Chemistry Reference
In-Depth Information
6.4 Application of TINS to Ligand Discovery
6.4.1 Soluble Targets
To date TINS has been applied to more than ten different soluble targets. We have immobil-
ized the target at a range of concentrations for the various screens, from as high as 500 μM
to as low as 100 μM solution equivalent. We now typically screen at 100-150 μM, which
represents an optimal balance between sensitivity, artifact suppression and protein con-
sumption. In all cases we have used the pH domain of AKT as the reference. Typically
we immobilize the target and reference on the activated Sepharose, Actigel ALD. The
efficiency of immobilization is monitored by UV absorption of the supernatant and visual
inspection to insure that no precipitation has occurred. If an enzymatic assay of the target is
available, we use it at this stage to confirm that the immobilized protein remains functional.
The derivatized supports are subsequently packed into the dual-cell sample holder under
pressure (0.5 bar per cell), connected to the solvent delivery lines from the sample handling
system and then placed in the magnet. In most cases a small number of known weak ligands
(up to six) are available to test whether the target has been functionally immobilized and
to demonstrate that we can indeed detect ligand binding. One of the known ligands is then
selected for use in monitoring the condition of the target during screening. We routinely
monitor the condition of the immobilized target through repeated injection of the known
ligand throughout the screen.
Once the immobilized target has been deemed functional, we carry out the actual screen.
The mixes are delivered in 1mL volumes in deep 96-well plates to the Gilson autosampler.
Sample handling is controlled byBruker HyStar software, which communicateswithBruker
TopSpin to acquire the NMR data. Using the Hadamard sampling experiment described
earlier, we currently acquire data for 30min with an additional 5 minutes for sample hand-
ling resulting in a cycle time of about 35min. In a recent screen, 324 experiments were run
in total to assess the binding of 1393 compounds from our fragment collection. This number
includes repeated assaying of the positive control to assess target condition and some over-
lap of compounds (e.g. compounds appear in two different mixtures). This design allows
us to assess the repeatability of the screening data. Such a screen was carried out without
human intervention in under 8 days. Finally, since the target and reference are immobilized,
it is possible to change the buffer conditions to match closely the crystallography condi-
tions without regard to protein stability. We routinely screen under solution conditions in
which the reference protein would precipitate if not immobilized. Nonetheless, its ligand
binding characteristics vary only very moderately from one set of solution conditions to
the next.
6.4.2 TINS Proof of Principle Application to a Bacterial Membrane
Protein
TINS is a comparative method, where detection of ligand binding to the immobilized target
is quantitated by comparison with an immobilized reference. With membrane proteins,
partitioning of ligands can occur on the native or synthetic lipids surrounding the target
present on the resin. An appropriate reference system had to be developed to control for
nonspecific binding of hydrophobic compounds to lipids or detergents used to solubilize
