Engineering galanin analogues that discriminate between GalR1 and GalR2 receptor subtypes and exhibit anticonvulsant activity following systemic delivery

Abstract

Galanin modulates seizures in the brain through two galanin receptor subtypes, GalR1 and GalR2. To generate systemically active galanin receptor ligands that discriminate between GalR1 and GalR2, the GalR1-preferring analogue Gal-B2 (or NAX 5055) was rationally redesigned to yield GalR2-preferring analogues. Systematic truncations of the N-terminal backbone led to [N-Me,des-Sar]Gal-B2, containing N-methyltryptophan. This analogue exhibited 18-fold preference in binding toward GalR2, maintained agonist activity, and exhibited potent anticonvulsant activity in mice following intraperitoneal administration.

Figure 1

Structures of the N-terminus and GalR2/GalR1 receptor preference for galanin analogues. Analogues generated using the backbone “atom shaving” technique. All Analogues maintain the Gal(2-13) pharmacophore and have the C-terminal KK(Lys-palmitoyl)K motif [R = TLNSAGYLLGPKK(Lys-palmitoyl)K-conh₂].

Figure 2

Representative binding curves for Analogues Gal-B2 (triangles) and [N-Me, des-Sar]Gal-B2 (circles) against hGalR1 (left) and hGalR2 (right). Each binding assay was performed in triplicate to generate one 10-point binding curve. Binding affinities are reported as the average of three independent binding curves. Binding affinities (K_I) for Gal-B2 are 3.5 nM hGalR1 and 52 nM hGalR2 and for [N-Me, des-Sar]Gal-B2 are 364 nM hGalR1 and 20 nM hGalR2.

Figure 3

(A) Agonist activity of Analogues determined by calcium mobilization assay (Millipore). The % response is normalized to galanin receptor agonists, Gal(1-30) for hGalR1 and Gal(2-29) for hGalR2. At 1.25 μM ligand concentration, Analogue [N-Me, des-Sar]Gal-B2 preferentially activates hGalR2 over hGalR1, to 83% ± 3.8% and 26% ± 1.9% respectively (n = 4, p < 0.01). (B) The dose response curve for Analogue [N-Me, des-Sar]Gal-B2; CF-1 mice (n = 8) were treated with the Analogue and challenged by with 6 Hz (32 mA) corneal stimulation 1 hour after i.p. administration.% animals protected is (#mice protected/#mice tested)*100.