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tolerated by the receptor as evidenced by only a seven-fold loss in receptor affinity (K i=0.54 nM) with
respect to the parent of the series, NPC 18545. This implies that the φ, ψ backbone dihedral angles about
Phe 5 are on the order of -60°, -60° in the biologically active conformation. This combination of dihedral
angles represents a helical twist or “kink” in the midsection of the peptide.
Since the original submission of the manuscript describing these constrained linear and cyclic peptides,
bradykinin B2 receptors have been cloned from other species including human [27,28]. Toward the goal
of designing a nonpeptide antagonist as a human therapeutic agent, it would be interesting to evaluate
these analogues against these newly reported receptor homologues. This would likely be fruitful and
valuable given that there is evidence, including that presented in this chapter, showing that guinea pig
and rat B2 receptors differ structurally from the human B2 receptor at the antagonist binding site. Hence,
a structure-activity relationship established against the guinea pig or rat receptors could be misleading in
the context of potential human therapeutics.
A systematic study of the relative importances of amides and side chains in a prototypical second
generation antagonist, NPC 18545 (DArg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9) has
recently been described [37,38]. The D-Arg0 and Ser6-DTic7-Oic8-Arg9 segments were left intact in all
peptides on the assumption that N-terminal positive charge(s) and a hydrophobic C-terminal β turn are
minimally required for binding. In a systematic fashion, the amino acids in the core of the peptide (Arg1- Pro2-Hyp3-Gly4-Phe5) were substituted with glycine, an amino acid bearing no chirality or side chain.
Binding assays, either in membranes from the guinea pig ileum or in membranes from a stable cell line
expressing the human B2 receptor, were performed on each peptide and the results compared with the
parent, NPC 18545, which has a Ki against [ 3H]-bradykinin of 0.08 nM. The elimination of all chirality
and sidechain moieties in the segment Arg1-Pro2-Hyp3-Gly4-Phe5 via replacement by Gly1-Gly2-Gly3- Gly4-Gly5 (NPC 18152), led to a peptide that no longer binds the receptor. This demonstrated that one or
more of the side chains in this segment are critical during ligand-receptor interaction. Incorporation of
either the Arg1 or Phe5 side chains led to improved potency (285 nM and 483 nM, respectively).
Constructing a peptide with side chains of both Arg1 and Phe5 in place (NPC 18149) yielded a peptide
with good affinity, Ki of 13.7 nM. Overall, this study shows that to maintain potency in the low
picomolar range, peptides in this series require Arg1, Phe5, and either Pro2 or Hyp3 (but not both). The Nterminal
charged moieties and the hydrophobic C-terminal β turn are also required. Potencies in the low
nanomolar range are attainable without including the side chains or chirality associated with Pro2 and
Pro3. These data have
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