Flexible fragment linking vs the transcription factor EthR

Transcription factors have a well-earned reputation for being extremely difficult targets. Although the literature is littered with inhibitors of various transcription factors, most of these turn out to be of questionable validity, to put it politely. A recent paper in Biochem. J. by Sachin Surade, Tom Blundell, and collaborators at the University of Cambridge and the Ecole Polytechnique Fédérale de Lausanne-EPFL reports what looks to be the real deal.

The researchers were interested in a protein called EthR, a transcription factor from Mycobacterium tubercuolosis involved in antibiotic resistance. Unlike many other transcription factors, this one contains an allosteric binding pocket known to bind lipophilic molecules. Armed with this knowledge, the researchers performed a thermal shift assay using a library of 1250 fragments at 10 mM each, which resulted in 86 hits that stabilized the protein by at least 1 °C. These were then tested for their ability to disrupt the interaction between EthR and DNA using surface plasmon resonance (SPR), and 45 of them showed greater than 10% inhibition at 0.5 mM. Reassuringly, only 1 of 45 fragments that had shown no stabilization in the thermal shift assay showed more than 10% inhibition here, suggesting that the thermal shift assay had a low false negative rate.

Confirmed hits were characterized by full dose-response curves and soaked into crystals of EthR, resulting in several co-crystal structures. Compound 1 was particularly interesting because two copies of it bound to the central hydrophobic channel, which was only possible due to conformational changes in the protein. Also, although the likely natural ligand of EthR appears to make only hydrophobic contacts to the protein, the carbonyl of compound 1 makes hydrogen bonds. In one of the two bound molecules, the interaction is with an asparagine residue of EthR; in the other, it is with a water molecule.

Swapping the cyclopentyl ring to a phenyl to yield compound 5 gave a slight loss in potency but simplifies subsequent modifications, and crystallography revealed that it binds in the same manner as compound 1. More significantly, linking two molecules of compound 5 via a disulfide bond (compound 9) improved the affinity by more than two orders of magnitude.

Of course, disulfides can react with cysteine residues in a protein – a fact that can be rather useful for finding inhibitors. Thus, it was essential to demonstrate that compound 9 was really binding non-covalently to the protein rather than acting through an unrelated mechanism. Happily, the researchers were able to determine the co-crystal structure of compound 9 bound to EthR, confirming that it binds in the same manner as the two molecules of compound 5, including the two hydrogen bonds. (Unfortunately though, none of the crystal structures appear to be deposited in the protein data bank.)

Compounds 1 and 9 were both tested for their activity to enhance the effect of the antibiotic ethionamide in Mycobacterium tubercuolosis cultures, and both were active, though with similar potencies despite their very different affinities to the isolated protein; it seems likely that the disulfide bond would be reduced in the bacterium. It will be interesting to replace this with a more stable linkage (amides were also tried but did not improve affinity).

One interesting conclusion is that “flexible fragments in the library can lead to a more efficient exploration of chemical space.” This is exemplified by the fact that floppy fragment 1 binds in two somewhat different conformations to the two sites on the protein. Having some flexibility in the early stage of a project can be useful, and another reason not to be too rigid in assembling a fragment library.