SYNTHESIS, BIOLOGICAL-ACTIVITY, AND CONFORMATIONAL-ANALYSIS OF PEPTIDOMIMETIC ANALOGS OF THE SACCHAROMYCES-CEREVISIAE ALPHA-FACTOR TRIDECAPEPTIDE

Biochemical and biophysical investigations on the Saccharomyces cerevi siae alpha-factor indicate that this tridecapeptide mating pheromone ( WHWLQLKPGQPMY) might adopt a type II beta-turn in the center of the pe ptide when it binds to its G protein-coupled receptor. To test this hy pothesis we synthesized analogues of ex-factor incorporating a (R or S )-gamma-lactam conformational constraint [3-(R or S)-amino-2-oxo-1-pyr rolidineacetamido] in place of the Pro-Gly at residues 8 and 9 of the peptide and tested their biological activities and receptor binding. A nalogues were purified to >99% homogeneity as evidenced by high-perfor mance liquid chromatography and capillary electrophoresis and characte rized by amino acid analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The restricted ex-factor analogue WHWLQL K[(R)-gamma-lactam] QP[Nle]Y was more active than its lactam-containin g diastereomeric homologue WHWLQLK[(S)-gamma-lactam] QP[Me] Y and abou t equally active with the [Nle(12)] ex-factor in growth arrest and FUS 1-lacZ gene induction assays. Both lactam analogues competed with trit iated [Nle(12)]-alpha-factor for binding to the ct-factor receptor (St e2p) with the (R)-gamma-lactam-containing peptide having 7-fold higher affinity than the (S)-gamma-lactam-containing homologue. Two-dimensio nal NMR spectroscopy and modeling analysis gave evidence that the (R)- gamma-analogue is a flexible peptide that assumes a transient gamma-tu rn structure around the lactam moiety. The results represent the first example of an alpha-factor analogue containing a peptidomimetic const raint that is as active as the native pheromone. The correlation betwe en activity and structure provides further evidence that the biologica lly active conformation of the molecule contains a turn in the middle of the pheromone. This study provides new insights into the structural basis of alpha-factor activity and adds to the repertoire of conforma tionally biasing constraints that can be used to maintain and even enh ance biological activity in peptide hormones.