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(Ser 20, Ser 277). This clearly demonstrated that Cys 20 and Cys 277 are the only sulfhydryls available for
crosslinking receptor-bound bradykinin. These results provided direct biochemical evidence that the Nterminus
of bradykinin, when bound to the B2 receptor, is adjacent to extracellular loop 3 and the Nterminal
domain in the receptor.
Further consideration of the model led to the hypothesis that agonist peptides may minimally require an
intact C-terminal β-turn structure with appropriate side chains in place and N-terminal amino and
guanidine groups for primary electrostatic interaction(s) with Asp286 and Asp268 in extracellular loop 3.
As a test of this hypothesis, the prototypical second generation antagonist, NPC 18545, (DArg0-Arg1- Pro2-Hyp3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9) was modified such that residues 2–5 were replaced by a
simple twelve carbon chain spacer (12-aminotridecanoic acid). The resulting compound, NPC 18325,
contains only the appropriately charged moieties at the N-terminus, separated by a simple organic spacer
moiety from a known β turn forming tetrapeptide [25,26]. This pseudopeptide was tested in the human
bradykinin B2 receptor binding assay and found to have a Ki of 44 nM against [ 3H]bradykinin binding
[50]. Functionally, this pseudopeptide was an agonist as measured by its ability to stimulate IP
production in a stable CHO cell line expressing the human B2 receptor and in WI-38 cells. Since it was
designed on the basis of an agonist site on the receptor, this result was not completely surprising despite
the incorporation of the DTic-Oic pair at the C-terminus that previously had been shown to be critical in
high affinity antagonists.
Subsequently the length of the linear carbon chain was varied to further explore the hypothesis that the
agonist site on the receptor had two domains, a hydrophilic site in the extracellular loop area and a
hydrophobic domain in the transmembrane area [50]. Presumably, the two terminal portions of NPC
18325 can only simultaneously interact with each putative domain of the receptor binding site when the
carbon chain is 12–13 methylenes long. But if the carbon chain length is shortened too far, this ligand
might be unable to simultaneously interact with both domains, resulting in an affinity loss. This series of
pseudopeptides and their respective human bradykinin B2 receptor affinities are presented in Table 3.
The data are consistent with the hypotyhesis since the receptor affinity decreases as the carbon chain
length is shortened. An alternative explanation of the data is that a certain hydrophobicity profile is
required of the compounds in this series for good receptor affinity. These results indicated that there
may be additional hydrophobic or flexibility prerequisites to binding in this series of pseudopeptides.
One noteworthy observation was that NPC 18325 showed divergent behavior when evaluated against
different species homologues of the bradykinin B2 receptor. Notably, in the guinea pig ileal membrane
preparation assay, the affinity for the receptor was approximately 10-fold less than what had been
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