Synthesis and biological properties of enzyme-resistant analogues of substance P.
Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance-P-degrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance-P-degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high-performance liquid chromatography reverse-phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogues described, an heptapeptide analogue of residues 5-11, less than Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2, showed almost complete resistance both towards substance-P-degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid-phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre-formed symmetrical anhydrides in dimethylacetamide. Fluorenylmethyloxycarbonyl was used as an alpha-amino protecting group in conjunction with t-butyloxycarbonyl as an epsilon-amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP-Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio-Rex 70 ion-exchanger. The peptides were finally purified on a semi-preparative reverse-phase column.
D000818:Animals; D001681:Biological Assay; D010450:Endopeptidases / Q000378:metabolism*; D006168:Guinea Pigs; D007031:Hypothalamus / Q000378:metabolism; D007082:Ileum / Q000187:drug effects; D007118:Immunoassay; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D008666:Metalloendopeptidases; D008722:Methods; D051381:Rats; D013373:Substance P / Q000031:analogs & derivatives* / Q000494:pharmacology; D013379:Substrate Specificity
D007202:Indicators and Reagents; D013373:Substance P; D010450:Endopeptidases; D008666:Metalloendopeptidases; C029610:substance P degrading enzyme