09 April 2013

Fast, competitive thermodynamic data

The importance of thermodynamics in drug discovery is often debated, with advocates arguing that ligands binding primarily through enthalpic interactions may be superior to those whose binding is driven by entropy. Some publications support these claims, though the data are rather sparse. This is at least partly because thermodynamic measurements are typically done using isothermal titration calorimetry (ITC), which consumes sizable amounts of protein and is not exactly high throughput. In a recent issue of J. Med. Chem., Jose Caaveiro, Kouhei Tsumoto, and their colleagues at the University of Tokyo and GE Healthcare Japan describe a new screening method that could speed things up.

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.


Anonymous said...

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.

Anonymous said...

So faster but uses the same amount of protein per test?

Anonymous said...

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

Anonymous said...

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