Chemistry Reference
In-Depth Information
To use SPR,
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]
a component of the biological interaction (e.g. the biomolecule, screening
target or reference compound) is attached to an optical sensor chip. The other component
is then flowed in solution past the chip. Binding of the ligand to the surface-bound target
causes a change in the effective mass and this is detected by a change in the refractive
index of the sensor. This experiment can be configured in many ways, including options for
which component is immobilized. In general, response sensitivity is related to the degree
of change in effective molecular mass due to ligand binding on the immobilized receptor.
Thus, immobilization of a small ligand to the surface and flowing past a much larger
biomolecule will result in greater sensitivity than the converse. We employed the peptide-
based, active-site directed inhibitor KTEEISEVN-statine-VAEF for immobilization.
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]
The
typical protocol is illustrated in Figure 11.4; flowing a solution of BACE results in a large
response when the biomolecule binds to the immobilized small peptidic inhibitor. If a test
compound, at a particular concentration (inhibitor 1 in Figure 11.4), is incubated with
BACE, the interaction between BACE and the immobilized reference compound will be
blocked in a manner that is dependent on the affinity of the test compound. If a higher
affinity test compound is used (e.g. inhibitor 2), the interaction between BACE and the
immobilized peptide will be further reduced. In this manner, the binding affinity can be
evaluated.
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To quantitate the binding affinity, the experiment can be repeated at multiple
inhibitor concentrations.
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]
Using these approaches, the affinity of
1
was determined as IC
50
2500 M. Next, we
selected 20 structurally related compounds from the corporate collection for analysis using
the same approach. Representatives
(2-5)
are shown in Figure 11.5. It is noteworthy that
the new leading compound
(5)
was screened in the original high throughput assays, but
was not detected because of its weak affinity.
Initial NMR
screening hit
H
H
H
H
2
N
H
2
N
O
O
H
N
HN
N
O
H
2
N
O
H
N
N
H
2
N
N
O
N
N
N
2
1
3
4
5
Inhib. at
500
µ
M
IC
50
17%
>5000
28%
2500
35%
nd
62%
nd
69%
570
µ
M
M
LE 0.32
µ
M
LE 0.28
µ
Figure 11.5
Binding affinity analysis using SPR. Based on the original NMR hit
(1)
, we iden-
tified structurally related compounds with improved binding. nd, not determined; LE, ligand
efficiency, explained in the text.
