What works for crystallography?

As a recent post emphasized, crystallography is a key technique for fragment-based lead discovery. We’ve occasionally touched on things that can go wrong in crystallography, but in a recent paper in Drug Discovery Today, Helena Käck and colleagues at AstraZeneca (Mölndal) put things in a more positive light by asking what factors lead to success.

The paper starts with a literature review of successful fragment structures published between 2012 and 2014 and summarizes some of the key findings. First is the need to easily generate robust crystals that diffract well and are stable for long periods of time. If the ligand-binding site is known, it is important that this is accessible and not occluded by protein or ligands. Finally, the crystals should be stable when soaked in high concentrations (> 10 mM) of ligand, ideally in the presence of 10% DMSO.

None of these factors will come as a surprise to experienced crystallographers, but the authors do a nice job of concisely summarizing them as well as providing solutions to common problems. For example, the use of surrogate proteins can help in cases where the target itself is hard to crystallize. Proteins can be grown in the presence of known ligands, which can then be soaked out. And various additives can also help.

All of this is nice, but what really makes this paper noteworthy is the second part, in which the authors discuss their own experience with soluble epoxide hydrolase (sEH), a potential cardiovascular and immune target that we’ve previously discussed here, here, and here. This protein seems to have all the hallmarks of technical success. Indeed, both HTS and fragment screens at AstraZeneca produced high hit rates, and 65% of hits taken into crystallography produced structures. In all, 55 structures were determined, with ligands ranging in size from 130 to 540 Da and affinities ranging between 0.003 and 600 µM. Of these, 38 could be considered fragments. As seen before, the protein is relatively rigid, and the ligands bind in a variety of subsites within the large lipophilic active site.

With so much data, the researches asked whether ligand properties could predict crystallographic success. The most robust correlation was seen with affinity: 94% of compounds with affinities below 0.1 µM produced structures, while only 36% of compounds with affinities above 100 µM did.

Ligand efficiency (LE) was also correlated with crystallographic success, though three small fragments (MW < 160 Da) with very high LE values did not produce structures – a phenomenon which has been noted by others.

In contrast to another recent study that compared many fragment-screening approaches, solubility did not predict success. The researchers suggest that this is because crystal conditions are so different from the conditions under which standard solubility measurements are run.

Admirably, the structures of 52 of the ligands are reported in the supplementary material – along with their measured affinities – and the resulting crystal structures have been deposited in the protein data bank. Some of the ligands bind at multiple sites and some have dual conformations; these ambiguities are noted. Moreover, a set of inactive analogs has also been included. Together with smaller sets of previously released structures, this provides a bonanza of structural and affinity data with which to benchmark computational docking programs. Hopefully we’ll see more of this public sharing of data.