21 September 2012

Fragments vs DDAH – covalent and noncovalent

Using biochemical assays to find fragments sidesteps the need for expensive biophysical instruments, but is fraught with difficulties. In a recent paper in Bioorg. Med. Chem., Thomas Linsky and Walter Fast at the University of Texas, Austin, describe how they used functional screening to discover fragment inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme involved in nitric oxide production.

The researchers used a 4000-member fragment library from ChemBridge, which they screened against two different isoforms of DDAH at 0.1 mM in a biochemical screen. This resulted in 79 hits against the human enzyme and 44 hits against a bacterial (P. aeruginosa) DDAH, 101 in total. 66 of these were then repurchased along with 41 analogs, and tested in a completely different biochemical assay against human DDAH-1; 31 showed >20% inhibition at 0.4 mM, including only 22 of the original hits, suggesting that many of the initial hits were indeed false positives. Further studies showed that 5 of the 31 compounds interfered with this secondary assay (ie, they showed “inhibition” even in the absence of enzyme), suggesting that they were false positives in both the primary and secondary biochemical assays.

DDAH contains an active-site cysteine, and the researchers wanted to exclude molecules that might be generically reactive, so they incubated the remaining 26 compounds with the low-molecular weight thiol glutathione and then retested them; this eliminated another 21 compounds.

Finally, the remaining 5 compounds were examined by mass-spectrometry, and one of these turned out to be a compound other than what was listed on the bottle! This left just 4 legitimate fragment hits.

Two of these compounds were 4-halopyridines, which, although not generally reactive with thiols, could covalently modify the active site cysteine of DDAH (see here for more details). The other two compounds were reversible, competitive inhibitors of human DDAH-1. Although low affinity (Ki values of 0.8 and 1.7 mM), they had respectable ligand efficiencies (0.38 and 0.29 kcal/mol/atom, respectively).

Interestingly, when Linksy and Fast retrospectively analyzed where the four validated fragments came from, they found that only the two halopyridines were detected in the primary screen; the two reversible fragment hits had been purchased for the secondary round of screening as analogs of primary hits.

This is a richly detailed and well-executed example of fragment-based screening in academia. It demonstrates once again that high-concentration biochemical screens can be used to find fragments, but be prepared to wade through a lot of junk: only about 2% of the original hits proved to be legitimate (see here for similar results from Vernalis on a different target). It also illustrates the utility of exploring analogs of initial fragment hits; in this case, even though most of the primary hits didn’t hold up, they nonetheless led to new fragments. Of course, this does raise the question of how spurious primary hits can lead to genuine inhibitors – what do you think?

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