Chemistry Reference
In-Depth Information
2.2 Target-Based NMR Screens
A target based screen focuses on changes in the protein (or other target) NMR
spectrum to identify a binding event. Typically, chemical shift perturbations (CSPs)
occur in the protein NMR spectrum upon ligand binding. The complexity and
severe peak overlap in a protein 1D
1
H NMR spectrum makes it impractical to
observe subtle CSPs for weak binding ligands. Instead, two-dimensional (2D)
heteronuclear NMR [
70
-
72
] experiments are typically used for target-based
NMR ligand affinity screens [
73
]. 2D
1
H-
13
C/
15
N HSQC/TROSY NMR experi-
ments require a significant increase in experiment time (
10 min) due to the
additional dimension and the need to collect a reference spectrum for the ligand-
free protein. Also, the protein needs to be
15
N and/or
13
C isotopically labeled.
Importantly, 2D
1
H-
13
C/
15
N HSQC/TROSY NMR experiments provide additional
information about the ligand binding site.
A binding ligand often results in the observation of CSPs of the resonances in a
2D
1
H-
15
N- or
1
H-
13
C-HSQC spectrum (Fig.
2a
). These CSPs are usually caused by
a change in the chemical environment for residues proximal to the bound ligand or
residues undergoing ligand-induced conformational changes. The availability of
the protein structure and the NMR sequence assignments (correlation of an NMR
resonance with a specific amino acid residue) allows for the CSPs to be mapped
onto a three-dimensional (3D) representation of the protein's surface. A cluster of
residues on the protein surface with observed CSPs often identifies the ligand-
binding site.
The ligand binding affinity or
K
D
is also routinely determined from CSPs
measured from a series of 2D
1
H-
13
C/
15
N HSQC/TROSY NMR experiments. The
magnitude of the CSPs at varying ligand concentrations is correlated to the
K
D
for
the protein-ligand complex using the following equation [
74
,
75
]:
>
q
K
D
þ
2
ð
K
D
þ
½þ
L
½
P
Þ
ð
½þ
L
½
P
Þ
ð
4
L
½
½
P
Þ
CSP
obs
¼
;
CSP
max
(1)
2
½
P
where [
P
] is the protein concentration, [
L
] is the ligand concentration, CSP
max
is the
maximum CSP observed for a fully bound protein, and CSP
obs
is the observed CSP
at a particular ligand concentration. A least squares fit of (
1
) to the experimental
CSP data is used to calculate a
K
D
(Fig.
2b
).
As previously mentioned, since target-based screens require the use of multidi-
mensional NMR experiments, data collection is significantly longer relative to
ligand-based NMR screens. Also target-based screens require higher protein
concentrations (
M). This severely limits the utility of
target-based NMR screens for the high-throughput analysis of large compound
libraries. Instead, the approach is typically used to validate hits from a high-
throughput screen or the analysis of relatively small fragment-based libraries
[
76
-
78
]. A fragment-based library consists of low molecular-weight compounds
(
>
50
m
M compared to
<
10
m
<
250-350 Da) that are fragments of known drugs or have drug-like properties
