Substance P (SP) has been implicated in a wide range of physiological responses, including pain transmission, inflammation, hypertension, smooth muscle contraction, and promotion of cell growth and would healing. This peptide binds to and activates, the NK1 G-protein coupled receptor, and to a lesser degree the NK2 and NK3 receptors as well. In view of the intense interest in SP and the multiple physiological effects attributed to it, an understanding of those structural requirements for receptor binding and activation by SP are of critical importance for the design of agonists and as well as an understanding of the action of antagonists. Both agonists and antagonists are expected to be of clinical importance in the treatment of related diseases.
The specific aims of this proposal are designed to provide structural information relating to agonists with high activity and better-defined conformations than SP. The first is to obtain NMR data on the conformation of beta-MeF substituted SP analogs and from that derive solution phase structures. These structures will be compared with those predicted by previous modeling. The second is to synthesize several new series of SP analogs. In peptides 1a - 1d the phenylalanine rings are linked, constraining their geometry. In peptides 2a - 2d, the N-terminus of the peptide is replaced by an alkyl spacer and the N-terminal Arg amino acid. NMR derived structures will be determined for these peptides. A series of biochemical and physiological assays will be carried out to evaluate the activities of these analog peptides as compared to SP. These include rabbit iris sphincter muscle contraction, cAMP and IP3 production and binding to the NK1 and NK2 receptors. These assays, when applied to this series of closely and structurally related peptides, will provide data regarding the origins of receptor selectivity as well as the active conformation of SP. It may also be discovered that there are differences in selectivity between species and/or tissues that will be seen when using these subtle probes that would be overlooked using more conventional probes.
The final aim of this proposal is to use the structural information derived in this work to design a new generation of SP agonists and antagonists.
