CYCLIC GALANIN-ANALOGS AND USES THEREOF

Abstract

The invention generally relates to the field of medicine and pharmacology. More particularly, it relates to novel analogs of galanin, and the use thereof in therapy. Provided is a cyclic peptide analog of galanin, wherein the galanin analog comprises a (methyl)Lanthionine bridge and wherein the analog has the general formula "X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X- 21-X22-", or a truncated variant thereof lacking X1 and/or up to 11 of the C-terminal residues, wherein two residues selected from the group consisting of X3, X4, X6, X7, X10 and X13-X19 together form a Lanthionine bridge of the structure Ala-S-Ala, or a methylLanthionine bridge of the structure Abu-S-Ala or Ala-S-Abu.

Description

[0001] This patent application is a divisional of U.S. patent application Ser. No. 15/557,273 filed Sep. 11, 2017 which is the U.S. National Stage of PCT/EP2016/055266, filed Mar. 11, 2016, which claims the benefit of priority from European Patent Application No. 15159102.1 filed Mar. 13, 2015, teachings of each of which are incorporated by reference herein in their entireties.

[0002] The invention generally relates to the field of medicine and pharmacology. More particularly, it relates to novel analogs of galanin, methods for producing them, and the use thereof in therapy.

[0003] Galanin is a neuropeptide consisting of a chain of 29-30 amino acids produced from the cleavage of a 123-amino acid protein known as preprogalanin, which is encoded by the GAL gene. It is widely expressed in the brain, spinal cord, and gut of humans as well as other mammals. Galanin has been implicated in many biologically diverse functions, including: nociception, waking and sleep regulation, cognition, feeding regulation, regulation of mood, regulation of blood pressure, it also has roles in development as well as acting as a trophic factor. Galanin is linked to a number of diseases including Alzheimer's disease, epilepsy as well as depression, eating disorders and cancer.

[0004] Galanin stimulates three receptor subtypes, GalR1, GalR2 and GalR3 [Mitsukawa K, 2010 Galanin, galanin receptors, and drug targets. EXS 102, 7-23]. There are differences in distribution and pathways between these subtypes.

[0005] GalR1 is expressed in basal forebrain, hypothalamus, spinal cord and colonic epithelial cells where it is upregulated in the case of inflammatory bowel disease (IBD). Not much is known about the effects following stimulation of GalR1. Stimulation of GalR1 seems to be involved in feeding [Saar I, 2011 Novel galanin receptor subtype specific ligands in feeding regulation. Neurochemistry Int 58, 714-20].

[0006] GalR2 is widely distributed in brain, but also occurs in pituitary gland and peripheral tissues. Stimulation of GalR2 has been reported to be neuroprotective in case of MS [Wraith D C, 2009 A role for galanin in human and experimental inflammatory demyelination PNAS 106, 15466-71; Zhang L, 2012 Galanin transgenic mice with elevated circulating galanin levels alleviate demyelination in a cuprizone-induced MS mouse model. PLoS One. 7 (3):e33901] and Alzheimer and to have anxiolytic, antidepressant and anticonvulsant effects.

[0007] GalR3 is widely distributed at low abundance [Lu X, 2005 Distribution and differential regulation of galanin receptor subtypes in rat brain: effects of seizure activity. Neuropeptides 39, 147-52; Mitsukawa K, 2010 Galanin, galanin receptors, and drug targets. EXS 102, 7-23; Hokfelt T, 2008 Galanin--25 years with a multitalented neuropeptide. Cell Mol Life Sci 65, 1793-5; Hokfelt T 2005 Galanin and its receptors: Introduction to the Third International Symposium, San Diego, Calif., USA, 21-22 October 2004. Neuropeptides 39, 125; Kuteeva E, 2008 Galanin, galanin receptor subtypes and depression-like behavior. Cell Mol Life Sci 65, 1854-63 Mol Life Sci 65, 1842-53]. Only little information is available on ligand receptor Gal3 interaction [Sollenberg U E, 2010 Binding of Chimeric Peptides M617 and M871 to Galanin Receptor Type 3 Reveals Characteristics of Galanin Receptor-Ligand Interaction. Int J Pept Res Ther 16, 17].

[0008] Taken together, while effects of stimulation of GalR1 and GalR3 seem unknown or are unbeneficial, agonistic stimulation of GalR2 appears effective in case of MS [Wraith D C, 2009 A role for galanin in human and experimental inflammatory demyelination. PNAS 106, 15466-71; Zhang L, 2012 Galanin transgenic mice with elevated circulating galanin levels alleviate demyelination in a cuprizone-induced MS mouse model. PLoS One. 7(3):e33901].

[0009] GalR2 stimulation leads to neuroprotection [Elliott-Hunt C R, 2007 Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage. J Neurochem 100, 780-9] and anticonvulsant activities [Lu X, 2010 GalR2-positive allosteric modulator exhibits anticonvulsant effects in animal models. PNAS 107, 15229-34]. Galanin appears to exert these anticonvulsant effects through both type 1 and type 2 receptors, with distinct downstream signaling cascades [Lerner J T, 2008 Galanin and epilepsy. Cell Mol Life Sci 65, 1864-71 Neurochem 100, 780-9]. The GalR2 linked G(o)2 mediates the inhibition of insulin release by galanin by regulating both K(ATP) and Ca(2+) channels in mice [Tang G, 2012 Go2 G protein mediates galanin inhibitory effects on insulin release from pancreatic .beta. cells. PNAS 109, 2636-41]. With respect to safety it seems that agonistic specificity for GalR2 and reduced agonistic specificity for GalR3 might be relevant. The main signaling for GALR2 is via Gq through the phospholipase C/protein kinase C pathway, which is not stimulated via GalR1 or GalR3. However, it has been demonstrated that GalR2 can also couple to Gi proteins to inhibit adenylate cyclase, like GalR1 and GalR3; and can also stimulate MAPK.

[0010] The present inventors recognized the need for galanin analogs that display differential pathway activation such that, according to needs, therapeutically relevant pathway(s) can be selectively stimulated. They therefore set out to design novel receptor subtype selective, systemically active, galanin receptor ligands. More specifically, they aimed at providing galanins agonistically acting via GalR2, and/or GalR1, while antagonistically, not or weakly agonistically acting via GalR3 in view of their therapeutic interest. In addition, it would be desirable to have galanins showing reduced capacity to induce receptor internalization, thus enhancing their (in vivo) efficacy.

[0011] It was surprisingly found that at least some of these goals could be met by the introduction of a lanthionine-ring in the galanin peptide. Without wishing to be bound by theory, it is thought that cyclization by the (methyl)lanthionine structure leads to a conformational constraint, which modulates galanine receptor subtype interaction. The (methyl)lanthionine can be introduced not only by replacing existing residues in the endogenous peptide sequence, but also by inserting amino acids.

[0012] Accordingly, the invention provides a cyclic peptide analog of galanin, wherein the galanin analog comprises a (methyl)lanthionine bridge and wherein the analog has the general formula "X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X- 21-X22 (SEQ ID NO:1), or a truncated variant thereof lacking X1 and/or up to 11 of the C-terminal residues,

[0013] wherein two residues selected from the group consisting of X3, X4, X6, X7, X10 and X13-X19 together form a Lanthionine bridge of the structure Ala-S-Ala, or a methylLanthionine bridge of the structure Abu-S-Ala or Ala-S-Abu, wherein:

[0014] X1 is pE, G, N, RGRG (SEQ ID NO:2), RGRGN (SEQ ID NO:3) or RGRGG (SEQ ID NO:4)

[0015] X2 is WN or WT

[0016] X3 is L, (me)lan, A, D, V, K, Q or N

[0017] X4 is N, T, Q or (me)lan

[0018] X5 is S or A

[0019] X6 is A or (me)lan

[0020] X7 is G, (me)lan, A or K

[0021] X8 is Y

[0022] X9 is L

[0023] X10 is L, (me)lan or A

[0024] X11 is G, A or (me)lan

[0025] X12 is P or A or (me)lan

[0026] X13 is H, (me)lan, V, Q, P, Q, E, K or A

[0027] X14 is A, (me)lan, L, Q, P, H, K or I

[0028] X15 is V, (me)lan, P, F, G, K or A

[0029] X16 is (me)lan, P, F, L, N or G

[0030] X17 is N, (me)lan, P, G, S, A or H

[0031] X18 is H, (me)lan, A, L, P, A or R

[0032] X19 is R, (me)lan, L, M, F or A

[0033] X20 is R, A,

[0034] X21 is L, and

[0035] X22 is A, in which

[0036] pE means pyroglutamate;

[0037] (me)lan means Lan or meLan, wherein Lan denotes the N- or C-terminal half of a Lanthionine (Ala-S-Ala) and meLan denotes the N- or C-terminal half of a methylLanthionine (Abu-S-Ala or Ala-S-Abu);

[0038] with the proviso that not more than two of X3, X4, X6, X7, X10 X11 and X13-X19 are (me)lan which together form a (methyl)Lanthionine bridge and wherein said (methyl)Lanthionine bridge is of the size i, i+3 or i, i+4 or i, i+5.

[0039] In another embodiment the invention provides a cyclic peptide analog of galanin, wherein the galanin analog comprises a (methyl)lanthionine bridge and wherein the analog has the general formula

TABLE-US-00001 (SEQ ID NO: 5) "X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14- X15-X16-X17-X18-X19-X20-X21-X22",

wherein two residues selected from the group consisting of X3, X4, X6, X7, X10 and X13-X19 together form a Lanthionine bridge of the structure Ala-S-Ala, or a methylLanthionine bridge of the structure Abu-S-Ala or Ala-S-Abu, and wherein:

[0040] X1 is pE, G, N, RGRG (SEQ ID NO:2), RGRGN (SEQ ID NO:3) or RGRGG (SEQ ID NO:4)

[0041] X2 is WN or WT

[0042] X3 is L, (me)lan, A, D, V, K or Q

[0043] X4 is N, T or (me)lan

[0044] X5 is S or A

[0045] X6 is A or (me)lan

[0046] X7 is G, (me)lan, A or K

[0047] X8 is Y

[0048] X9 is L

[0049] X10 is L, (me)lan or A

[0050] X11 is G, A or (me)lan,

[0051] X12 is P or A

[0052] X13 is H, (me)lan, V, Q, P, Q, E, K or A

[0053] X14 is A, (me)lan, L, Q, P, H, K or I

[0054] X15 is V, (me)lan, P, F, G, K or A

[0055] X16 is (me)lan, P, F, L or G,

[0056] X17 is N, (me)lan, P, G, S or A,

[0057] X18 is H, (me)lan, A, L, P or A,

[0058] X19 is R, (me)lan, L, M, F or A

[0059] X20 is R or A,

[0060] X21 is L

[0061] X22 is A or a truncated variant thereof lacking X1 and/or up to 10 of the C-terminal residues,

[0062] in which pE means pyroglutamate (pGlu); (me)lan means Lan or meLan, wherein Lan denotes the N- or C-terminal half of a Lanthionine (Ala-S-Ala) and meLan denotes the N- or C-terminal half of a methylLanthionine (Abu-S-Ala or Ala-S-Abu);

[0063] with the proviso that not more than two of X3, X4, X6, X7, X10, X11 and X13-X19 are (me)lan which together form a (methyl)Lanthionine bridge and wherein said (methyl)Lanthionine bridge is of the size i, i+3; i, i+4 or i, i+5.

[0064] Cyclic peptide analogs of galanin are known in the art. Carpenter et al. (Biochem. 1999, 38, 15295) constructed a cyclized analog of Gal(1-6) which possessed a lactam bridge between the side-chains of Asp4 and Lys8. This resulted in an analog that exhibited low nanomolar receptor binding affinity and a low nanomolar EC50 for the GalR2 receptor subtype.

[0065] WO03/099863 discloses a modified galanin peptide sequence that can serve as exemplary precursor for introducing a posttranslational lanthionine ring at position Ser6 and Cys10. Nothing is mentioned about the properties of the resulting cyclic analog.

[0066] Green et al. (Bioorg Med Chem. 2013 Jan. 1; 21 (1):303-10) used hydrocarbon stapling as a strategy to stabilize the helical conformation of bioactive peptides. Disclosed is a stapled galanin analog wherein Ala-7 and Leu-11 of the full-length galanin was substituted with (S)-2-(4-)pentenyl) alanine. The analog retained agonist activities towards GalR1 and GalR2, and suppressed seizures in a mouse model of epilepsy. No alteration of receptor specificity was observed.

[0067] A cyclic galanin-analog of the invention, herein also referred to as "lanthi-galanin", is characterized by a (methyl)lanthioninestructure formed by not more than two of X3, X4, X6, X7, X10, X11 and X13-X19 which together form a (methyl)Lanthionine bridge and wherein said (methyl)Lanthionine bridge is of the size i, i+3 or i, i+4 or i, i+5.

[0068] In one embodiment, a galanin analog is of the formula X1-X22, i.e. it contains all of X1 through X22 as defined herein above. In another embodiment, it is a C-terminally truncated variant lacking up to 10 C-terminal residues. For example, it is of the formula X1-X12, lacking all 10 residues X13 through X22. Other truncated variants include X1-X13, X1-X14, X1-X15, X1-X16, X1-X17, X1-X18, X1-X19, X1-X20 and X1-X21. As will be understood by the skilled person, the annotation above refers to an analog including the stretch of contiguous residues e.g. X1-X13 refers to an analog consisting of X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13. Variants lacking residues X20-X22 are preferred.

[0069] X1 can be selected from pE, G, N, RGRG (SEQ ID NO:2), RGRGN (SEQ ID NO:3) and RGRGG (SEQ ID NO:4). In a preferred aspect, X1 is pE or G, more preferably pE. Introduction of pyroglutamate at position 1 can protect the analog against N-terminal hydrolysis. In addition, it was surprisingly found to shift the biological activity of the analog towards a preference for signaling via the Gal2R. Pyroglutamic acid (also known as PCA, 5-oxoproline, pidolic acid, or pyroglutamate for its basic form) is an uncommon amino acid derivative in which the free amino group of glutamic acid or glutamine cyclizes to form a lactam. It is a metabolite in the glutathione cycle that is converted to glutamate by 5-oxoprolinase. Pyroglutamate is found in many proteins including bacteriorhodopsin. N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamate.

[0070] In another embodiment, the lanthi-galanin lacks the N-terminal X1, optionally in combination with a C-terminal truncation of up to 10 residues. Accordingly, also provided is a galanin analog of the formula X2-X22. In a further aspect, the invention provides a variant comprising X2-X12, X2-X13, X2-X14, X2-X15, X2-X16, X2-X17, X2-X18, X2-X19, X2-X20, X2-X21 i.e. containing both an N-terminal and a C-terminal truncation of the general formula X1-X22.

[0071] X2 is WN or WT.

[0072] Preferably, X2 is WN.

[0073] X3 is L, (me)lan, A, D, V, K, Q

[0074] X4 is N, T or (me)lan

[0075] X5 is S or A

[0076] X6 is A or (me)lan

[0077] X7 is G, (me)lan, A or K

[0078] In one aspect, X3 is L, X4 is N, X5 is A, X6 is A, and/or X7 is G.

[0079] X8 is always Y, and X9 is always L

[0080] X10 is L, (me)lan or A

[0081] X11 is G, A, (me)lan,

[0082] X12 is P or A

[0083] X13 is H, (me)lan, V, Q, P, E, K, A

[0084] X14 is A, (me)lan, L, Q, P, H, K, I

For example, X10 is L, X11 is G, X12 is P, X13 is H, and/or X14 is A.

[0085] X15 is V, (me)lan, P, F, G, K, A

[0086] X16 is (me)lan, P, F, L or G,

[0087] X17 is N, (me)lan, P, G, S, A,

[0088] X18 is H, (me)lan, A, L, P, A,

[0089] X19 is R, (me)lan, L, M, F, A

[0090] X20 is R, A,

[0091] X21 is L,

[0092] X22 is A,

[0093] A lanthi-galanin of the invention contains a (methyl)Lanthionine bridge of the size i, i+3 or i, i+4 or i, i+5. Thus, the ring size is limited to those wherein the residues involved in ring formation are spaced by either two, three or four residues. Larger ring sizes were found to be detrimental to the activity. In one embodiment, the (methyl)Lanthionine bridge is of the size i, i+3 or i, i+4. In a specific aspect, the (methyl)Lanthionine bridge is of the size i, i+3. For example, the ring is formed between residues X3 and X6, X4 and X7, X5 and X8, X6 and X9, X7 and X10 or X8 and X11.

[0094] Furthermore, it was surprisingly found that ring introduction from position 13 onwards can be done without significant loss of activity while allowing for altered receptor specificity as compared to wildtype galanin. More in particular, introducing a (methyl)lanthionine in the C-terminal half of the peptide was found to give a shift towards GalR2 preference. Accordingly, in one embodiment, two residues selected from the group consisting of X13-X19 together form a (me)Lanthionine bridge. Suitable pairs of residues forming the ring structure include X13 and X16, X13 and X17, X14 and X17, X14 and X18, X15 and X18, and X15 and X19. Exemplary analogs are GalM50, GalM51, GalM52 and GalM75. In a preferred embodiment, residues X20-X22 are absent and two residues selected from the group consisting of X13-X19 together form a (me)Lanthionine bridge.

[0095] The C-terminal carboxyl group of a galanin analog according to the invention can be in the free (COOH) or amide (CONH.sub.2) form. In one embodiment, the analog is protected by a C-terminal amidation.

Depending on its structure, a galanin analog of the invention can have one or more specific biological activitie(s). In one embodiment, the invention provides an analog that is capable of inhibiting cAMP production in cells expressing a galanin R1 receptor (GalR1). Preferably, cAMP production is inhibited with an EC50 of less than 200 nM.

[0096] Alternatively, the galanin analog is capable of inducing beta arrestin recruitment in cells expressing GalR1 with an EC50 of less than 400 nM.

[0097] The galanin analog is capable of inducing calcium efflux in cells expressing a galanin R2 receptor with an EC50 of less than 100 nM and of inducing beta arrestin recruitment with an EC50 of less than 5 .mu.M.

[0098] In a preferred embodiment, the invention provides a cyclic galanin analog having an enhanced capacity to stimulate GalR2 compared to GalR1. The term "GALR2-specific agonist" indicates an analog capable of triggering a response in a cell as a result of the activation of GALR2 by the substance, but which does not activate (or activates with less potency) GALR1 and/or GALR3. Methods of identifying whether or not a compound is an agonist of a galanin receptor are known in the art, for example, Botella et al. (1995) Gastroenterology 108 3-11 and Barblivien et al. (1995) Neuroreport 6 1849-1852. Exemplary GalR2-specific analogs include GalM54 and GalM54tr. The analog may bind to GalR2 with a binding affinity of between 0 and 100 .mu.M and/or it has a specificity of greater than 30-fold for GalR2 over GalR1. Preferably, it has a specificity of greater that 50 fold for GalR2 over GalR1, more preferably, of greater than 100 fold for GalR2 over GalR1.

[0099] In a specific embodiment, the galanin analog is selected from the group consisting of:

TABLE-US-00002 (SEQ ID NO: 6) GWNmelanNAmelanGYLLGPHAVGNHR (SEQ ID NO: 7) GWNmelanNAmelanGYLLGPHAVGNH (SEQ ID NO: 8) GWNLNAAmelanYLmelanGPHAVGNHR (SEQ ID NO: 9) GWNLNAAmelanYLmelanGPHAVGNH (SEQ ID NO: 10) GWNLNAAlanYLLlanGPHAVGNHR (SEQ ID NO: 11) GWNLNAAlanYLLlanGPHAVGNH (SEQ ID NO: 12) GWNLNAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 13) GWNLNAAGYLLGAmelanAVmelanNH (SEQ ID NO: 14) GWNLNAAGYLLGAlanAVlan (SEQ ID NO: 15) GWNLNAAGYLLGAlanAVlanNHR (SEQ ID NO: 16) GWNLNAAGYLLGAlanAVIanNH (SEQ ID NO: 17) GWNLNAAGYLLGAmelanAVGmelanHR (SEQ ID NO: 18) GWNLNAAGYLLGAlanAVGlan (SEQ ID NO: 19) GWNLNAAGYLLGAlanAVGlanHR (SEQ ID NO: 20) GWNLNAAGYLLGAlanAVGNlanR (SEQ ID NO: 21) GWNLNAAGYLLGAlanAVGNlan (SEQ ID NO: 22) GWNLNAAGYLLGAlanAVGNHlan (SEQ ID NO: 23) GWNLNAAGYLLGPHmelanVGmelanHR (SEQ ID NO: 24) GWNLNAAGYLLGPHlanVGlan (SEQ ID NO: 25) GWNLNAAGYLLGPHAmelanGNmelanR (SEQ ID NO: 26) GWNLNAAGYLLGPHAlanGNlan (SEQ ID NO: 27) GWNLNAAGYLLGASlanVGNHlan (SEQ ID NO: 28) pEWNLNAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 29) pEWNLNAAGYLLGAmelanAVGmelanHR (SEQ ID NO: 30) pEWNLNAAGYLLGAmelanAVmelanNH (SEQ ID NO: 31) pEWNLNAAGYLLGAlanAVlan (SEQ ID NO: 32) pEWNLNAAlanYLLlan (SEQ ID NO: 33) pEWNLNAAGYLLGPHmelanVGmelanHR (SEQ ID NO: 34) GWNLNAAGYLLGmelanHAmelanG (SEQ ID NO: 35) GWNLNAAGYLLGPmelanAVmelan (SEQ ID NO: 36) GWNLNAAGYLLAmelanHAmelanG (SEQ ID NO: 37) GWNLNAAGYLLGAmelanAVmelan (SEQ ID NO: 38) pEWNLNAAGYLLAmelanHAmelanG (SEQ ID NO: 39) pEWNLNAAGYLLGAmelanAVmelan (SEQ ID NO: 40) pEWNLNAAGYLLAlanHAlanG (SEQ ID NO: 41) pEWNLNAAGYLLAmelanHAmelanGNHR (SEQ ID NO: 8) GWNLNAAmelanYLmelanGPHAVGNHR (SEQ ID NO: 42) pEWNLNAAGYLLGPHAmelanGNmelanR (SEQ ID NO: 36) GWNLNAAGYLLAmelanHAmelanG (SEQ ID NO: 38) pEWNLNAAGYLLAmelanHAmelanG (SEQ ID NO: 37) GWNLNAAGYLLGAmelanAVmelan (SEQ ID NO: 39) pEWNLNAAGYLLGAmelanAVmelan (SEQ ID NO: 43) GWNVNAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 44) GWNDNAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 45) GWNNNAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 46) GWNLVAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 47) GWNLKAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 48) GWNLDAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 49) GWNLQAAGYLLGAmelanAVmelanNHR (SEQ ID NO: 50) pEWNLNAAGYLLGPlanAVlan (SEQ ID NO: 51) pEWTLNAAlanYLLlan (SEQ ID NO: 52) pEWNLNAAGYLLGPmelanAVmelan (SEQ ID NO: 53) pEWTLNAAmelanYLLmelan

[0100] Specifically preferred analogs include:

TABLE-US-00003 (GalM50b) (SEQ ID NO: 41) pEWNLNAAGYLLAmelanHAmelanGNHR, (GalM89) (SEQ ID NO: 36) GWNLNAAGYLLAmelanHAmelanG, (GalM89b) (SEQ ID NO: 38) pEWNLNAAGYLLAmelanHAmelanG, (GalM54) (SEQ ID NO: 28) pEWNLNAAGYLLGAmelanAVmelanNHR, (GalM54tr) (SEQ ID NO: 30) pEWNLNAAGYLLGAmelanAVmelanNH (GalM74b) (SEQ ID NO: 42) pEWNLNAAGYLLGPHAmelanGNmelanR (GalM82) (SEQ ID NO: 33) pEWNLNAAGYLLGPHmelanVGmelanHR (4178B isomer B) (SEQ ID NO: 50) pEWNLNAAGYLLGPlanAVIan and (4179B isomer B) (SEQ ID NO: 51) pEWTLNAAlanYLLlan (SEQ ID NO: 52) pEWNLNAAGYLLGPmelanAVmelan (SEQ ID NO: 53) pEWTLNAAmelanYLLmelan

[0101] In a specific embodiment, the galanin analog is selected from the group consisting of:

TABLE-US-00004 (GalM50b) (SEQ ID NO: 41) pEWNLNAAGYLLAmelanHAmelanGNHR, (GalM89) (SEQ ID NO: 36) GWNLNAAGYLLAmelanHAmelanG, (GalM89b) (SEQ ID NO: 38) pEWNLNAAGYLLAmelanHAmelanG, (GalM54) (SEQ ID NO: 28) pEWNLNAAGYLLGAmelanAVmelanNHR, (GalM54 tr) (SEQ ID NO: 30) pEWNLNAAGYLLGAmelanAVmelanNH (GalM74b) (SEQ ID NO: 42) pEWNLNAAGYLLGPHAmelanGNmelanR (GalM82) (SEQ ID NO: 33) pEWNLNAAGYLLGPHmelanVGmelanHR (4178B isomer B) (SEQ ID NO: 50) pEWNLNAAGYLLGPlanAVlan and (4179B isomer B) (SEQ ID NO: 51) pEWTLNAAlanYLLlan (SEQ ID NO: 52) pEWNLNAAGYLLGPmelanAVmelan (SEQ ID NO: 53) pEWTLNAAmelanYLLmelan

[0102] Also provided is a pharmaceutical composition comprising at least one lanthi-galanin according to the invention. A further embodiment relates to a galanin analog according to the invention for use as medicament. A further embodiment relates to a galanin analog selected from the group consisting of:

TABLE-US-00005 (GalM50b) (SEQ ID NO: 41) pEWNLNAAGYLLAmelanHAmelanGNHR, (GalM89) (SEQ ID NO: 36) GWNLNAAGYLLAmelanHAmelanG, (GalM89b) (SEQ ID NO: 38) pEWNLNAAGYLLAmelanHAmelanG, (GalM54) (SEQ ID NO: 28) pEWNLNAAGYLLGAmelanAVmelanNHR, (GalM54tr) (SEQ ID NO: 30) pEWNLNAAGYLLGAmelanAVmelanNH (GalM74b) (SEQ ID NO: 42) pEWNLNAAGYLLGPHAmelanGNmelanR (GalM82) (SEQ ID NO: 33) pEWNLNAAGYLLGPHmelanVGmelanHR (4178B isomer B) (SEQ ID NO: 50) pEWNLNAAGYLLGPlanAVlan and (4179B isomer B) (SEQ ID NO: 51) pEWTLNAAlanYLLlan, for use as medicament. (SEQ ID NO: 52) pEWNLNAAGYLLGPmelanAVmelan (SEQ ID NO: 53) pEWTLNAAmelanYLLmelan

[0103] Also provided is a pharmaceutical composition comprising a galanin analog selected from the group consisting of:

TABLE-US-00006 (GalM50b) (SEQ ID NO: 41) pEWNLNAAGYLLAmelanHAmelanGNHR, (GalM89) (SEQ ID NO: 36) GWNLNAAGYLLAmelanHAmelanG, (GalM89b) (SEQ ID NO: 38) pEWNLNAAGYLLAmelanHAmelanG, (GalM54) (SEQ ID NO: 28) pEWNLNAAGYLLGAmelanAVmelanNHR, (GalM54tr) (SEQ ID NO: 30) pEWNLNAAGYLLGAmelanAVmelanNH (GalM74b) (SEQ ID NO: 42) pEWNLNAAGYLLGPHAmelanGNmelanR (GalM82) (SEQ ID NO: 33) pEWNLNAAGYLLGPHmelanVGmelanHR (4178B isomer B) (SEQ ID NO: 50) pEWNLNAAGYLLGPlanAVlan and (4179B isomer B) (SEQ ID NO: 51) pEWTLNAAlanYLLlan. (SEQ ID NO: 52) pEWNLNAAGYLLGPmelanAVmelan (SEQ ID NO: 53) pEWTLNAAmelanYLLmelan

[0104] The medicament is advantageously used for the prevention or treatment of brain injury, damage or disease. The brain injury or damage is caused by one of : embolic, thrombotic or haemorrhagic stroke; direct or indirect trauma or surgery to the brain or spinal cord; ischaemic or embolic damage to the brain during cardiopulmonary bypass surgery or renal dialysis; reperfusion brain damage following myocardial infarction; brain disease; immunological damage, chemical damage or radiation damage. The brain disease can be one of Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis or variant Creutzfeld Jacob Disease.

[0105] For example, a galanin analog finds its use as anxiolyticum, antidepressant, anticonvulsant or for neuroprotection. Specific preferred conditions to be treated are Multiple Sclerosis, Alzheimer's disease and Inflammatory Bowel Disease.

[0106] Also provided herein is a method for providing a cyclic galanin analog according to the invention. In one embodiment, the lanthioninegalanine analog can be obtained by baseassisted sulfur extrusion of a cyclic disulfide-bridge-containing "parent" analog, in particular disulfide bridged D-Cys-L-Cys analogs. For example, the parent analog is pEWNLNAAdCYLLC (SEQ ID NO:54), pEWNLNAAGYLLGAdCAVC (SEQ ID NO:55), pEWNLNAAGYLLGPHdCVGC (SEQ ID NO:56) or pEWNLNAAGYLLGPHAdCGNC (SEQ ID NO:57). Wherein a disulfide bridge is formed by dC and the C-terminal C.

[0107] In another embodiment, the method comprises the steps of (i) providing a peptide (i.e. the linear precursor peptide) selected from the group consisting of GWNTNACGYLLGPHAVGNHR (SEQ ID NO:58),

TABLE-US-00007 (SEQ ID NO: 59) GWNTNACGYLLGPHAVGNH, (SEQ ID NO: 60) GWNLNAATYLCGPHAVGNHR, (SEQ ID NO: 61) GWNLNAATYLCGPHAVGNH, (SEQ ID NO: 62) GWNLNAASYLLCGPHAVGNHR, (SEQ ID NO: 63) GWNLNAASYLLCGPHAVGNH, (SEQ ID NO: 64) GWNLNAAGYLLGATAVCNHR, (SEQ ID NO: 65) GWNLNAAGYLLGATAVCNH, (SEQ ID NO: 66) GWNLNAAGYLLGASAVC, (SEQ ID NO: 67) GWNLNAAGYLLGASAVCNHR, (SEQ ID NO: 68) GWNLNAAGYLLGASAVCNH, (SEQ ID NO: 69) GWNLNAAGYLLGATAVGCHR, (SEQ ID NO: 70) GWNLNAAGYLLGASAVGC, (SEQ ID NO: 71) GWNLNAAGYLLGASAVGCHR, (SEQ ID NO: 72) GWNLNAAGYLLGASAVGNCR, (SEQ ID NO: 73) GWNLNAAGYLLGASAVGNC, (SEQ ID NO: 74) GWNLNAAGYLLGASAVGNHC, (SEQ ID NO: 75) GWNLNAAGYLLGPHTVGCHR, (SEQ ID NO: 76) GWNLNAAGYLLGPHSVGC, (SEQ ID NO: 77) GWNLNAAGYLLGPHATGNCR, (SEQ ID NO: 78) GWNLNAAGYLLGPHASGNC, (SEQ ID NO: 74) GWNLNAAGYLLGASAVGNHC, (SEQ ID NO: 79) QWNLNAAGYLLGATAVCNHR, (SEQ ID NO: 80) QWNLNAAGYLLGATAVCNH, (SEQ ID NO: 81) QWNLNAAGYLLGASAVC, (SEQ ID NO: 82) QWNLNAASYLLC, (SEQ ID NO: 83) QWNLNAAGYLLGPHTVGCHR, (SEQ ID NO: 84) GWNLNAAGYLLGTHACG, (SEQ ID NO: 85) GWNLNAAGYLLGPTAVC, (SEQ ID NO: 86) GWNLNAAGYLLATHACG, (SEQ ID NO: 87) GWNLNAAGYLLGATAVC, (SEQ ID NO: 88) QWNLNAAGYLLATHACG, (SEQ ID NO: 89) pEWNLNAAGYLLGATAVC, (SEQ ID NO: 90) QWNLNAAGYLLASHACG, (SEQ ID NO: 91) QWNLNAAGYLLATHACGNHR, (SEQ ID NO: 60) GWNLNAATYLCGPHAVGNHR, (SEQ ID NO: 92) QWNLNAAGYLLGPHATGNCR, (SEQ ID NO: 86) GWNLNAAGYLLATHACG, (SEQ ID NO: 88) QWNLNAAGYLLATHACG, (SEQ ID NO: 87) GWNLNAAGYLLGATAVC, (SEQ ID NO: 93) QWNLNAAGYLLGATAVC, (SEQ ID NO: 94) GWNVNAAGYLLGATAVCNHR, (SEQ ID NO: 95) GWNDNAAGYLLGATAVCNHR, (SEQ ID NO: 96) GWNNNAAGYLLGATAVCNHR, (SEQ ID NO: 97) GWNLVAAGYLLGATAVCNHR, (SEQ ID NO: 98) GWNLKAAGYLLGATAVCNHR, (SEQ ID NO: 99) GWNLDAAGYLLGATAVCNHR, (SEQ ID NO: 100) GWNLQAAGYLLGATAVCNHR, (SEQ ID NO: 101) QWNLNAAGYLLGPTAVC, (SEQ ID NO: 102) QWTLNAATYLLC, (SEQ ID NO: 103) QWNLNAAGYLLGPSAVC, (SEQ ID NO: 104) QWTLNAASYLLC

[0108] (ii) inducing dehydration of a Ser or Thr residue of said peptide; and

[0109] (iii) inducing ring closure by coupling the dehydrated Ser or Thr to the thiol group of the Cys residue of said peptide.

[0110] Step (iii) may comprise inducing ring closure by chemical or enzymatic means. Preferably, it comprises enzymatic ring closure. E.g. by exploiting the lanthipeptide enzyme machinery of a (bacterial) host cell according to methods known in the art.

[0111] In case the N-terminal residue of the peptide is glutamine (Q), the method preferably further comprises conversion of Q to pE according to methods known in the art, for example as described in Rink et al. (Journal of Pharmacological and Toxicological Methods 61 (2010) 210-218)

[0112] Specifically preferred linear peptide sequences for providing a lanthi-galanin include

QWNLNAAGYLLATHACGNHR (SEQ ID NO:91) (e.g. to produce GalM50b), GWNLNAAGYLLATHACG (SEQ ID NO:86) (e.g. to produce GalM89), QWNLNAAGYLLATHACG (SEQ ID NO:88) (e.g. to produce GalM89b), QWNLNAAGYLLGATAVCNHR (SEQ ID NO:79) (e.g. to produce GalM54), QWNLNAAGYLLGPHATGNCR (SEQ ID NO:92) (e.g. to produce GalM74b)QWNLNAAGYLLGPHTVGCHR (SEQ ID NO:83) (e.g. to produce GalM82), QWNLNAAGYLLGPSAVC (SEQ ID NO:103) (e.g. to produce 4178B isomer B) and QWTLNAASYLLC (SEQ ID NO:104) (e.g. to produce 4179B isomer B). QWNLNAAGYLLGPTAVC (SEQ ID NO:101) (to produce 4178) QWTLNAATYLLC (SEQ ID NO:102) (to produce 4179)

[0113] Also provided is an isolated nucleic acid sequence encoding a precursor peptide recited above, and a vector comprising said nucleic acid sequence, which nucleic acid is genetically fused to a nucleic acid sequence encoding a lanthipeptide-leader sequence.

[0114] Preferred nucleic acid sequences are those encoding one of the following peptide sequences:

TABLE-US-00008 (SEQ ID NO: 58) GWNTNACGYLLGPHAVGNHR, (SEQ ID NO: 59) GWNTNACGYLLGPHAVGNH, (SEQ ID NO: 60) GWNLNAATYLCGPHAVGNHR, (SEQ ID NO: 61) GWNLNAATYLCGPHAVGNH (SEQ ID NO: 62) GWNLNAASYLLCGPHAVGNHR, (SEQ ID NO: 63) GWNLNAASYLLCGPHAVGNH (SEQ ID NO: 64) GWNLNAAGYLLGATAVCNHR, (SEQ ID NO: 65) GWNLNAAGYLLGATAVCNH, (SEQ ID NO: 66) GWNLNAAGYLLGASAVC, (SEQ ID NO: 67) GWNLNAAGYLLGASAVCNHR, (SEQ ID NO: 68) GWNLNAAGYLLGASAVCNH, (SEQ ID NO: 69) GWNLNAAGYLLGATAVGCHR, (SEQ ID NO: 70) GWNLNAAGYLLGASAVGC, (SEQ ID NO: 71) GWNLNAAGYLLGASAVGCHR, (SEQ ID NO: 72) GWNLNAAGYLLGASAVGNCR, (SEQ ID NO: 73) GWNLNAAGYLLGASAVGNC, (SEQ ID NO: 74) GWNLNAAGYLLGASAVGNHC, (SEQ ID NO: 75) GWNLNAAGYLLGPHTVGCHR, (SEQ ID NO: 76) GWNLNAAGYLLGPHSVGC, (SEQ ID NO: 77) GWNLNAAGYLLGPHATGNCR, (SEQ ID NO: 78) GWNLNAAGYLLGPHASGNC (SEQ ID NO: 74) GWNLNAAGYLLGASAVGNHC, (SEQ ID NO: 79) QWNLNAAGYLLGATAVCNHR, (SEQ ID NO: 80) QWNLNAAGYLLGATAVCNH, (SEQ ID NO: 81) QWNLNAAGYLLGASAVC, (SEQ ID NO: 82) QWNLNAASYLLC, (SEQ ID NO: 83) QWNLNAAGYLLGPHTVGCHR, (SEQ ID NO: 84) GWNLNAAGYLLGTHACG, (SEQ ID NO: 85) GWNLNAAGYLLGPTAVC, (SEQ ID NO: 86) GWNLNAAGYLLATHACG, (SEQ ID NO: 87) GWNLNAAGYLLGATAVC, (SEQ ID NO: 88) QWNLNAAGYLLATHACG, (SEQ ID NO: 89) pEWNLNAAGYLLGATAVC, (SEQ ID NO: 90) QWNLNAAGYLLASHACG, (SEQ ID NO: 91) QWNLNAAGYLLATHACGNHR, (SEQ ID NO: 60) GWNLNAATYLCGPHAVGNHR, (SEQ ID NO: 92) QWNLNAAGYLLGPHATGNCR, (SEQ ID NO: 86) GWNLNAAGYLLATHACG, (SEQ ID NO: 88) QWNLNAAGYLLATHACG, (SEQ ID NO: 87) GWNLNAAGYLLGATAVC, (SEQ ID NO: 93) QWNLNAAGYLLGATAVC, (SEQ ID NO: 94) GWNVNAAGYLLGATAVCNHR, (SEQ ID NO: 95) GWNDNAAGYLLGATAVCNHR, (SEQ ID NO: 96) GWNNNAAGYLLGATAVCNHR, (SEQ ID NO: 97) GWNLVAAGYLLGATAVCNHR, (SEQ ID NO: 98) GWNLKAAGYLLGATAVCNHR, (SEQ ID NO: 99) GWNLDAAGYLLGATAVCNHR, (SEQ ID NO: 100) GWNLQAAGYLLGATAVCNHR, (SEQ ID NO: 101) QWNLNAAGYLLGPTAVC, (SEQ ID NO: 102) QWTLNAATYLLC, (SEQ ID NO: 103) QWNLNAAGYLLGPSAVC, (SEQ ID NO: 104) QWTLNAASYLLC.

[0115] Specifically preferred nucleic acids encoding linear peptide sequences for providing a lanthi-galanin include

QWNLNAAGYLLATHACGNHR (SEQ ID NO:91) (e.g. to produce GalM50b), GWNLNAAGYLLATHACG (SEQ ID NO:86) (e.g. to produce GalM89), QWNLNAAGYLLATHACG (SEQ ID NO:88) (e.g. to produce GalM89b), QWNLNAAGYLLGATAVCNHR (SEQ ID NO:79) (e.g. to produce GalM54), QWNLNAAGYLLGPHATGNCR (SEQ ID NO:92) (e.g. to produce GalM74b) QWNLNAAGYLLGPHTVGCHR (SEQ ID NO:83) (e.g. to produce GalM82) QWNLNAAGYLLGPSAVC (SEQ ID NO:103) (e.g. to produce 4178B isomer B) QWTLNAASYLLC (SEQ ID NO:104) (e.g. to produce 4179B isomer B). QWNLNAAGYLLGPTAVC (SEQ ID NO:101) (to produce 4178) and QWTLNAASYLLC (SEQ ID NO:102) (to produce 4179).

[0116] Further embodiments relate to a food-grade bacterial host cell, preferably a L. lactis host cell, comprising a vector according to the invention, additionally comprising lanthionine-introduction enzymes. For example, the host cell endogenously contains the lantibiotic enzyme machinery, or it is provided with a plasmid encoding the maturation enzymes NisB and NisC, and the translocation enzyme NisT. Such host cells are advantageously used as an oral delivery system for the in situ production of lanthi-galanins in the gut.

[0117] Hence, the invention also relates to a pharmaceutical composition comprising viable host cells comprising a nucleic acid sequence encoding a precursor peptide recited above, and a pharmaceutically acceptable carrier, diluent or vehicle. Preferably, the composition is formulated for oral administration.

[0118] The host cells capable of producing the lanthi-galanin in situ are suitably used in a method of treating or alleviating the symptoms associated with inflammatory bowel disease, for instance wherein the bowel disease is Crohn's disease or ulcerative colitis (UC).

[0119] Therapeutic uses of GalR1 and GalR3 antagonists of the invention are the following. Galanin is thought to play a potentially key role in IBD. Gal1R is the only gal receptor found in colonic tissue. Cloning of the Gal1R gene has revealed multiple recognition sites for the inflammation-associated transcription factor NF kappa B. A GalR1-specific antagonist may result in new pharmacologic therapies for the treatment of diarrhea resulting from the inflammation [Gross 2007 Role of Inflammatory Bowel Disease. Inflamm Bowel Disease 13, 918-32].

[0120] Antagonism of GalR3 has been reported to reduce pancreatitis [Barreto S G, 2011 Galanin receptor 3 a potential target for acute pancreatitis therapy. Neurogastroenterol Motil 23 (3):e141-51]. GalR3 antagonists seem furthermore to act as antidepressant [Swanson C J, 2005 Anxiolytic- and antidepressant-like profiles of the galanin-3 receptor (Gal3) antagonists SNAP 37889 and SNAP 398299. PNAS 102, 17489-94].

BRIEF DESCRIPTION OF THE FIGURES

[0121] FIG. 1: Exemplary analog GalM51-induced .beta.-arrestin recruitment via GalR1

[0122] FIG. 2: Exemplary analog GalM54 preferentially stimulates .beta.-arrestin recruitment via GalR2

[0123] FIG. 3: Exemplary analogs GalM51 and GalM52 inhibit cAMP production via GalR1

EXPERIMENTAL SECTION

Example 1

Synthesis of Lanthionine-Stabilized Galanin Analogs (Lanthi-Galanins)

[0124] Lanthionine containing galanin variants were made according to established procedures described for example in Kluskens 2005 Post-translational Modification of Therapeutic Peptides by NisB, the Dehydratase of the Lantibiotic Nisin. Biochemistry 44, 12827-12834; Kluskens 2009 Angiotensin-(1-7) with thioether-bridge: an ACE-resistant, potent Ang-(1-7) analogue. J. Pharmacol. Exper. Ther. 328, 849-854; Rink 2007c NisC, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed non-lantibiotic peptides. Biochemistry 46, 13179-13189.

[0125] Briefly, Lactococcus lactis comprising a two plasmid system was used. The first plasmid encoded the leader peptide of the lantibiotic nisin MSTKDFNLDLVSVSKKDSGASPR (SEQ ID NO: 105) genetically fused at its C-terminus to the precursor peptide of the aimed for (methyl)lanthionine-galanin which peptide contains at position [i] a serine/threonine and at position [i+3], [i+4] or [i+5] a cysteine. The plasmid encoding the fusion peptide comprising the nisin leader and the (methyl)lanthionine galanin precursor was co-expressed in L. lactis with the second plasmid pIL3BTC plasmid, encoding the maturation enzymes NisB and NisC, and the translocation enzyme NisT. NisB dehydrated the serine or threonine to yield dehydroalanine and dehydrobutyrine, respectively. Subsequently, the cyclase NisC covalently coupled the dehydroamino acid to a cysteine, yielding a lanthionine (lan) or methyllanthionine (melan) respectively. The second plasmid encodes the nisin modification and export enzymes NisBTC.

[0126] The practical procedure for production (Kluskens 2005 Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin. Biochemistry 44, 12827-34 ; Rink 2005 Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes. 44, 8873-82), isolation and purification of the modified galanin variants was as follows:

[0127] The culture Lactococcus lactis NZ9000 pIL3BTC pNZ-GalM'X' was grown overnight in M17 broth (Difco) supplemented with 0.5% glucose and the antibiotics chloramphenicol (5 .mu.g/ml) and erythromycin (5 .mu.g/ml). Next day 1 ml culture was diluted in 100 ml minimal medium (Jensen P R, Hammer K. 1993 Minimal Requirements for Exponential Growth of Lactococcus lactis. Appl Environ Microbiol 59, 4363-6; Rink 2005 Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes. 44, 8873-82) supplemented with nisin (1 .mu.g/ml). The culture was further grown for 24 - 48 hours. Peptides from cell-free supernatant were precipitated in 10% trichloroacetic acid (TCA). Subsequently the galanin peptide was liberated from the leader peptide by digestion of the fusion peptide, dissolved in 40 mM Tris (pH=8), 100 mM NaCl, 2 mM CaCl.sub.2, with the protease Factor Xa (5 .mu.g) for 18 h at 30.degree. C. The digestion mixture was applied on a Phenomenex C12 250.times.4.6 mm.times.4 micron column using a HP1050 HPLC system or a JASCO PU-1580 HPLC system. The peptides were eluted with a gradient using as buffer A (0.1% trifluoracetic acid (TFA) in milliQ) and buffer B (0.1% TFA in acetonitrile). The used gradient was 10% to 50% buffer B with a slope of 1.14%/min. Peaks were collected and dried using a speed-vacuum apparatus. Peptides were analyzed by mass spectrometry after incubation with or without 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). An addition of CDAP results in a shift of +25 Dalton and is present when there is no ring closure (Rink. 2007c NisC, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed non-lantibiotic peptides. Biochemistry 46, 13179-13189). When needed, an N-terminal pyroglutamate (pE) was introduced by incubation of the purified galanin peptide containing an N-terminal glutamine for 18 h at 50.degree. C. in 50 mM phosphate buffer (pH=7.6). A second HPLC run was performed for isolation of the galanin variant with the N-terminal pyroglutamate. Quantification of the purified galanin variants was performed by comparing the peak area of the compound in the HPLC chromatogram detected at 280 nm with the area of a known amount of reference Galanine-(1-15). Peptides with a methyllanthionine were enzymatically, D,L stereospecifically cyclized by NisC. Introduction of a lanthionine (lan) into a peptide the stereospecificity has not been established here since , due the reactivity the coupling of the highly reactive dehydroalanine can take place not only via catalysis by NisC but also spontaneously.

Example 2

Activity of Lanthionine-Stabilized Galanin Analogs (Lanthi-Galanins)

[0128] The biological activity of the (lanthi)galanins was determined in CHO-K1 cell lines that express either the GalR1, or GalR2 receptor. Activity measurements were performed using commercial kits from DiscoverX for determining arrestin recruitment (GalR1, GalR2) and inhibition of cAMP synthesis (GalR1). In addition, HEK293 cell lines that express the GalR2 receptor have been used for methods from Molecular Probes to measure calcium efflux (GalR2).

TABLE-US-00009 TABLE 1 GalR1 GalR2 Ratio EC5Os relative to Gal-(1-15) .beta. .beta. Galanin Arrestin cAMP Arrestin Ca.sup.2+ GWNLNAAGYLLGPHAVGNHRSFSDKNGLTS ND ND ND ND (SEQ ID NO: 106) Wild type galanine-(1-30) Bersani 1991 FEBS Letters 283, 189-194 Lanthi-galanin precursors. T..C and S..C have been enzymatically converted into methyllanthionine (melan) and lanthionine (lan), purified and activites measured Gal1-15: GWTLNSAGYLLGPHA (SEQ ID NO: 107) 1 1 1 1 Diaz-Cabiale 2005 Neuropeptides 39, 185-190 T3N-Gal-(1-22); termed: GalWtb 1.7 1.6 3.8 2.3 GVVNLNAAGYLLGPHAVGNHR (SEQ ID NO: 108) GalM50: GWNLNAAGYLLATHACGNHR 3.8 14 0.5 2.8 (SEQ ID NO: 109) GalM50b: pEWNLNAAGYLLATHACGNHR ND no 6.7 4.8 (SEQ ID NO: 110) activity GalM51: GWNLNAAGYLLGATAVCNHR 1.5 3.6 0.6 2.9 (SEQ ID NO: 64) GalM52: GWNLNAAGYLLGPHTVGCHR 8.3 7.5 0.6 1.9 (SEQ ID NO: 75) GalM54: pEVVNLNAAGYLLGATAVCNHR >1000 >1000 1.4 3.4 (SEQ ID NO: 111) GalM54tr: pEVVNLNAAGYLLGATAVCNH >1000 16 1 1.2 (SEQ ID NO: 112) GalM60: GWNLNAATYLCGPHAVGNHR (SEQ ID NO: 60) 12.7 >1000 >1000 8.7 GalM73: GWNLNAASYLLCGPHAVGNHR 21.2 no 45 5.0 (SEQ ID NO: 62) activity GalM74: GWNLNAAGYLLGPHATGNCR 2.3 4.4 2.6 5.0 (SEQ ID NO: 77) GalM74b: pEWNLNAAGYLLGPHATGNCR 129 No 4.2 2.6 (SEQ ID NO: 113) activity GalM75: GWNLNAAGYLLGASAVCNHR 4.3 1.6 1.8 3 (SEQ ID NO: 67) GalM76: GVVNLNAAGYLLGATAVGCHR 1 0.6 1.0 0.9 (SEQ ID NO: 69) GalM76b: pEWNLNAAGYLLGATAVGCHR 9.6 12.2 0.5 1 (SEQ ID NO: 114) GalM77: GVVNLNAAGYLLGASAVGCHR 1.6 2 3.8 2.6 (SEQ ID NO: 71) GalM78: GWNLNAAGYLLGASAVGNCR 1.6 1.6 2.7 2.1 (SEQ ID NO: 72) GalM79: GWNLNAAGYLLGASAVGNHC 1.6 ND 0.7 1.9 (SEQ ID NO: 74) GalM82: pEWNLNAASYLLC ND ND 493 12.9 (SEQ ID NO: 115) GalM83: pEWNLNAAGYLLGPHTVGCHR 123 no 78 7.3 (SEQ ID NO: 116) activity GalM84: GWNLNAAGYLLGTHACG 35 ND >1000 6.3 (SEQ ID NO: 84) GalM85: GVVNLNAAGYLLGPTAVC 18 11.6 6.2 4.7 (SEQ ID NO: 85) GalM87: pEWNLNAAGYLLGPTAVC 260 no 4.8 3 (SEQ ID NO: 117) activity GalM89: GWNLNAAGYLLATHACG 148 no 3 2.6 (SEQ ID NO: 86) activity GalM89b: pEWNLNAAGYLLATHACG no no 2.7 2.1 (SEQ ID NO: 118) activity activity GalM90: GWNLNAAGYLLGATAVC 2.7 4.9 3.2 4.6 (SEQ ID NO: 87) GalM91: pEWNLNAAGYLLGATAVC 3 >1000 1.5 1.3 (SEQ ID NO: 89) GalM92: GWNVNAAGYLLGATAVCNHR 10.1 ND 3.3 4.4 (SEQ ID NO: 94) GalM93: GVVNDNAAGYLLGATAVCNHR 34.6 ND 141 7.2 (SEQ ID NO: 95) GalM94: GWNNNAAGYLLGATAVCNHR 70.3 ND ND 7.1 (SEQ ID NO: 96) GalM95: GWNLVAAGYLLGATAVCNHR ND ND ND 107 (SEQ ID NO: 97) GalM96: GWNLKAAGYLLGATAVCNHR ND ND ND 534 (SEQ ID NO: 98) GalM97: GWNLDAAGYLLGATAVCNHR ND ND ND 502 (SEQ ID NO: 99) GalM98: GWNLQAAGYLLGATAVCNHR ND ND ND 6.7 (SEQ ID NO: 100) GalWtb: GWNLNAAGYTJPrPHAVGNHR 1.7 1.6 3.8 2.7 (SEQ ID NO: 108) GalM92: GWNVNAAGYLLGATAVCNHR 5.3 6.1 1.8 4.4 (SEQ ID NO: 94) GalM93: GWNDNAAGYLLGATAVCNHR 30 41 141 7.2 (SEQ ID NO: 95) GalM94: GWNNNAAGYLLGATAVCNHR 46 41 5.4 7.1 (SEQ ID NO: 96) GalM95: GWNLVAAGYLLGATAVCNHR no no >1000 107 (SEQ ID NO: 97) activity activity GalM96: GWNLKAAGYLLGATAVCNHR NA no no 534 (SEQ ID NO: 98) activity activity GalM97: GWNLDAAGYLLGATAVCNHR no no >1000 502 (SEQ ID NO: 99) activity activity GalM98: GWNLQAAGYLLGATAVCNHR 23 28 53 6.7 (SEQ ID NO: 100) GalM101: GWNLNKAGYLLGPHAVGNHR 365 no no 118 (SEQ ID NO: 119) activity activity GalM102: GWNLNDAGYLLGPHAVGNHR NA no no no (SEQ ID NO: 120) activity activity activity GalM103: GWNLNLAGYLLGPHAVGNHR >1000 14.1 no no (SEQ ID NO: 121) activity activity GalM104: GWNLNWAGYLLGPHAVGNHR NA NA NA NA (SEQ ID NO: 122) GalM105: GWNLNAAKYLLGPHAVGNHR no no 29 45 (SEQ ID NO: 123) activity activity GalM106: GVVNLNAADYLLGPHAVGNHR NA no no no (SEQ ID NO: 124) activity activity activity GalM107: GWNLNAAQYLLGPHAVGNHR no no >1000 31 (SEQ ID NO: 125) activity activity GalM108: GVVNLNAAPYLLGPHAVGNHR NA no >1000 no (SEQ ID NO: 126) activity activity

Results

[0129] The results are summarized in Table 1. Values indicate the EC50 of the analog expressed relative to that observed for linear galanin (1-15), which is set at a value of 1.0. ND indicates `not determined`.

[0130] It was surprisingly found that introduction of a (methyl)lanthionine in the C-terminus of the galanin peptide gives a slight shift towards GalR2 preference. Especially GalM50b, GalM89b, GalM54, GalM87, GalM74b have a preference for stimulating GalR2. The presence of an N-terminal pE seems to contribute to the preference for GalR2 of some analogs (e.g. GalM50b, GalM89b, GalM74b), but not of others (e.g. GalM76b).

[0131] Furthermore, a preferred ring position with respect to activity appears to be at the C-terminal part. Preferably, the ring is positioned after X12. GalM73, despite having the ring more central, has retained some activity. GalM82 is of particular interest because, despite its low activity, it is small and hydrophobic which most likely favors passage over the blood brain barrier (BBB). Moreover, both the N-terminus and C-terminus are protected, and it can be easily synthesized chemically.

[0132] In addition GalM92 and GalM94 which have a L4V and L4N substitution, respectively, have comparable efficacy via GalR2 like GalWtb.

Example 3

Chemically Synthesized Lanthionine Analogs of Galanine Obtained by Base Assisted Sulfur Extrusion of Disulfide Bridge Variants

[0133] Two peptides were ordered at Pepscan as crude material: 1) Sym-4178: H-QWNLNAAGYLLGPcAVC-OH (SEQ ID NO:127) This peptide is related to GalM87. However, while the biologically synthesized GalM87 likely is D,L lanthionine containing, the chemical synthesis leads to more than one isomer. Interestingly one of the lanthionine-containing isomers 4178B (Table 2) is highly active. 2) Sym-4179: H-QWTLNAAcYLLC-OH (SEQ ID NO:128).

[0134] This peptide is related to GalM82 (Table 1). However, while the biologically synthesized GalM82 likely is D,L lanthionine containing, the chemical synthesis leads to more than one isomer. Most interestingly one of these, 4179B (Table 2) seems to have high activity.

[0135] Peptide was dissolved in 0.3% ammonia. Peptides were incubated o/n at 37.degree. C. for the introduction of the lanthionine via base assisted sulfur extrusion of the disulfide bridged peptide. Different isomers could be isolated by HPLC applying the same method as was used as for the biologically produced peptides. Different isomers were purified and lyophilized. Dry peptides were dissolved in 100 mM phosphate buffer pH=7.6 and peptides were incubated o/n at 50.degree. C. After HPLC purification peptides with a pE formation were selected and used in the different Galanin receptor activation assays.

Results

TABLE-US-00010

[0136] TABLE 2 Lanthi-galanin variants chemically obtained by base- Ratio EC5Os relative to Gal-(1-15) assisted sulfur extrusion GalR1 GalR2 4178 similar to M87 but possibly a .beta.- cAMP .beta.- Ca different lanthionine Arrestin inhibition Arrestin efflux 4178 pEWNLNAAGYLLGPlanAVlan >1000 no activity 137 11 A (SEQ ID NO: 50) isomer A 4178 pEVVNLNAAGYLLGPlanAVlan 39 no activity 2.3 1.4 B (SEQ ID NO: 50) isomer B 4179 similar to M82 but likely a different lanthionine isomer 4179 pEWTLNAAlanYLLlan no no activity NA 94 A (SEQ ID NO: 51) activity isomer A 4179 pEWTLNAAlanYLLlan 720 no activity 3.8 2.6 B (SEQ ID NO: 51) isomer B 4179 pEWTLNAAlanYLLlan ND ND ND 248 C (SEQ ID NO: 51) isomer C Isomers A, B, C are the different unidentified isomers which could be isolated after the thioether introduction; ND = not determined. Lan..lan is 1 lanthionine (Ala-S-Ala).

Sequence CWU 1

1

128127PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"VARIANT(1)..(5)/replace="pyroGlu" or "Gly" or "Asn" or "Arg-Gly-Arg-Gly" or "Arg-Gly-Arg-Gly-Gly"MISC_FEATURE(1)..(5)/note="This region may encompass 1-5 residues"VARIANT(7)..(7)/replace"Thr"VARIANT(8)..(8)/replace="Abu" or "Ala" or "Asp" or "Val" or "Lys" or "Gln" or "Asn"VARIANT(9)..(9)/replace="Thr" or "Gln" or "Abu" or "Ala"VARIANT(10)..(10)/replace="Ala"VARIANT(11)..(11)/replace="Abu"VARIAN- T(12)..(12)/replace="Abu" or "Ala" or "Lys"VARIANT(15)..(15)/replace="Abu" or "Ala"VARIANT(16)..(16)/replace="Ala" or "Abu"VARIANT(17)..(17)/replace="Ala" or "Abu"VARIANT(18)..(18)/replace="Abu" or "Val" or "Gln" or "Pro" or "Glu" or "Lys" or "Ala"VARIANT(19)..(19)/replace="Abu" or "Leu" or "Gln" or "Pro" or "His" or "Lys" or "Ile"VARIANT(20)..(20)/replace="Abu" or "Pro" or "Phe" or "Gly" or "Lys" or "Ala"VARIANT(21)..(21)/replace="Abu" or "Phe" or "Leu" or "Asn" or "Gly" or "Ala"VARIANT(22)..(22)/replace="Abu" or "Pro" or "Gly" or "Ser" or "Ala" or "His"VARIANT(23)..(23)/replace="Abu" or "Ala" or "Leu" or "Pro" or "Arg"VARIANT(24)..(24)/replace="Abu" or "Leu" or "Met" or "Phe" or "Ala"VARIANT(25)..(25)/replace="Ala"MISC_FEATURE(1)..(27)/note="Positions 8, 9, 11, 12 and 15-24 may encompass a single methylLanthionine or Lanthionine bridge of the structure Abu-S-Ala, Ala-S-Abu or Ala-S-Ala"MISC_FEATURE(1)..(27)/note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions"source/note="See specification as filed for detailed description of substitutions and preferred embodiments" 1Arg Gly Arg Gly Asn Trp Asn Leu Asn Ser Ala Gly Tyr Leu Leu Gly1 5 10 15Pro His Ala Val Pro Asn His Arg Arg Leu Ala 20 2524PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 2Arg Gly Arg Gly135PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 3Arg Gly Arg Gly Asn1 545PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 4Arg Gly Arg Gly Gly1 5527PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"VARIANT(1)..(5)/replace="pyroGlu" or "Gly" or "Asn" or "Arg-Gly-Arg-Gly" or "Arg-Gly-Arg-Gly-Gly"MISC_FEATURE(1)..(5)/note="This region may encompass 1-5 residues"VARIANT(7)..(7)/replace"Thr"VARIANT(8)..(8)/replace="Abu" or "Ala" or "Asp" or "Val" or "Lys" or "Gln"VARIANT(9)..(9)/replace="Thr" or "Abu" or "Ala"VARIANT(10)..(10)/replace="Ala"VARIANT(11)..(11)/replace="A- bu"VARIANT(12)..(12)/replace="Abu" or "Ala" or "Lys"VARIANT(15)..(15)/replace="Abu" or "Ala"VARIANT(16)..(16)/replace="Ala" or "Abu"VARIANT(17)..(17)/replace="Ala"VARIANT(18)..(18)/replace="Abu" or "Val" or "Gln" or "Pro" or "Glu" or "Lys" or "Ala"VARIANT(19)..(19)/replace="Abu" or "Leu" or "Gln" or "Pro" or "His" or "Lys" or "Ile"VARIANT(20)..(20)/replace="Abu" or "Pro" or "Phe" or "Gly" or "Lys" or "Ala"VARIANT(21)..(21)/replace="Abu" or "Phe" or "Leu" or "Gly" or "Ala"VARIANT(22)..(22)/replace="Abu" or "Pro" or "Gly" or "Ser" or "Ala"VARIANT(23)..(23)/replace="Abu" or "Ala" or "Leu" or "Pro"VARIANT(24)..(24)/replace="Abu" or "Leu" or "Met" or "Phe" or "Ala"VARIANT(25)..(25)/replace="Ala"MISC_FEATURE(1)..(27)/note="Positions 8, 9, 11, 12, 15, 16 and 18-24 may encompass a single methylLanthionine or Lanthionine bridge of the structure Abu-S-Ala, Ala-S-Abu or Ala-S-Ala"MISC_FEATURE(1)..(27)/note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions"source/note="See specification as filed for detailed description of substitutions and preferred embodiments" 5Arg Gly Arg Gly Asn Trp Asn Leu Asn Ser Ala Gly Tyr Leu Leu Gly1 5 10 15Pro His Ala Val Pro Asn His Arg Arg Leu Ala 20 25620PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(4),(7)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 4 and 7 6Gly Trp Asn Xaa Asn Ala Xaa Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 20719PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(4),(7)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 4 and 7 7Gly Trp Asn Xaa Asn Ala Xaa Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His820PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(8),(11)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 8 and 11 8Gly Trp Asn Leu Asn Ala Ala Xaa Tyr Leu Xaa Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 20919PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(8),(11)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 8 and 11 9Gly Trp Asn Leu Asn Ala Ala Xaa Tyr Leu Xaa Gly Pro His Ala Val1 5 10 15Gly Asn His1021PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(8),(12)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 8 and 12 10Gly Trp Asn Leu Asn Ala Ala Xaa Tyr Leu Leu Xaa Gly Pro His Ala1 5 10 15Val Gly Asn His Arg 201120PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(8),(12)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 8 and 12 11Gly Trp Asn Leu Asn Ala Ala Xaa Tyr Leu Leu Xaa Gly Pro His Ala1 5 10 15Val Gly Asn His 201220PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 12Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 201319PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 13Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His1417PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 17 14Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa1520PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 17 15Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 201619PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 17 16Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His1720PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(18)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 18 17Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Gly Xaa His Arg 201818PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(18)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 18 18Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Gly Xaa1920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(18)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 18 19Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Gly Xaa His Arg 202020PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(19)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 19 20Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Gly Asn Xaa Arg 202119PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(19)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 19 21Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Gly Asn Xaa2220PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(20)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 20 22Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Gly Asn His Xaa 202320PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(15),(18)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 15 and 18 23Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Xaa Val1 5 10 15Gly Xaa His Arg 202418PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(15),(18)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 15 and 18 24Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Xaa Val1 5 10 15Gly Xaa2520PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(16),(19)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 16 and 19 25Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Xaa1 5 10 15Gly Asn Xaa Arg 202619PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(16),(19)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 16 and 19 26Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Xaa1 5 10 15Gly Asn Xaa2720PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(15),(20)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 15 and 20 27Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Xaa Val1 5 10 15Gly Asn His Xaa 202820PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 28Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 202920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14),(18)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 18 29Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Gly Xaa His Arg 203019PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 30Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His3117PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14),(17)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 17 31Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa3212PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(8),(12)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 8 and 12 32Glu Trp Asn Leu Asn Ala Ala Xaa Tyr Leu Leu Xaa1 5 103320PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(15),(18)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 15 and 18 33Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Xaa Val1 5 10 15Gly Xaa His Arg 203417PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(13),(16)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 13 and 16 34Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Xaa His Ala Xaa1 5 10 15Gly3517PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 35Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Xaa Ala Val1 5 10 15Xaa3617PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(13),(16)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 13 and 16 36Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Xaa His Ala Xaa1 5 10 15Gly3717PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic

peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 37Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa3817PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(13),(16)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 13 and 16 38Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Xaa His Ala Xaa1 5 10 15Gly3917PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 39Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa4017PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(13),(16)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 13 and 16 40Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Xaa His Ala Xaa1 5 10 15Gly4120PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(13),(16)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 13 and 16 41Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Xaa His Ala Xaa1 5 10 15Gly Asn His Arg 204220PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(16),(19)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 16 and 19 42Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Xaa1 5 10 15Gly Asn Xaa Arg 204320PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 43Gly Trp Asn Val Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 204420PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 44Gly Trp Asn Asp Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 204520PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 45Gly Trp Asn Asn Asn Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 204620PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 46Gly Trp Asn Leu Val Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 204720PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 47Gly Trp Asn Leu Lys Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 204820PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 48Gly Trp Asn Leu Asp Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 204920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 49Gly Trp Asn Leu Gln Ala Ala Gly Tyr Leu Leu Gly Ala Xaa Ala Val1 5 10 15Xaa Asn His Arg 205017PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14),(17)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 14 and 17 50Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Xaa Ala Val1 5 10 15Xaa5112PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(8),(12)Xaa = Lanthionine. A single Lanthionine residue represents Ala-S-Ala and is incorporated into the peptide chain at positions 8 and 12 51Glu Trp Thr Leu Asn Ala Ala Xaa Tyr Leu Leu Xaa1 5 105217PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14),(17)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 14 and 17 52Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Xaa Ala Val1 5 10 15Xaa5312PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(8),(12)Xaa = methylLanthionine. A single methylLanthionine residue represents Abu-S-Ala or Ala-S-Abu and is incorporated into the peptide chain at positions 8 and 12 53Glu Trp Thr Leu Asn Ala Ala Xaa Tyr Leu Leu Xaa1 5 105412PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(8)..(8)D-Cys 54Glu Trp Asn Leu Asn Ala Ala Cys Tyr Leu Leu Cys1 5 105517PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(14)..(14)D-Cys 55Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Cys Ala Val1 5 10 15Cys5618PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(15)..(15)D-Cys 56Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Cys Val1 5 10 15Gly Cys5719PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-GluMOD_RES(16)..(16)D-Cys 57Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Cys1 5 10 15Gly Asn Cys5820PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 58Gly Trp Asn Thr Asn Ala Cys Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 205919PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 59Gly Trp Asn Thr Asn Ala Cys Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His6020PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 60Gly Trp Asn Leu Asn Ala Ala Thr Tyr Leu Cys Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 206119PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 61Gly Trp Asn Leu Asn Ala Ala Thr Tyr Leu Cys Gly Pro His Ala Val1 5 10 15Gly Asn His6221PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 62Gly Trp Asn Leu Asn Ala Ala Ser Tyr Leu Leu Cys Gly Pro His Ala1 5 10 15Val Gly Asn His Arg 206320PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 63Gly Trp Asn Leu Asn Ala Ala Ser Tyr Leu Leu Cys Gly Pro His Ala1 5 10 15Val Gly Asn His 206420PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 64Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 206519PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 65Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His6617PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 66Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Cys6720PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 67Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Cys Asn His Arg 206819PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 68Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Cys Asn His6920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 69Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Gly Cys His Arg 207018PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 70Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Gly Cys7120PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 71Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Gly Cys His Arg 207220PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 72Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Gly Asn Cys Arg 207319PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 73Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Gly Asn Cys7420PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 74Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Gly Asn His Cys 207520PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 75Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Thr Val1 5 10 15Gly Cys His Arg 207618PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 76Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ser Val1 5 10 15Gly Cys7720PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 77Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Thr1 5 10 15Gly Asn Cys Arg 207819PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 78Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Ser1 5 10 15Gly Asn Cys7920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 79Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 208019PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 80Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His8117PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 81Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Ser Ala Val1 5 10 15Cys8212PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 82Gln Trp Asn Leu Asn Ala Ala Ser Tyr Leu Leu Cys1 5 108320PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 83Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Thr Val1 5 10 15Gly Cys His Arg 208417PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 84Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Thr His Ala Cys1 5 10 15Gly8517PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 85Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Thr Ala Val1 5 10 15Cys8617PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 86Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Thr His Ala Cys1 5 10 15Gly8717PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 87Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys8817PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 88Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Thr His Ala Cys1 5 10 15Gly8917PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 89Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys9017PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 90Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Ser His Ala Cys1 5 10 15Gly9120PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 91Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Thr His Ala Cys1 5 10 15Gly Asn His Arg 209220PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 92Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Thr1 5 10 15Gly Asn Cys Arg 209317PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 93Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys9420PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 94Gly Trp Asn Val Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 209520PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 95Gly Trp Asn Asp Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 209620PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 96Gly Trp Asn Asn Asn Ala Ala

Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 209720PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 97Gly Trp Asn Leu Val Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 209820PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 98Gly Trp Asn Leu Lys Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 209920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 99Gly Trp Asn Leu Asp Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 2010020PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 100Gly Trp Asn Leu Gln Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 2010117PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 101Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Thr Ala Val1 5 10 15Cys10212PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 102Gln Trp Thr Leu Asn Ala Ala Thr Tyr Leu Leu Cys1 5 1010317PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 103Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Ser Ala Val1 5 10 15Cys10412PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 104Gln Trp Thr Leu Asn Ala Ala Ser Tyr Leu Leu Cys1 5 1010523PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 105Met Ser Thr Lys Asp Phe Asn Leu Asp Leu Val Ser Val Ser Lys Lys1 5 10 15Asp Ser Gly Ala Ser Pro Arg 2010630PRTUnknownsource/note="Description of Unknown Galanin polypeptide" 106Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg Ser Phe Ser Asp Lys Asn Gly Leu Thr Ser 20 25 3010715PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 107Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala1 5 10 1510820PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 108Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2010920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 109Gly Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Thr His Ala Cys1 5 10 15Gly Asn His Arg 2011020PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 110Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Thr His Ala Cys1 5 10 15Gly Asn His Arg 2011120PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 111Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His Arg 2011219PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 112Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Cys Asn His11320PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 113Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Ala Thr1 5 10 15Gly Asn Cys Arg 2011420PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 114Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Ala Thr Ala Val1 5 10 15Gly Cys His Arg 2011512PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 115Glu Trp Asn Leu Asn Ala Ala Ser Tyr Leu Leu Cys1 5 1011620PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 116Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro His Thr Val1 5 10 15Gly Cys His Arg 2011717PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 117Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Thr Ala Val1 5 10 15Cys11817PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(1)..(1)pyro-Glu 118Glu Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Ala Thr His Ala Cys1 5 10 15Gly11920PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 119Gly Trp Asn Leu Asn Lys Ala Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012020PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 120Gly Trp Asn Leu Asn Asp Ala Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012120PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 121Gly Trp Asn Leu Asn Leu Ala Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012220PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 122Gly Trp Asn Leu Asn Trp Ala Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012320PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 123Gly Trp Asn Leu Asn Ala Ala Lys Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012420PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 124Gly Trp Asn Leu Asn Ala Ala Asp Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012520PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 125Gly Trp Asn Leu Asn Ala Ala Gln Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012620PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 126Gly Trp Asn Leu Asn Ala Ala Pro Tyr Leu Leu Gly Pro His Ala Val1 5 10 15Gly Asn His Arg 2012717PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(14)..(14)D-Cys 127Gln Trp Asn Leu Asn Ala Ala Gly Tyr Leu Leu Gly Pro Cys Ala Val1 5 10 15Cys12812PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide"MOD_RES(8)..(8)D-Cys 128Gln Trp Thr Leu Asn Ala Ala Cys Tyr Leu Leu Cys1 5 10

Claims

1. An isolated nucleic acid sequence encoding a peptide selected from the group consisting of TABLE-US-00011 (SEQ ID NO: 58) GWNTNACGYLLGPHAVGNHR (SEQ ID NO: 59) GWNTNACGYLLGPHAVGNH (SEQ ID NO: 60) GWNLNAATYLCGPHAVGNHR (SEQ ID NO: 61) GWNLNAATYLCGPHAVGNH (SEQ ID NO: 62) GWNLNAASYLLCGPHAVGNHR (SEQ ID NO: 63) GWNLNAASYLLCGPHAVGNH (SEQ ID NO: 64) GWNLNAAGYLLGATAVCNHR (SEQ ID NO: 65) GWNLNAAGYLLGATAVCNH (SEQ ID NO: 66) GWNLNAAGYLLGASAVC (SEQ ID NO: 67) GWNLNAAGYLLGASAVCNHR (SEQ ID NO: 68) GWNLNAAGYLLGASAVCNH (SEQ ID NO: 69) GWNLNAAGYLLGATAVGCHR (SEQ ID NO: 70) GWNLNAAGYLLGASAVGC (SEQ ID NO: 71) GWNLNAAGYLLGASAVGCHR (SEQ ID NO: 72) GWNLNAAGYLLGASAVGNCR (SEQ ID NO: 73) GWNLNAAGYLLGASAVGNC (SEQ ID NO: 74) GWNLNAAGYLLGASAVGNHC (SEQ ID NO: 75) GWNLNAAGYLLGPHTVGCHR (SEQ ID NO: 76) GWNLNAAGYLLGPHSVGC (SEQ ID NO: 77) GWNLNAAGYLLGPHATGNCR (SEQ ID NO: 78) GWNLNAAGYLLGPHASGNC (SEQ ID NO: 74) GWNLNAAGYLLGASAVGNHC (SEQ ID NO: 79) QWNLNAAGYLLGATAVCNHR (SEQ ID NO: 80) QWNLNAAGYLLGATAVCNH (SEQ ID NO: 81) QWNLNAAGYLLGASAVC (SEQ ID NO: 82) QWNLNAASYLLC (SEQ ID NO: 83) QWNLNAAGYLLGPHTVGCHR (SEQ ID NO: 84) GWNLNAAGYLLGTHACG (SEQ ID NO: 85) GWNLNAAGYLLGPTAVC (SEQ ID NO: 86) GWNLNAAGYLLATHACG (SEQ ID NO: 87) GWNLNAAGYLLGATAVC (SEQ ID NO: 88) QWNLNAAGYLLATHACG (SEQ ID NO: 93) QWNLNAAGYLLGATAVC (SEQ ID NO: 90) QWNLNAAGYLLASHACG (SEQ ID NO: 91) QWNLNAAGYLLATHACGNHR (SEQ ID NO: 60) GWNLNAATYLCGPHAVGNHR (SEQ ID NO: 92) QWNLNAAGYLLGPHATGNCR (SEQ ID NO: 86) GWNLNAAGYLLATHACG (SEQ ID NO: 88) QWNLNAAGYLLATHACG (SEQ ID NO: 87) GWNLNAAGYLLGATAVC (SEQ ID NO: 93) QWNLNAAGYLLGATAVC (SEQ ID NO: 94) GWNVNAAGYLLGATAVCNHR (SEQ ID NO: 95) GWNDNAAGYLLGATAVCNHR (SEQ ID NO: 96) GWNNNAAGYLLGATAVCNHR (SEQ ID NO: 97) GWNLVAAGYLLGATAVCNHR (SEQ ID NO: 98) GWNLKAAGYLLGATAVCNHR (SEQ ID NO: 99) GWNLDAAGYLLGATAVCNHR (SEQ ID NO: 100) GWNLQAAGYLLGATAVCNHR (SEQ ID NO: 103) QWNLNAAGYLLGPSAVC (SEQ ID NO: 104) QWTLNAASYLLC (SEQ ID NO: 101) QWNLNAAGYLLGPTAVC and (SEQ ID NO: 102) QWTLNAATYLLC.

2. A vector comprising an isolated nucleic acid sequence according to claim 1, wherein said nucleic acid is genetically fused to a nucleic acid sequence encoding a lanthipeptide-leader sequence.

3. A food-grade bacterial host cell comprising a vector according to claim 2.

4. The food-grade bacterial host cell of claim 3 comprising a L. lactis host cell.

5. The food-grade bacterial host cell of claim 3 further comprising lanthionine introduction enzymes.

6. An oral delivery system for production of lanthi-galanins in the gut, said s system comprising the food-grade bacterial host cell of claim 3.

7. A pharmaceutical composition comprising viable host cells according to claim 3 and a pharmaceutically acceptable carrier, diluent or vehicle.

8. The pharmaceutical composition of claim 7 formulated for oral administration.

9. A method of treating or alleviating symptoms associated with inflammatory bowel disease, said method comprising administering to a subject in need thereof a pharmaceutical composition according to claim 7.

10. The method of claim 9 wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis (UC).