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This initial stage of the docking process was used to reduce the computational difficulties that would be
inherent in “tumbling” a complete bradykinin molecule (which has great flexibility) about the receptor
in a similar fashion. However, following this initial stage, insight into those regions of the receptor
capable of accommodating the C-terminal portion of the bradykinin molecule was obtained. On the basis
of energetics, and as qualitatively shown in Figure 4, those particular regions are clustered in the central
part of the receptor near to the extracellular domain. Using this information as a steering device to limit
the size of the problem, an exhaustive conformational search was performed using the entire nineresidue
sequence of bradykinin as a probe molecule, again enforcing a C-terminal β turn via dihedral
angle constraints. Specifically, 24 unique geometric orientations (eight on each of three axes) of the
bradykinin molecule were sampled at each of 100 grid points identified during the initial stage as likely
zones to bind the tetrapeptide probe molecule. Bradykinin-receptor complexes within the lowest 150
kcal mol -1 interaction energy with respect to the lowest found (17 complexes out of 2400) were grouped
into sets of related conformational families, of which there were five. Computationally, each of the five
complexes were presumed to be equally likely. All of these simulations were accomplished using
custom routines written using the program CHARMm [21].
To guide the selection of which of the five bradykinin-receptor complexes to consider a “lead” model,
supporting experimental evidence was sought from site-directed mutagenesis experiments. This support
was taken primarily from work describing bradykinin binding assays performed of mutant rat B2
receptors [44,45]. The underlying strategy of the mutation studies was based on the hypothesis that,
since bradykinin has positive charges at either end of its sequence (Arg 1 and Arg 9), separated by a group
of rather hydrophobic amino acids (Pro 2-Pro 3-Gly 4-Phe 5-Ser 6-Pro 7-Phe 8), it was likely that some acidic
residues in the receptor participated during ligand binding. Several mutant receptors were made such
that each contained either a point mutation or a small cluster of point mutations, wherein native residues,
having negatively charged side chains (Asp, Glu), were replaced by alanine(s). Table 2 lists the initial
cluster mutations (rat) that were prepared as well as the follow-up single point mutations (rat).
Figure 5 shows a stereoview of the selected ligand-receptor complex chosen on the basis of best
agreement with the results of these mutagenesis studies. None of the other four putative complexes were
in agreement with this experimental data and were not considered further. Of particular significance in
this work was that the trans-membrane residue Glu 49, when mutated to alanine, showed no adverse
effect on bradykinin receptor affinity with respect to rat wild type. A similar result was reported for the
Glu 196 rarrow.gif Ala 196 mutation. These residues are remotely situated with respect to the proposed site
of bradykinin
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