Design of chimeric peptide ligands to galanin receptors and substance P receptors

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Abstract
Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1)D(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD ∼ 1 nm) or its N-terminal fragment galanin-(1-13) (KD∼ 1,nm), which constitutes the A′-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD= 0.4μm), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm) or spantide (1 μm). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N-terminal galanin-(1-13) fragment or of its analogs, linked to the C-terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high-affinity ligands to peptide receptors can be produced by linking biologically active N-terminal and C-terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N- and C-terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).