Protein-templated click chemistry – just add copper

We’ve written previously about protein-templated chemistry (here and here), in which a protein catalyzes the formation of a more potent inhibitor from two lower affinity fragments. Of course, proteins aren’t the only things that can catalyze reactions: copper is well-known to promote the cycloaddition between azides and alkynes. Like peanut butter and chocolate, it turns out that copper in the context of a protein can be even better than either alone, as reported in a recent issue of Angew. Chem. by an international team of researchers from Japan and the US.

The researchers were interested in using in situ click chemistry to discover inhibitors of histone deacetylases (HDACs), and they decided to see if they could use an activity assay to detect the formation of inhibitors formed in situ. They incubated two different hydroxamic-containing alkynes (known HDAC inhibibitors) with 15 different azides in the presence of HDAC8 and looked for enhanced inhibition of the enzyme. Of these 30 combinations, they found a single hit: the reaction of compound 1b with compound 2o (see figure).


However, there were several oddities. First, the linked compound (anti-3) is no more potent than the initial hydoxamic-containing fragment. Second, only the anti isomer was formed, despite the fact that the syn isomer is almost 10-fold more potent. Finally, the yields of anti-3 were much higher than typically observed in these sorts of experiments. This made the researchers suspicious, and after a series of experiments they determined that trace amounts of copper, most likely introduced in the synthesis of 1b, had incorporated into the active site of HDAC8 and were serving to accelerate the reaction. A small amount of copper in the absence of protein was unable to catalyze the reaction, nor was the protein alone when copper was carefully removed.

There are a number of interesting implications from this paper, but one in particular is rather sobering: in situ assembly screening does not necessarily yield the most potent inhibitor. I suspect this is a general feature of kinetically-guided methods of inhibitor discovery, but what do you think?