Kisspeptin Antagonists - Kisspeptin Signaling in Reproductive Biology - page 163

These results imply that Asn 2 interacts with the receptor to assist binding, probably
via hydrogen bonding, and that the presence and positioning of the side chain is
critical to this interaction. The results also suggest that a charged residue is needed
at this position for receptor binding to take place. Finally, the same substitutions
were made for Trp 3 (peptide 231 with D -Trp 3 and peptide 235 with D -Ala 3 ). Peptide
231 and peptide 235 could not bind to KISS1R (Fig. 8.1 ). This suggests that Trp 3
may also form interactions with the receptor and that the presence and positioning
of a side chain is again important for this interaction and binding to occur.
These results from examining the effects of residue substitutions within full-length
KP-10 suggest that the main binding residues lie within the C-terminal domain via
Phe 6 , Arg 9 , and Phe 10 , as was proposed within truncated analogues, and within the
N-terminal domain via Asn 2 and Trp 3 . This would account for the reduced binding
in truncated peptides where Asn 2 and Trp 3 were removed. The study of the full-
length analogues also suggests that the functional peptide conformation requires
fl exibility in the centre of the peptide, as bulky residues at position 5 ablate receptor
binding. The introduction of rigidity via D -Trp 8 within the C-terminus is tolerated
well, suggesting that this residue does not contribute to receptor binding interac-
tions or that the D-Trp makes new interactions with the receptor to compensate for
a loss of interaction from L-Leu. Similar results were seen with Tyr 1 substitutions,
again suggesting this residue has no involvement in receptor binding or that the new
substitutions make new contacts.
Although further analogues were made, these were used to investigate receptor
activation and to screen for KP antagonists, since most residues within KP-10 have
been tested for effects on binding to human KISS1R in the above research.
Functional Tests of KP-10 Peptide Analogues In Vitro
Active KP-10 Peptide Analogues Inhibit KP-10-Induced Inositol
Phosphate Production and Intracellular Calcium Release
Analogues were tested for receptor activation via phospholipase C-mediated inositol
phosphate (IP) production and antagonistic properties by their ability to inhibit kiss-
peptin-stimulated IP production and intracellular calcium release. A full antagonist
binds to the receptor, but does not activate it, and inhibits kisspeptin-stimulated IP
production or intracellular calcium release by >80%. KP activates the receptor with a
mean EC 50 of 3.9 × 10 −9 M; therefore a dose of 10nM was used for antagonist studies.
Effects on Inositol Phosphate Production
Truncated peptides all activated KISS1R at high doses except peptides 201 and 206, 7,
which had substitutions of Leu 8 . Truncating the ligand to fi ve amino acids retained its