Biomedical Engineering Reference
In-Depth Information
structure of a macromolecule: if the structure of one of the ligands bound to
the protein is available, then an unknown ligand can be oriented on the
protein. A particularly impressive example of this was the recent elucidation of
the binding orientation of a number of ligands of the GPCR GPR40.
30
Although not strictly a magnetisation-transfer method, Interligand NOEs
(ILOEs) measures direct NOEs between two ligands that simultaneously bind
a protein and are within 5-7
˚
of each other. The method can be used to screen
for pairs of ligands that can be subsequently elaborated based on the relative
orientation provided by the NOE information.
38
In the latter publication,
fragment screening identified two ligands that simultaneously bound to
pantothenate synthetase from Mycobacterium tuberculosis. However, no
structural information was available and the hydrophobic ligands aggregated
and bound the protein specifically and non-specifically. Chemical modification
of the fragments addressed the aggregation and using the ILOE experiment the
orientation of the two fragments was established. Subsequent linking yielded
an 860 nM inhibitor from fragments with dissociation constants of 800 mM
and 1 mM, suggesting that the binding of the fragments was not perturbed in
the linked compound.
11.2.2.3 Fluorinated Molecules
19
F NMR has proven to be a useful tool in drug discovery.
19
F NMR is
sensitive (the c is similar to that of
1
H) and the spins resonate across a broad
spectrum. Although direct
19
F screening of fragments has been proposed,
39
the
method clearly requires a fluorine atom in each member of the library and
thereby limits the number and type of compounds available. However, when
known ligands
40
or enzyme substrates are available,
41
incorporation of
fluorine into these molecules can lead to a high-throughput, reliable assay
that selects for ligands for a specific binding site (via competition binding) or
with biochemical activity. More recently the combined use of cryoprobe
technology and optimised pulse sequences has led to considerable increases in
the throughput (up to 10-fold) of this experimental approach.
42
The group of
Giralt has taken the approach of simultaneously screening for inhibitors of two
different proteases by labelling substrates specific for each with
19
F and mixing
them.
43
In addition to throughput considerations,
19
F NMR can be used to
characterise hits from fragment screening. In one interesting case,
19
F-labelled
ATP was used to help characterise fragments binding to the kinase PDK1 and
a number of allosteric activators were discovered.
73
11.3 Hit Prioritisation
High-throughput screening (HTS) has been widely used in drug discovery to
screen large libraries (up to 3 or 4 million compounds) for (typically) biological
modulation of a target.
44
HTS screens may result in a few hundred to many
thousands of positives. Among these 'hits' many are not the result of a specific,
