EthR revisited: fragment growing this time

A few months ago we described a fragment linking approach against the protein EthR, a transcriptional repressor from Mycobacterium tubercuolosis responsible for resistance to the second-line tuberculosis drug ethionamide. In a new paper in J. Med. Chem., a different team led by Benoit Deprez and Nicolas Willand (Université Lille Nord de France and Institut Pasteur) describe work on the same target using fragment growing and merging.

The researchers started with a fragment (compound 3) they had previously made as part of an in-situ click chemistry effort. A thermal shift assay revealed that this compound marginally stabilized EthR. More convincingly, it displayed mid-micromolar inhibition of EthR binding to DNA, with respectable ligand efficiency.

Interestingly, when compound 3 was cocrystallized with the protein, it bound at two different locations within the binding site. (In the work we highlighted previously this year, a different fragment also bound at two sites, and in that case the researchers linked fragments bound at each site to create a tighter binder.) In the current paper, the researchers focused on fragment growing.

Compound 3 is a sulfonamide that can be readily constructed from amines and sulfonyl chlorides, and the researchers started by constructing a 976-member virtual library of larger sulfonamides. These were then screened in silico against the protein, and many of the top-scoring hits resulted from an isopentylamine building block (such as compound 4). Ten of these were made and tested, and indeed, compounds 4 and 8 were more effective than compound 3 at stabilizing EthR in the thermal shift assay. Moreover, not only did compound 8 show low micromolar activity in the DNA-binding assay (IC₅₀ = 4.9 µM), it also showed low micromolar activity in sensitizing M. tubercuolosis to ethionamide (EC₅₀ = 5.7 µM).

Crystallography of compound 8 bound to EthR revealed that the isopentyl substituent was binding in a hydrophobic part of the pocket, and adding a few fluorine atoms (compound 17) gave a satisfying increase in potency as well as solubility. Replacing the sulfonamide with an amide (compound 19) further improved potency.

The researchers also made a couple compounds in which a second copy of compound 3 was merged with compound 19, and although this approach did produce a compound with nearly the same potency, it was also larger and less soluble.

This team has been pursuing EthR for some time, and they were able to use information from previous structures both in the computational screening as well as in the optimization. In that sense, this is an example of fragment-assisted drug discovery. It is also another nice example of fragment work in academia.