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action on the binding of antagonist peptides. In addition, it has been shown that bradykinin can be
covalently crosslinked to the B2 receptor while antagonists cannot. These observations have fostered the
belief that the agonist and antagonist binding sites of the receptor are not the same. At best, they may be
partially overlapping, although there is no direct evidence for this. The ultimate identification of the
amino acid residues that make up the antagonist site would be another important step toward the goal of
structure-based design of novel nonpeptide antagonists.
As described in a previous section of this chapter, characterizations of the bradykinin B2 receptors from
rat and human using NPC 17410 (Figure 3) revealed different pharmacologies. Specifically, it showed a
higher affinity for the human B2 receptor than it did for the rat B2 (human IC 50 = 0.95 nM, rat IC 50 =
48.0). This ligand “tool” provided a means for evaluating a series of bradykinin rat/human B2 receptor
chimeras [51–53]. Several different chimeras were prepared in a systematic fashion and the affinity of
NPC 17410 was determined for each. The chimeras are depicted schematically in Figure 6 together with
the IC 50 values determined for NPC 17410. Chimeras I through III sample the N-and C-terminal sections
of the receptor for any contributions to an antagonist binding site. The remaining chimeras sample the
core transmembrane domains of the receptor. Each chimera was shown to induce a membrane
depolarization similar to wild type receptor in response to bradykinin when expressed in Xenopus
oocytes. For each NPC 17410 assay, [ 3H]-NPC 17731 was used as the radioligand.
From this systematic approach, specific groups of contiguous residues within the receptor were
identified as possible contributors to an antagonist binding site. The NPC 17410 binding to chimeras III,
IV, and VIII showed rat-like pharmacology (low NPC 17410 affinity). The NPC 17410 binding to
chimeras I, II, VI, and VII showed human-like NPC 17410 pharmacology (high receptor affinity).
Binding to chimeras V and VIII, however, was similar to rat-like NPC 17410 pharmacology, but the
affinity of the compound was slightly shifted back toward human-like results. Comparisons of rat and
human receptor sequences in the regions sampled by the chimeras reveals that only two clusters of
residues differ between rat and human B2 receptors. Specifically, TM2 has the same sequence in rat and
human receptors so it is unlikely that the differential pharmacology associated with NPC 17410 binding
can be attributed to residues there. However, TM3 has a cluster of 3 residues that differ (rat rarrow.gif
human: Thr110 rarrow.gif Ala108, Met111 rarrow.gif Ile109, Tyr113 rarrow.gif Ser111) and TM6 has a
cluster of 5 residues that differ (rat rarrow.gif human: Phe259 rarrow.gif Leu257, Leu256 rarrow.gif
Ile254, Val255 rarrow.gif Ile253, Gly252 rarrow.gif
Leu 250, Ala 249 rarrow.gif Val 247) in rat and human receptors. These differences represent important
targets for follow-up point (and cluster) mutation experiments. Our current thinking is that the largest
effects on NPC 17410 pharmacology, if any, might be derived from the TM3 mutants since
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