Biomedical Engineering Reference
In-Depth Information
schematic representation of a prototypical NAAG-based inhibitor and several
inhibitor examples are shown in Figure 3.3.
3.7.1.2.2 S1 Pocket in Inhibitor Design. The strict enzymological defini-
tion would restrict the S1 pocket to the arginine patch. However, the 3D
structures of various GCPII/inhibitor complexes reveal a shape-shifting 'S1
funnel' (or even several S1 funnels) with a narrow base (
0.8 nm at the
position of the active site zincs) that widens towards the surface of the pro-
tein. The rim of the S1 funnel is
B
2 nm away from the base and has an
approximate diameter of 2 nm. Depending on the conformation of the
entrance lid, the S1 funnel can be either continuous with the external space
(the entrance lid open) or 'plugged' by the entrance lid helix, effectively
restricting its size and shape.
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This structural feature is much less restrictive
and more amenable for modifications of the distal functionalities (D-moi-
eties) of GCPII-specific inhibitors. This positional and structural variability
can be seen by superimposing several diverse GCPII-specific inhibitors in
the GCPII specificity pocket. While the P1
0
parts of inhibitors overlap very
well, the D-parts probe a vast area of the S1 funnel. Additionally, interac-
tions between the D-part of an inhibitor and GCPII can lead to pronounced
structural rearrangements of the enzyme, resulting in the appearance of
'remote binding sites' (see below) that can contribute further to the chemi-
cal diversity exploitable for inhibitor design.
The existence of 'remote binding sites' (RBSs) interacting with distal groups
of inhibitors was inferred from early SAR studies, and these observations were
later upheld by structural studies. In general, such RBSs are found in the
vicinity of the S1 funnel and are revealed only upon favorable interaction with
the D-moiety of an inhibitor. The common denominator of isolated RBSs is
their very low anity and specificity for a D-moiety of an inhibitor, but the
avidity effects are responsible for a substantial decrease in GCPII binding
constants for a given inhibitor.
So far, two remote binding sites have been identified by X-ray crystal-
lography, and more detailed and extensive studies of diversified inhibitors will
likely expand the existing RBS portfolio.
The first RBS was revealed in a series of X-ray structures of GCPII in complex
with four urea-based compounds featuring variable D-moieties.
80,83
The study
led to the identification of a hydrophobic pocket adjacent to the S1 site with
approximate dimensions of 0.85
0.7
0.9 nm. The bottom of the pocket is
defined by amino acid segments Arg463-Asp465 and Arg534-Arg536, while the
walls are shaped by the side chains of Glu457, Arg463, Asp465, Arg534, and
Arg536. The formation of the pocket is enabled by the flexibility of the side
chains of Arg536 and Arg463 and their simultaneous 'atypical' positioning,
elicited by the binding of the DCIBzl inhibitor. The engagement of the distal
inhibitor moiety contributes to tighter inhibitor binding (410-fold increase).
70
The second RBS, termed the 'arene-binding cleft,' participates in an aromatic
stacking interaction with distal part of inhibitors.
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Although the arene-binding
B
