmol) was injected 5 min before M35 contained compounds with 125I-galanin-displacing activity su- administration, 7 min of PPS induced seizures, which lasted significantly perior to galnon, but we did not discover them because of their less, than in M35-only treated rats. P Ͻ 0.05: *, vs. control; †, vs. M35. DMSO low abundance (caused by inefficient coupling between their and saline-treated groups were pooled in one group, because there were building blocks and losses in cleavage and separation steps). no differences among them. Open bars: time in seizures; filled bars; time of the last seizure. Galnon was further studied in the adenylate cyclase assay to elucidate whether it has agonist- or antagonist-like properties at galanin receptor(s). It inhibited basal and forskolin- identification of nonpeptide ligands for several neuropeptide stimulated adenylate cyclase activity with a pattern similar receptors (12). Intriguingly, such approaches have been largely to galanin, indicating that it acts as an agonist at galanin unsuccessful in the case of galanin. At the present time, galanin receptors. itself is still the best agonist for the three cloned galanin receptor To test the effects of galnon in vivo, we used the pentylenetet- subtypes. Several chimeric galanin-based peptides (M35, M40) razole model of epileptic seizure in mice. Galnon applied i.p have been shown to act as high affinity, selective antagonists at lowered the maximal seizure score, increased seizure latency, galanin receptors in tissues and͞or in vivo assays (24). and blocked the proconvulsant effect of ICV M35, hence we can

7140 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.102163499 Saar et al. Downloaded by guest on October 5, 2021 assume that it penetrated the blood–brain barrier to act at spinal cord (27), suggesting that PNA1 may have caused down- galanin receptors located centrally. It must be noted that PTZ- regulation of GalR1 expression. It was also demonstrated by induced convulsions represent a model for generalized absence immunoprecipitation analysis that the same PNA oligomer seizures (25), whereas a model of status epilepticus used in this down-regulates the expression of GalR1 in vitro (20). In line with study is a model of focal limbic seizures. From this perspective, these results, we studied the effect of galnon on SSSE in galnon shows a broad-spectrum anticonvulsant activity, which PNA1-pretreated rats. Galnon completely failed to alter SSSE in makes it a promising agent for managing multiple forms of the rats with GalR1 down-regulation but successfully blocked epilepsy. SSSE in rats treated with PNAscr. This finding suggests that Experiments in rats showed that galnon also possessed strong galnon’s anticonvulsant effects may be mediated by means of anticonvulsant effect against SSSE, a particularly severe form of GalR1. epileptic seizures that is resistant to conventional antiepileptic In conclusion, we report that galnon, 7-((9-fluorenylmethoxy- drugs (26). No currently available anticonvulsant in the United carbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin, a non- States pharmacopoeia acts through peptide receptors, and one peptide ligand for galanin receptors, possesses agonist properties major drawback of current agents is that they all work through in vitro and in vivo and strong anticonvulsant properties in vivo. a few, very similar mechanisms. The potency of galnon in the present studies provides evidence that a systemically active We thank Dr. Anders Unde´n, Dr. Anders Jure´us, Dr. Kalle Kaljuste, galanin agonist can be an effective anticonvulsant in rodents and Dr. Ello Karelson, and Dr. Khadijeh Rezaei for advice and help. This deserves evaluation in a broad spectrum of seizure and epilepsy work was supported by a grant from AstraZeneca and The Swedish models. Natural and Medical Research Councils and Marianne and Marcus We have shown previously that intrathecal administration of Wallenberg’s Foundation, the Research Service of the Veterans 21-mer PNA targeting the region 18–38 of the coding sequence Health Administration, and Grant NS 13515 from the National of rat GalR1 mRNA (PNA1) decreased 125I-galanin binding in Institute of Neurological Disorders and Stroke.

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