Kisspeptin Antagonists - Kisspeptin Signaling in Reproductive Biology - page 156

to be one of the most potent regulators of this system known to date, with KP doses as
low as 1 fmol (i.c.v.) capable of stimulating GnRH secretion in vivo. KP and
KISS1R have been shown to have a role in puberty onset, regulation of the pre-
ovulatory LH surge, and integration of other regulatory pathways that affect the
reproductive axis, such as steroid hormone feedback and seasonal breeding stimuli
[ 4 , 5 ]. The discovery of the KP system arose from the observation that mutations in
KISS1R caused delayed or absent puberty, with similar results evident in knockout
mice [ 6 , 7 ]. Sex steroids negatively regulate Kiss1 mRNA levels within the ARC in
various species [ 8 - 12 ]. Oestrogen can also positively regulate Kiss1 mRNA within
either the ARC in sheep and primates or the AVPV in rodents at the time of the
pre-ovulatory LH surge, which initiates ovulation [ 13 - 15 ]. Kiss1 gene expression
is also regulated by metabolic and nutritional status, as well as photoperiod. This
regulation of Kiss1 is covered in depth in other chapters in this volume.
Most of the data concerning the physiological role of the KP system to date is
derived from the measurement of changes in mRNA levels or altered patterns of
immunohistochemical staining of KP peptide. To directly elucidate the role of KP
and KISS1R in physiological processes, the development of KP antagonists is
needed. To develop these antagonists, the structure of kisspeptin needs to be
assessed. The KiSS1 gene encodes a polypeptide consisting of 145 amino acids,
known as the precursor peptide. This precursor peptide gives rise to a secretory
precursor protein of 126 amino acids that is proteolytically cleaved and modifi ed to
form a C-terminal amide moiety, KP-54 [ 16 ]. KP-54 is then cleaved further to
smaller fragments of 14, 13, and 10 amino acids in length. These fragments have
subsequently been shown to bind to and activate the KISS1R receptor with equal
potency [ 17 ]. Therefore, the fi nal ten amino acid fragments of the C-terminus [ 18 ]
have been used to analyse the structure of KP and to design KP antagonists. KP-10
contains the following ten amino acids: Tyr 1 , Asn 2 , Trp 3 , Asn 4 , Ser 5 , Phe 6 , Gly 7 ,
Leu 8 , Arg 9 , and Phe 10 . It is thought that the activation domain is within the fi ve most
N-terminal residues, where there are many hydrophobic residues, and that the bind-
ing domain is within the fi ve most C-terminal residues, which contain both hydro-
phobic and charged residues to form bonds with the receptor (Fig. 8.2a ).
These ten amino acids provide multiple targets for amino acid substitution when
designing antagonists. Several strategies for peptide analogue development have
been utilised. Alanine screening involves each residue being systematically changed
to alanine, which has no functional side chain. This was reported by two groups and
both revealed that alanine substitutions at Phe 6 and Phe 10 of KP-10 signifi cantly
reduced binding to and activation of KISS1R in vitro and in vivo, making them criti-
cal to KP peptide activity [ 19 , 20 ]. Orsini et al. also showed, via alanine screening,
that substitution of Arg 9 also signifi cantly decreased binding and activation, as did
substitutions of Leu 8 . They concluded that Phe 6 , Arg 9 , and Phe 10 create a binding
pharmacophore with the two phenyl rings of Phe 6 and Phe 10 on top of each other,
fl anked by Arg 9 with Leu 8 on the opposite side of the peptide [ 19 ]. The second
approach is to make more intuitive amino acid changes based on knowledge of the
peptide structure of KP-10. This involves making either conservative changes at
positions of interest, such as changing the side chain but not the overall charge of a
residue, or changing L-amino acids to D-amino acids to assess the positioning of