Crystallographic screening of soluble epoxide hydrolase

Last year we highlighted a paper from Yasushi Amano and colleagues at Astellas in which they performed fragment-screening on soluble epoxide hydrolase (sEH), a potential target for inflammation and hypertension. A new paper from the same group in Bioorg. Med. Chem. builds on that work and provides some interesting comparisons.

In the first paper, the researchers performed an enzymatic screen with fragments at high concentrations, resulting in a hit rate of around 7.3%, of which 126 of the 307 hits resulted in crystal structures. However, despite this bounty of hits, only 2 new scaffolds were found that bind to the catalytic triad of the enzyme.

Given the success rate with crystallography, the new paper focused on crystallographic screening as the primary fragment-finding method. The researchers chose 800 fragments from their in-house collection with molecular weights between 151-250 Da. To identify new scaffolds, fragments containing amide or urea moieties – known catalytic-site binders – were excluded. The fragments were then pooled into cocktails of 10 and soaked into crystals of sEH, with each fragment at a final concentration of 1 mM. X-ray diffraction data of the soaked crystals resulted in 8 hits. To ensure that nothing was missed, cocktails of the remaining 9 fragments from pools with a hit were retested, but nothing new came up. Although the researchers do not comment on the lower hit rate compared with the original screen, this could be because they were looking specifically for new scaffolds.

Despite the 1% hit rate, the fragments identified were quite interesting, with IC₅₀ values ranging from 52 to 2200 µM. Most of the fragments formed hydrogen bonds to the catalytic triad, but the details differed from reported inhibitors. For example, several fragments contained secondary amines. Fragment 1 (cyan) in particular was well-positioned to reach into two sub-pockets on either side of the catalytic center, so 14 analogs were chosen for screening, resulting in molecules with significantly increased activity, such as compound 9 (magenta).

The crystal structure of compound 9 bound to sEH reveals that it binds in a similar manner as fragment 1. However, the added hydroxyl group is able to make new interactions that were unavailable to fragment 1, and the larger adamantyl group of compound 9 is able to make more hydrophobic interactions than the smaller phenyl ring.

This is a lovely illustration of the gains in both affinity and ligand efficiency that can be had by scaffold-hopping. It is also a nice example of using fragments to explore new chemical space. Finally, it is laudable that all the structural information is deposited in the protein data bank.