Assessing ligandability by thermal scanning

Ligandability refers to the ability to find small-molecule leads against a target. A protein might be ligandable but not druggable if, for example, potent inhibitors of the target do not affect a disease state. But knowing in advance whether a target is ligandable can be useful, both to decide whether to embark on a campaign and to plan the resources it will likely require. Fragment screens by NMR have been shown to be good predictors of ligandability, but not everyone has access to this technology. Computational methods (such as FTMap) are also useful, but require a structure of the target. In a recent paper in J. Med. Chem., Stefan Geschwindner and colleagues at AstraZeneca describe high-throughput thermal scanning (HTTS) for assessing ligandability.

Thermal scanning (alternately called, as the researchers note, thermal shift, differential scanning fluorimetry (DSF), or thermofluor) relies on the preferential binding of a fluorescent dye to protein that is heat-denatured. Since ligands generally stabilize a protein against denaturation, an increase in melting temperature (Tm) is taken as an indication of binding. The assays can be plate-based and thus very fast.

The researchers chose 16 diverse targets (mostly enzymes) and screened their 763-ligandability fragment set (described here) at 1 mM by HTTS. Hits were defined as compounds that increased  thermal stability at least 3-fold above the standard deviation of controls. Targets were then categorized as follows:

Low ligandability: hit rate < 1.5%

Medium ligandability: hit rate between 1.5 and 4.5%

High ligandability: hit rate > 4.5%

Nine targets ranked low, and all of these failed high throughput screening (HTS), while 5 out of the 7 targets ranked medium or high by HTTS yielded useful HTS hits. Of course, failure in an HTS does not preclude target advancement by other means – including FBLD. Ultimately all but three targets (including all of those ranked medium or high and 6 of 9 ranked low) went on to enter hit-to-lead optimization programs.

Encouragingly, HTTS and NMR agreed perfectly for low and high ligandability targets, but NMR assigned three targets as medium where HTTS assigned them as low. The researchers thus set out to increase the sensitivity of HTTS.

It turns out that entropically-driven binders tend to cause greater thermal shifts than enthalpically driven binders. The observation that most fragments bind largely enthalpically, and with low affinity too, makes them particularly challenging to detect. To try to shift the balance, the researchers repeated the HTTS assay for three of the low-scoring targets in D₂O instead of H₂O, which enhances entropic interactions at the expense of enthalpic interactions. Indeed, all three targets showed enhanced hit rates, and two moved from low to medium ligandability.

Another way to improve sensitivity of a thermal shift assay is to add urea, which destabilizes proteins by lowering the unfolding enthalpy. Adding non-denaturing amounts of urea (0.8 to 2.4 M concentration) to the three low-scoring targets above did indeed increase the hit rate for two of them.

One interesting tidbit is the observation that particularly stable targets, with unfolding temperatures >70 °C, tend to produce lower hit rates in HTTS than less stable targets. This could account for the very different experiences people have had with the technique.

This is a nice paper, and the approach may be worth implementing, as the researchers note has already happened at AstraZeneca. Although HTTS is unlikely to ever be as robust as SPR, NMR, or crystallography, it is hard to beat the low cost and high speed.