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within the N-terminal region that appeared important for receptor activation and
antagonism.
Next, to test whether antagonism was due to a combination of all three substituted
residues or whether one was more critical, we took the two position 1 analogues and
removed or changed the Gly
5
or D -Trp
8
substitution to examine the effect on the
antagonistic properties of the peptide. For analogues with D -Tyr
1
, modifi cation of
either of these residues completely ablated any antagonistic effect (peptides 237, 8),
indicating that the D -Tyr
1
has relatively little effect on the receptor antagonism alone.
However, for D -Ala
1
analogues, conversion of Gly
5
back to Ser
5
(peptide 247) slightly
reduced the antagonism to 78% and changing of D -Trp
8
to L -Trp
8
(peptide 248)
reduced antagonism further to 65%. However, D -Leu
8
at
this position (peptide 276)
increased antagonism to 85%, suggesting a D-amino acid is needed at this position for
antagonism. However, peptide 276 does have intrinsic agonist activity (Fig. 8.6). The
results indicate that D -Ala
1
and D -Trp
8
are the most important residues for antagonism.
However, Gly
5
must also play a role by keeping the peptide fl exible, since other sub-
stitutions at this position within peptide 234 have drastic effects except when D -Ser
5
(peptide 273) is substituted here, keeping antagonism high at 80% with an IC
50
of
1 × 10
−10
M. Replacement with Pro
5
(peptide 274), D - Pro
5
(peptide 275), or D -Ala
5
(peptide 277) reduces antagonism to 24%, 45%, and 24%, respectively (Fig. 8.6 ).
Therefore, fl exibility, possibly to allow receptor engagement appears to be the key factor
in this position. Since substitution of position 1, 5, and 8 had been shown to produce
antagonists, combinations of these with position 2 substitutions were tested (peptides
239, 240, 241, 242). However, all of these reduced antagonism. Also, introduction of
D -Trp
6
into peptides 230 and 234 to increase steric hindrance at the C-terminal region
(peptides 245 and 246) also reduced antagonism to around 60% (Fig. 8.6). The above
data shows that amino acid changes to D -Ala
1
, Gly
5
, and D -Trp
8
(peptide 234) are the
most critical for antagonism. However, other residues are tolerated at these positions
such as D -Trp
1
in peptide 230, D -Ser
5
in peptide 273, or D -Leu
8
in peptide 276.
Effects of Peptide Analogues on Mobilisation
of Intracellular Calcium
To further characterise inhibition of KP-10 signalling by these antagonists, stimula-
tion of intracellular calcium release in model cells was measured. Peptide 234 did
not stimulate calcium release alone, but did antagonise KP-stimulated calcium
release by 89%, with an IC
50
of 1 × 10
−10
M, further confi rming its potency. Peptide
273 could also antagonise intracellular calcium release to 63%, with no intrinsic
stimulation, and peptide 276 completely inhibited intracellular calcium secretion
(Figs. 8.6 and
8.8
). To confi rm these were specifi c effects, three analogues that had
reduced antagonism of IP were also tested. Peptides 274, 275, and 277 only antago-
nised by 28%, 27%, and 22%, respectively, confi rming the effects above are specifi c
(Fig. 8.6). Overall, four antagonists have been created (peptide 230, 234, 273, and
276) from this research and identifi ed the residues important for receptor binding
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