Biomedical Engineering Reference
In-Depth Information
Among target-based NMR techniques, the heteronuclear single quantum
coherence (HSQC) spectrum of a
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N- or
13
C-labelled protein is, by far, the
most commonly used to detect ligand binding. This technique monitors
chemical shift perturbations (CSPs) in the [
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N,
1
H] HSQC spectrum upon
ligand binding. An application of this method to BACE-1, a protease that is a
target for Alzheimer's disease, was recently published.
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Since the location of
BACE is within the brain, small-molecule inhibitors must first traverse the
blood-brain barrier. This extra requirement places further restrictionson
BACE inhibitors than those of other proteases, a class of targets already
considered challenging. The group of Daniel Wyss developed an efficient
scheme to produce labelled protein from inclusion bodies and used the protein
to screen approximately 10 000 fragments before the sequential assignment was
available. By first titrating known peptide analogues, the relevant CSPs were
determined and hits from the screen were selected by their effect on these
resonances. When the sequential assignment became available (via triple
labelling and through-bond methods),
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binding to the active site was
confirmed for nine independent classes of compounds. This latter publication
also contains many suggestions for successful elaboration of initial hits
discovered using NMR spectroscopy. The most promising fragment exhibited
instability and therefore the HSQC experiment was used to screen for stable
isosteres that also bound the active site aspartates of BACE-1. Although this
search was not ultimately fruitful,
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structure-activity relationship (SAR)
information gathered led to the conceptualisation of a new core which was also
characterised by NMR. The ability of the HSQC experiment to quickly discern
different binding modes of the BACE inhibitors proved to be critical for the
project. The approach has successfully yielded a compound that is now in
clinical trials.
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In an interesting twist to screening using HSQC spectra, Holak and co-
workers
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developed the antagonist-induced dissociation assay (AIDA) screen
for inhibitors of protein-protein interactions. This method monitors changes
that occur when a large protein (more than 30 kDa) binds to a smaller protein
(less than 20 kDa). When the complex is formed, the resonances broaden due
to increased transverse relaxation. In its initial incarnation, the smaller protein
(the N-terminal p53 binding domain of MDM2) was isotopically labelled and
mixed with the larger protein (the N-terminal 350 amino acid residues of p53)
resulting in the disappearance of most of the MDM2 peaks. The addition of
molecules that disrupt the protein-protein interaction restore the spectrum of
MDM2. In contrast, if compounds simply bind one partner, the spectrum is
unchanged. Variants of this technique have been recently implemented: a one-
dimensional (1D) proton version of AIDA
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and SEI-AIDA.
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The 1D proton
NMR version of AIDA monitors ligand binding though the effect on
tryptophan proton resonances, alleviating the requirement for labelled protein.
The SEI-AIDA (SEI for Selective Excitation Inversion) combines the 1D
proton technique with a selective excitation of protein resonances which
enables shorter relaxation delays.
