Fragments vs Mycobacterium tuberculosis InhA

Though this could change in a bad way, tuberculosis is currently the deadliest infectious disease worldwide, causing nearly 1.7 million deaths per year. Multidrug-resistant and extensively drug-resistant strains are widespread. Some approved drugs work by blocking the mycolic acid pathway essential for mycobacterial envelope formation. One member of the pathway, the enzyme InhA, is the target of isoniazid and ethionamide. Both of these molecules are prodrugs, and a major mechanism of resistance shuts down their bioactivation. To sidestep this problem, Mohamad Sabbah, Chris Abell, and collaborators at University of Cambridge and Comenius University in Bratislava have targeted InhA directly, as they describe in a recent open-access J. Med. Chem. paper. (See here for a previous FBLD effort against this target.)

The researchers began with a differential scanning fluorimetry (DSF) screen of 800 fragments, each at 5 mM. Forty-two fragments stabilized InhA by at least 3 °C and were tested at 1 mM in three ligand-based NMR assays: CPMG, WaterLOGSY, and STD. All 18 fragments that hit in at least two of these confirmatory assays were soaked into InhA crystals at 20 mM, yielding 5 hits.

None of the fragments inhibited enzymatic activity at 2 mM, but compound 1 was chosen for optimization based on an attractive growth vector into a hydrophobic region of the binding pocket. The carboxylic acid was replaced with an isosteric sulfonamide to yield compound 6, which has measurable activity. Various substituents were tested around the new phenyl ring, with a significant boost in activity caused by an aminomethyl moiety. Cyclizing the molecule and further medicinal chemistry ultimately led to compound 23, with high nanomolar activity. A crystal structure revealed that compound 23 bound as expected and that the primary amine was making interactions with the enzyme cofactor as well as an ordered water molecule.

Unfortunately, although compound 23 slightly inhibited the growth of M. tuberculosis, it did not inhibit synthesis of mycolic acids, suggesting that activity was through a different mechanism. The researchers suggest that the molecules may not be sufficiently cell permeable or that they are effluxed or metabolized. However, it may be that they just aren’t potent enough. Perhaps further medical chemistry will improve affinity by another couple orders of magnitude and achieve pathway inhibition. Regardless, this is a nice example of a robust biophysical assay cascade followed by fragment growing and structure-based design.