Fragments vs CHK2: high-concentration screening comes through

Checkpoint Kinase 2 (CHK2) is an oncology target that has been kicking around for years. Its relevance is still debated, so having more small molecule inhibitors would go a long way toward assessing its therapeutic potential. In a recent paper in PLoS One, Rob van Montfort and colleagues at The Institute of Cancer Research (UK) present their fragment-based efforts on CHK2.

The researchers describe the design of their screening library in some detail, starting with a series of typical computational filters on commercially available molecules. Although most of the molecules had MW < 300, molecular weights up to 320 Da were allowed for fragments containing F, Cl, or SO₂ moieties. Also, all fragments were required to have at least 10 heavy atoms, which is on the high-side for a minimum. A total of 1869 fragments were purchased. All of these were analyzed for solubility and purity (by nephelometry and LC-MS, respectively), though unfortunately the researchers do not provide pass rates.

Having assembled the library, the researchers then screened each fragment at 300 micromolar against CHK2 in a biochemical assay (AlphaScreen). This led to 45 hits, but 25 of these showed some interference with the AlphaScreen assay itself. However, the remaining 20 all showed dose-response curves in a different assay format, giving IC₅₀ values from 2.7 to 944 micromolar.

In parallel, the researchers screened CHK2 using a thermal shift assay, with each fragment present at 2 mM. Perhaps not surprisingly given the higher concentrations used, this led to 63 hits.

Where things got interesting – and encouraging – was when the researchers compared hits identified using the two methods. In contrast to others' experiences, there was reasonable overlap; of the 14 hits from both assays, 12 yielded measurable IC₅₀ values when assessed using a microfluidic functional assay. Most of the AlphaScreen hits that didn’t produce thermal shifts came from the set of 25 that had previously been flagged as interfering with the AlphaScreen assay itself, and several were also insoluble. Of the 49 thermal shift hits that did not show up in the AlphaScreen assay, 13 were insoluble. Regarding the remaining 36, the researchers propose that they may bind to CHK2 outside its active site and thus don’t inhibit enzymatic activity.

Next, the researchers attempted to characterize the binding modes of the fragments crystallographically. Of the nine fragments that produced structures, eight came from the set of fragments confirmed using both AlphaScreen and thermal shift. Significantly, the only fragment to yield a structure that was identified solely from the thermal shift assay also produced the worst IC₅₀ value (228 micromolar) and the lowest ligand efficiency. All nine fragments bind to the so-called hinge region of the kinase.

One interesting observation was that, although the library did contain larger molecules, 6 of the 9 fragments characterized crystallographically had MW < 200, and the other 3 were well under 300 Da. This is exactly what you would expect according to the concept of molecular complexity, and suggests that adding larger fragments to your library may actually lower your hit rate (though admittedly it may be a stretch to conclude too much from this one study).

Another interesting note is that co-crystallization was used in all cases. Folks sometimes believe that you need to grow vats of crystals for fragment soaking experiments, but co-crystallizing worked fine here, and in some cases may allow the protein to adopt different conformations than if grown in the absence of small molecules.

Overall, the paper presents some nice starting points against CHK2. Perhaps more important, this is a thorough, well-written, and open access account of fragment screening that is well worth perusing by anyone embarking on a fragment campaign.