Kisspeptin Antagonists - Kisspeptin Signaling in Reproductive Biology - page 158

Fig. 8.1 Diagram of amino acid substitution effects on receptor binding. Flowchart for amino acid
substitutions within truncated and full-length peptide analogues showing the effects of each
substitution on binding. The IC 50 for analogues that exhibit binding is shown in brackets and
peptide number in blue
study [ 23 ]. Next, a D -Arg 9 was substituted in place of the normal L -Arg 9 in conjunc-
tion with the Trp 10 substitution (peptide 200). This substitution of both residues of
the RFamide motif ablated kisspeptin's ability to bind to the receptor. These results
confi rm the importance of both the residues within this motif for receptor binding
interactions and that it is part of an essential pharmacophore (Fig. 8.1 ) [ 19 ].
Thus, Arg 9 and Phe 10 are important for receptor binding in truncated peptides,
and one further residue was also shown to be involved in this interaction within the
N-terminus (peptides 202, 203). The fi rst residue of the truncated fi ve amino acid
analogues is a bulky aromatic and hydrophobic Phe 6 . This is usually within the core
of the peptide structure and may evade exposure to an aqueous environment.
Substitutions of Phe 6 with Ala (peptide 202) and D-Phe (peptide 203) were made but
both had poor binding (Fig. 8.1 ), suggesting that the side chain of phenylalanine is
important for KP-10 to interact with KISS1R, along with Arg 9 and Phe 10 , possibly
via hydrophobic interactions.
As none of the above truncated peptides could bind KISS1R with the same or
higher affi nity than KP-10, this confi rms that more than fi ve residues are needed to
fully bind to the receptor. Therefore, further studies with truncated peptides were
abandoned and full-length ten-residue analogues were designed and tested. For the
fi rst full-length substitutions, the six most C-terminal residues were substituted, as it