Highly Active Analogs of α-Factor and Their Activities Against Saccharomyces cerevisiae

Abstract

Thirteen analogs of tridecapeptide α-factor (WHWLQLKPGQPMY) of Saccharomyces cerevisiae with C-or N-terminal Trp extension and isosteric replacement by Aib at position 8 and 11, Trp at position 13, D-Ala at position 9, and Orn and Glu at position 6 were synthesized and assayed for their biological activity. Receptor binding assay was carried out using our newly developed spectrophotometric method with detector peptide 14. C-or N-terminal extended analogs, α-factor-[Trp] n (n =1-5) 1-5 and [N-Trp] 1 -α-factor 6, were all less active than native α-factor and gradual decreases in both activity and receptor affinity were observed with greater Trp extension. Trp-substituted analog at position 13, [Trp 13 ]α-factor 7, exhibited about 2-fold reductions in both activity and receptor affinity. Aib-substituted analogs, [Aib 8 ]α-factor 8 and [Aib 11]α-factor 9, showed 5-to 10-fold reduction in activity as well as 3-fold reduction in receptor affinity compared to native α-factor. [Orn 6]α-factor 10 demonstrated strong potency with a 7.0-fold increase in halo activity as well as 1.8-fold increase in receptor affinity compared to native α-factor. ]α-factor 11 exhibited 15-fold higher halo activity as well as nearly 3-fold higher receptor affinity compared to native α-factor.

Spectrophotometric Determination of Affinities of α‐Factors for Their G Protein‐Coupled Receptors in Saccharomyces cerevisiae

“…Saturation of each detector was assessed by examining binding as a function of increasing detector concentrations at a cell concentration of 2.5 × 10 11 cells/mL. Scatchard plots were derived from the saturation binding data . The straight line of the Scatchard plot indicates that the interaction of each detector with its receptor can be characterized by a single equilibrium dissociation constant ( K D ).…”

“…To address this problem, we adopted the modified backbones containing either Orn or Arg at position 6 and d ‐Ala at position 9. Incorporation of a positively charged residue at position 6 generally increases biological activity, whereas incorporation of a negatively charged residue such as Glu at the same position reduces halo activity up to 50% as well as affinity by 25% compared to a positively charged analog . Therefore, we did not consider a negatively charged analog at position 6.…”

“…Strain Y 7925 ( MAT a ) was grown at 30 °C with shaking (200 rpm/s) to early log phase in YEPD broth (yeast extract: 1%, peptone: 2%, dextrose: 2%) supplemented with 2% glucose . Cells were then harvested by centrifugation at 6000 rpm/s (IBRD IB4; Hanil Science Centrifuge, Seoul, Korea), washed twice with saline water, resuspended in YEPD broth to 4.0 × 10 6 cells/mL, and placed on ice.…”

“…To select the optimum strain for affinity study, the following S. cerevisiae a ‐cells were tested; Y 7925, Y 7296, Y 7932, and Y 7951 . Cells were grown and harvested as described above with the following changes.…”

“…Competition for bound detectors, [Orn 6 ]α‐factor‐[Cys] 3 1 and [Arg 6 , d ‐Ala 9 ]α‐factor‐[Cys] 3 3 , by nonchromogenic competitive peptides was measured by the following protocol . In general , strain Y 7925 was grown to 1 × 10 7 cells/mL and harvested by centrifugation at 3000 g for 15 min at 4 °C.…”