14 March 2016

EthR revisited again: fragment merging this time

Fragment linking, growing, and merging: these are the main methods for enhancing affinity. Two years ago we highlighted a fragment screening effort against the tuberculosis target EthR, which involved fragment linking. A few months later we discussed fragment growing against the same target by a different group. Now the first group, led by Chris Abell at the University of Cambridge, has published a new paper in Org. Biomol. Chem. describing fragment merging.

In the original paper, a thermal shift assay had led to the discovery of a few dozen fragments, several of which were characterized crystallographically bound to EthR. In some cases, two molecules of the same fragment could bind in the large lipophilic cavity of the protein and block binding to DNA, as assessed by SPR. Capitalizing on this, two copies of compound 1 were linked together to generate a micromolar binder.

In the new paper, the researchers tried merging compound 1 with another fragment, compound 2, which also binds at two positions within the protein. Several merging strategies were attempted, and although they all stabilized the protein against thermal denaturation and could be characterized crystallographically bound to the protein, most were no better at blocking DNA binding than the original fragments. Compound 5, however, did show enhanced activity, and was the subject of additional SAR. This led to compound 15, which showed low micromolar binding by isothermal titration calorimetry (ITC) and functional activity. (Oddly, compound 1 appeared to bind considerably more tightly by ITC than suggested by its functional activity, perhaps a result of having two binding sites.) The crystal structure of the optimized, merged compound bound to EthR revealed that compound 15 binds as expected (gray), overlaying with one copy each of compound 2 (magenta) and compound 1 (cyan). 

Unfortunately, aside from compound 1, none of the molecules showed activity in a cell-based assay. The researchers propose that this is due to poor permeability across the notoriously impenetrable envelope of the mycobacterial envelope. All in all this is a nice story, as well as a sobering reminder that while potency is important, it is just one of many properties that need to be optimized.

As to the question of whether one should apply growing. linking, or merging, a single case study does not really permit generalization. However, it is satisfying that all three techniques can lead to early leads.

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