Kisspeptin Antagonists - Kisspeptin Signaling in Reproductive Biology - page 157

side chains. Finally, radical structural changes can also be made to change the
charge or fl exibility of the analogue. This approach is described in detail in this
chapter, both in vitro and in vivo. In addition to peptide antagonists, some small
molecule non-peptide antagonists have also been designed for KP, as they have
more potential for being taken forward into human trials, and these results are also
discussed later in this chapter. Any antagonists identifi ed could be very useful tools
to investigate whether blockade of the system directly affects GnRH/LH secretion,
puberty onset, steroid feedback, and the pre-ovulatory LH surge. Antagonists may
also be useful medicines, with potential application as novel contraceptives, and
potential for the management of many reproductive disorders which are often associ-
ated with irregular LH levels and pulse frequency.
Development of KP Peptide Antagonists and Receptor Binding
KP analogues were synthesised based on the structure of human KP-10, as this is
the smallest fragment needed to bind to and activate the receptor [ 21 , 22 ]. Analogues
were tested using CHO cells stably expressing human KISS1R for their ability to
bind to the receptor.
Experiments tested whether all ten amino acids of KP-10 were required for bind-
ing to the receptor using N-terminally truncated peptides possessing fi ve or seven
amino acid residues. These analogues were named peptides 188 (acFGLRF) and
186 (acNSFGLRF), respectively (for continuity, the amino acid residues are num-
bered to correspond to positions in the full length KP-10). Each of these truncated
peptides had a reduced binding affi nity for the receptor compared to KP-10
(Fig. 8.1 ). The reduction in binding affi nity seen for the 5aa- and 7aa-truncated
analogues suggests that more than 7aa are needed to bind effectively to KISS1R.
Therefore, this implies that all 10aa are needed for binding to human KISS1R and
that at least one of the N-terminal residues must be involved in this process.
Nevertheless, amino acids within the fi ve amino acid-truncated peptides were
substituted to see if the binding affi nities could be increased. These are peptides
189, 190, 191, and 200, 201, 202, 203, and 206, 207. All of these analogues had
reduced affi nity for human KISS1R (Fig. 8.1 ); however, these analogues did high-
light some residues important for binding interactions with the receptor.
As the RFamide motif at the C-terminus is hypothesised to be critical for receptor
binding, we fi rstly investigated substitutions at these two residues. Trp 10 was intro-
duced into peptide 189 to assess the effects of introducing a bulkier and polar side
chain into the RFamide motif. The use of Trp 10 also introduces rigidity to the phenyl
ring and some steric hindrance, thus reducing fl exibility of the C-terminus. Peptide
189 (acFGLRW) had an IC 50 of 9.5 × 10 −7 M for human KISS1R (Fig. 8.1 ). The
results imply that introducing a more rigid polar residue into the RFamide motif
decreases the ability to bind the receptor. This contrasts with a study by Niida et al.
that showed that small peptide analogues with FW-amide at the c-terminus were
potent agonists. However, these analogues had Guanidino or bis[(2-pyridinyl)
methyl] added at the n-terminus, and receptor binding was not investigated in this