Historically the most popular
method for finding fragments has been ligand-detected NMR. Preliminary results
of our current poll (to the right) suggest crystallography has pulled ahead.
(Please do vote if you haven’t already done so.) However, NMR has many uses
beyond finding fragments, as illustrated in a recent J. Med. Chem. paper by Sacha Larda, Steven LaPlante, and colleagues at INRS-Centre
Armand-Frappier Santé Biotechnologie, NMX, and Harvard.
Among the many artifacts that can occur in screening for small molecules, one of the most insidious is
aggregation. A distrubing number of small molecules form aggregates in water,
and these aggregates give false positives in multiple assays.
Unfortunately, determining whether aggregation is occurring is not always
straightforward. The new paper provides a simple NMR-based tool to do just
that.
All molecules tumble in solution,
but small fragment- or drug-sized molecules tumble more rapidly than large
molecules such as proteins. The “relaxation” of proton resonances is faster in
slower tumbling molecules, and in the NMR experiment called spin-spin
relaxation Carr-Purcell-Meiboom-Gill (T2-CPMG) various delays
are introduced and slower tumbling molecules show loss of resonances. Indeed,
this technique has frequently been used in fragment screening: if a fragment
binds to a protein, it will tumble more slowly, resulting in loss of signal.
The researchers recognized that
an aggregate could behave like a large molecule, and they confirmed this to
be the case for known aggregators, while non-aggregators did not. The
experiment is relatively rapid (~30 seconds), and has been used to profile a
5000-compound library to remove aggregators.
One of the frustrations of
aggregators is that it is currently impossible to predict whether a molecule
will aggregate, and indeed, the researchers show several examples of
closely related compounds in which one is an aggregator while the other is not.
Even worse, the phenomenon can be buffer-dependent: the researchers show a
fragment that aggregates in one buffer but not in another, even under the same
pH.
Many fragment screens are done
with pools of compounds, and the researchers find that molecules can show a
“bad apple effect”, whereby previously well-behaved molecules appear to be
recruited to aggregates.
The limit of detection for T2-CPMG
is said to be single-digit micromolar concentration of small molecule, though the researchers note that double-
or triple-digit micromolar concentrations are more practical, which is more
typical of fragment screens anyway. And some compounds may show rapid
relaxation due to non-pathological mechanisms, such as tautomerization or
various conformational changes.
Still, this approach seems like a
powerful means to rapidly assess hits, and pre-screening a library makes sense.
Another NMR technique using interligand nuclear Overhauser effect (ILOE) has also
been used to test for aggregation, though not to my knowledge so
systematically. For the NMR folks out there, which methods do you think are
best to weed out aggregators?
Posts
1 comment:
I remember seeing similar "bad apple effect" with different combinations of fragments to make pools. Indeed, a compound behaves well in one pool may become an aggregator in another pool.
If using a T2-CPMG like NMR method for fragment screening, the aggregators probably would show weak or no signals in the control assays where the bio-target (e.g. protein) is absent, due to loss of resonances as this paper suggested. So control experiments could really help in weeding out aggregators. This would be the case for other NMR screening methods like STD-NMR where the aggregators behave like large bio-molecules and give rise to saturation transfer even in the absence of real bio-molecules.
Post a Comment