Kisspeptin Antagonists - Kisspeptin Signaling in Reproductive Biology - page 161

Due to the small hydrogen atom in place of a side chain, this allows glycine to
create fl exibility within the peptide, to promote conformational changes. This
substitution retained binding to the receptor with an IC 50 of 1.2×10 −7 M
(Fig. 8.1 ). As this replacement was well-tolerated, it was included in the majority
of subsequent analogues. Next, amino acid changes at Leu 8 were attempted.
Leu 8 is a hydrophobic and aliphatic residue found in the peptide core, likely to
be involved in interactions with other hydrophobic residues. Peptide 209 had a
D -Leu 8 substitution, to check if the orientation was important for binding.
Peptide 210 and 228 had D -Trp 8 in place of Leu 8 to test the effect of orientation
and steric hindrance due to a bulky side chain on binding. In combination with
Gly 5 , this potentially allows fl exibility within the central region. Peptide 228
also had an acetyl group added to the N-terminus to try to convey resistance to
amino peptidases and extend the half-life of the peptide. Peptide 209 had
decreased ability to bind the receptor, with an IC 50 of 2.8 × 10 −6 M, but exhibited
similar capacity to displace radiolabelled ligand compared to KP-10. Peptide
210 was, however, able to bind the receptor with an IC 50 close to that of KP-10 at
6 × 10 −9 M. The results were similar to peptide 228, although the IC 50 was higher
at 1 × 10 −11 M (Fig. 8.1 ). Therefore, it appears that changes within residue 8 that
change side chain orientation and increase steric hindrance do not signifi cantly
affect the binding properties of KP-10.
The contribution of Phe 6 was next investigated in the full-length analogues, since
changes to this residue had ablated binding in the truncated peptides. Two substitu-
tions were made at this position in conjunction with Gly 5 . In peptide 211, replace-
ment with D-Phe 6 as in the truncated peptides reduced binding affi nity, and the same
was true with D-Trp substitution in peptide 212. This suggests that Phe 6 is critical
for receptor binding via interactions between its side chain and KISS1R (Fig. 8.1 ).
The above results examining the effects of substitutions of C-terminal residues
suggest that binding involves Phe 6 , Arg 9 , and Phe 10 . This is in line with data pub-
lished describing the pharmacophore created by these residues for binding to
KISS1R [ 19 ].
Following the demonstration that the C-terminal residues of KP are involved in
binding to receptor, attention was turned to the fi ve N-terminal residues to examine
if receptor binding also involved N-terminal interactions (Fig. 8.4). As peptide 210
could bind with a similar affi nity to KP-10, the D -Trp 8 substitution was incorporated
into all further analogues. Because position 5 could tolerate a glycine substitution to
promote fl exibility, the next changes examined if other residues would also be
acceptable. Three more substitutions were studied. These were D -Trp 5 (Peptide
229), D -Ala 5 (peptide 233), and D -Ser 5 (Peptide 273). Peptide 229 was unable to
bind to KISS1R; suggesting fl exibility is needed in this region. For peptides 233 and
273, the displacement of radiolabelled ligand is lower (Fig. 8.1 ), implying that only
glycine at this position allows high affi nity binding of KISS1R and that side chain
interactions are not important within this residue. Therefore, fl exibility is more
important at this position than side chain interactions, and Gly 5 was incorporated
into all subsequent analogues.