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.
3 comments:
While it is a very good example of a fragment-screening approach, this paper showed nothing new for EthR. Alain Baulard and his group have extensively shown EthR is druggable, with a lead compound of 400nM IC50, and the supposed novel cavities were already known.
So, if I read this correctly, 86 actives by thermal shift, 45 confirmed by SPR, leading to "several" crystal structures? That means that half of the TS actives did not confirm; a low false negative rate but a high false positive rate?
Disulfide is hardly an innocent linker, I wonder how good DTT is at inhibiting EthR
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