Pocket binding site

In the original fragment screening campaign we noticed that most of the hits were in 3 areas: substrate binding site, the ATP binding site and another area we termed the "pocket".

We found that the minifrag succinimide (PDB: 7B6J) also binds to this pocket. In a follow-on study compounds were designed based on the original fragment hits and the minifrag and 4 crystals structures were obtained (PDB: 7B6L, 7B6M, 7B9W and 7B6N).

Becca Steventon, in Chris Dowson's group, tested these compounds in an enzymatic assay.

Compounds were incubated with MurE for 10 minutes at 37c at 1mM (Compound 16 was tested at 0.5mM due to interference with the assay at 1mM). Ligase activity was then determined.

Compounds that were able to reduce the activity of the ligase by more than 35% were carried forward, and their effect on the coupling reaction was determined. Final activity levels were then determined for compounds that were true inhibitors.

3 compounds were identified which were able to reduce enzyme activity ~23% at 1 mM . However, the results were inconsistent across the series and compounds that were shown to inhibit enzyme activity don't have crystal structures. Taking into account that the compounds are basically brickdust and the lack of structural confirmation we have to conclude that there isn't enough evidence at present to support the hypothesis that the "pocket" is a newly discovered allosteric site.