The approach, which is essentially a competition screen, is
called single-injection thermal extinction, or SITE. In a conventional
calorimetric assay, a ligand is added to the protein in a calorimeter, and the
measured heat change upon binding is used to calculate enthalpy. In SITE, a
protein is first incubated with the fragment to be tested in the calorimeter,
and a known positive control binder is then added. If the fragment binds at the
same site as the known binder, addition of the positive control will cause a
smaller change in temperature, and this difference should reflect the enthalpy
of binding of the fragment compared to the positive control.
To validate the system, the researchers tested the
steroid-processing enzyme ketosteroid isomerase (KSI). They first ran an SPR
screen of 2000 fragments at 0.2 mM. To weed out false positives, they used two
different types of SPR chips, and looked closely at the binding curves; the
paper has a nice summary of some of the pathologies that can occur. A total of
129 hits were identified, of which 44 were then tested in SITE and
characterized more fully with SPR.
Interestingly, most of the most potent compounds – as
assessed by SPR – also gave the strongest signals in SITE, suggesting that
these compounds are binding largely through enthalpic interactions. A few of
the best compounds were further characterized by conventional ITC, and these did
in fact have better enthalpic efficiencies than the positive control (they had
better ligand efficiencies too).
It would be interesting to know how SITE behaves with
allosteric inhibitors or ligands that bind to different sites on the protein.
And of course, the jury is still out on whether enthalpic binders really do
make superior leads, and even whether it is possible to use thermodynamics
prospectively in lead optimization. But with a 9-fold drop in protein
consumption and an increase in speed, this technique may make it easier to get
the data to answer these questions.
4 comments:
One of the shortcomings of this approach is the fact, that the binding occupancy of the fragment will impact directly on the enthalpy readout. A fragment with less negative enthalpy but higher binding occupancy will be ranked similar to a fragment that displays a much more negative enthalpy but experiences less binding occupancy. This has direct consequences for selecting enthalpy-favoured fragments, as this approach is still based on a combination of enthalpy and affinity and does not give a direct measure of the binding enthalpy.
So faster but uses the same amount of protein per test?
It´s both speed and quantities that are positively affected. One needs about 1/4 of the usual protein amounts, which brings it into a similar protein concentration regime as standard NMR screening methods
The amount of protein for each assay strongly depends on the protein under scrutiny. For example, the concentration used to evaluate fragments with KSI was relatively high (50 uM) because of the low enthalpy of binding of the positive control, whereas for the protein ERK2 5 uM did suffice (supplemental information).
SITE cannot distinguish purely high affinity from purely high enthalpy as explained in the manuscript, and also pointed out by the comments here. But SITE reduces the number of potentially useful fragments to just a handful of them. These few compounds are now suitable for a in-depth analysis by multi-injection ITC. In the examples published in our report, as well as other unpublished data, high SITE effect is consistently correlated with high affinity (sub-mM range) and large EE values.
Ultimately, we hope SITE will be thoroughly tested by multiple laboratories and found it to be a useful methodology.
The authors
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