Chemistry Reference
In-Depth Information
the ligand alone in solution, (2) analyzing in the presence of BACE and (3) adding the
high-affinity active site directed peptidic inhibitor OM99-2
[
33
]
to the mixture to assess
if it displaces the ligand. Many hits from the initial screen were eliminated during the
confirmation step either because they were not displaced by addition of OM99-2 or because
the ligand was self-aggregating (aggregation causes NMR signal inversion under these
experimental conditions and thus appears as a false positive). Representatives among the
confirmed hits are shown in Figure 11.3. Although there was considerable diversity among
the hits, typical features included a hydrogen bonding group tethered to a hydrophobic
region.
H
OH
H
2
N
N
O
OH
O
O
N
N
S
O
1
IC
50
2500
m
M
LE
=
0.32
Figure 11.3
Representatives among the hits in the NMR screen. The affinity of
1
was
determined by SPR spectroscopy (below).
Because of the number and diversity of the hits from NMR screening, it was critical
to establish an efficient protocol to focus our efforts. Ideally, hits would be prioritized
according to detailed understanding of the binding mechanism using, for example, NMR
structural elucidation or crystallography.As our programprogressed, we increasingly relied
on crystallography for hit evaluation (discussed below). However, during the early phase
of our program we did not have access to these methods.
In the absence of detailed structural knowledge of the ligand bindingmode (other than the
observation that it was displaced by OM99-2), each NMR screening hit was progressed by
evaluating a set of structurally and/or chemically related analogs. This served two purposes.
First, it helped validate the hit as being representative of a potential series. Second, it helped
to define SAR and assess our likelihood of progressing the hit. Typically, we tested 10-25
compounds related to each. In cases where suitable analogs were not available in the
collection, we synthesized them. Many initial hits were eliminated in this process.
11.4 Determining Binding Affinities Using Surface Plasmon
Resonance Spectroscopy
According to our experimental methods, NMR spectroscopy could detect compounds that
bound to BACE, but did not provide a quantitative measure of the binding affinity, which, of
