Kisspeptin Antagonists - Kisspeptin Signaling in Reproductive Biology - page 164

agonistic properties (peptide 188-9). Changes to position 6 (peptide 202, 3) had no
antagonistic effects and changes to position 9/10 (peptide 200) could not bind, acti-
vate, or antagonise the receptor. However, position 8 changes did not intrinsically
activate IP production and had weak antagonistic properties. Peptide 201, possess-
ing Ala 8 , could antagonise the KP-10 stimulation by 67%, with an IC 50 of 7 × 10 −9 M.
Peptide 206, with a D -Trp 8 , could also antagonise KP-10 stimulation by 71%, with
an IC 50 of 5 × 10 −9 M, but peptide 207 with a D -Phe 8 could only antagonise by 53%,
with an IC 50 of 3 × 10 −9 M (Fig. 8.5 ). These results suggest that Leu 8 is important for
activation of KISS1R and that residues with bulky side chains are the most effective
substitutions for antagonism. However, as none of these truncated KP-10 analogues
could antagonise by more than 70%, they were not tested further and attentions
were turned to the full-length analogues.
Amino acid changes within the C-terminal region were focused on, to determine
residues within this region involved in receptor activation or creation of antagonism
(Fig. 8.7a-d ). Ser 5 to Gly 5 substitution in KP-10 (peptide 208) inhibited KP-10
stimulation of IP by 54% with an IC 50 of 1 × 10 −8 M (Fig. 8.6). Therefore, this change
was incorporated into subsequent analogues. However, as this analogue could still
stimulate IP release with an EC 50 of 4.5 × 10 −8 M, it implies that this substitution
alone is insuffi cient for antagonism. Analogues comprising Gly 5 and substitutions at
Phe 6 exhibited some antagonism but could still activate the receptor at high concen-
trations. Peptide 211 containing D -Phe 6 and peptide 212 containing D - Trp 6 could not
antagonise any further than peptide 208 (Fig. 8.6). This implies that a Phe 6 substitu-
tion is not useful for antagonist activity.
As Leu 8 had been shown to create antagonism in the truncated peptides, changes
at this residue were tested next in combination with Gly 5 . D -Leu 8 (peptide 209) did
not activate the receptor but could not antagonise any further than Gly 5 alone,
whereas D -Trp 8 (peptides 210 and 228) again elicited no intrinsic IP release but
caused further antagonism to 64%, with an apparent IC 50 of <1 × 10 −10 M. The acety-
lated version of this peptide could also inhibit KP-10 stimulation of IP by 69%.
Combination of changes at positions 6 and 8 with Gly 5 (peptide 213) appeared to
disrupt the structural conformation of the peptide, as it could no longer bind, acti-
vate, or antagonise the receptor (Fig. 8.6). This suggests that Leu 8 is important for
receptor activation and that appropriate substitutions in the C-terminal region pro-
mote antagonism. Therefore, D -Trp 8 was incorporated into further analogues along
with Gly 5 substitution. To test if glycine was the best residue for antagonism at
position 5, changes were made in combination with D -Trp 8 . D -Trp 5 (peptide 229)
slightly decreased the antagonism, however, D -Ala 5 (peptide 233) increased the
antagonism to 71% but the IC 50 was reduced to 7 × 10 −7 M. Therefore, Gly 5 is the
best substitution for this position (Fig. 8.6).
The fi ndings above indicated that Gly 5 in combination with D -Trp 8 could antago-
nise receptor activation and that Leu 8 was involved in receptor activation. In an
attempt to enhance this antagonism, substitutions within the N-terminal region were
tested in combination with these C-terminal region modifi cations (Fig. 8.7e-h ).
Changes were made at positions 1, 2, and 3. All of these analogues (peptides 230,
231, 232, 233, 234, 235, 236 and 243, 4) failed to stimulate IP release and did not
have intrinsic agonist activity. Position 1 substitution to D -Tyr 1 (peptide 230)