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.

Getting misled by NMR: ILOE artifacts

We’ve pointed out potential pitfalls with crystallography (here, here, and here) as well as with biochemical screening, but NMR has escaped attention– until now.

NMR has of course been a mainstay of fragment discovery methods since the original SAR by NMR paper. There have been plenty of developments since, but one that is particularly intriguing relies on the “interligand nuclear Overhauser effect,” or ILOE. In the “SAR by ILOE” approach, a 2D NMR experiment is used to detect when two small molecule ligands bind to a protein next to one another. There are some attractive features of this method. First, only ligands that bind in relatively close proximity to each other will generate a signal, thereby allowing researchers to identify fragments close enough to allow productive linking. Second, the technique can be applied to proteins that are too large to study by other NMR methods. In fact, it can be used even in the complete absence of structure. So what’s the problem?

In a new paper in J. Am. Chem. Soc., Chris Abell and colleagues at the University of Cambridge applied the approach to pantothenate synthetase (PtS) from M. tuberculosis. They previously did rigorous fragment screening followed by both linking and growing on this enzyme, which we discussed last year. Initial NMR experiments with compounds 1 and 2 (see figure) in the presence of PtS showed strong ILOE signals; the problem was that signals were seen between all the protons of compound 1 and all the protons of compound 2. This suggests non-specific binding: if the two molecules were binding next to each other in a single orientation you would expect that some protons from compound 1 would be closer to some protons in compound 2 than others, and there would thus be differences in signal intensities.

Adding a methyl group to compound 1 to give compound 4 didn’t help. In fact, there were ILOE signals from both the methyl groups of compound 4 to all the aromatic protons of compound 2, again suggesting non-specific binding. Even more damning, adding the substrates ATP and pantoate failed to significantly diminish the ILOE signals as expected; because crystallography showed these fragments bind in the active site, they should have been readily displaced by substrates.


Reasoning that the hydrophobic nature of compound 4 might be causing it to aggregate at high concentrations, the researchers appended a carboxyl group to give compound 5. NMR experiments with this compound in the presence of compound 2 and the protein now revealed specific ILOE signals between the 2-methyl group of compound 5 and H2 of compound 2. Moreover, this signal could be competed by adding ATP and pantoate.

Happily, linking these two fragments together resulted in compound 6, which bound to the enzyme three orders of magnitude more tightly than either of the starting fragments. The compound was also well-behaved mechanistically, showing competitive inhibition with ATP, and a crystal structure revealed that it binds as expected given the structures of the individual fragments.

Overall then this is a success story. However, it does suggest that the ILOE method may be more prone to aggregation artifacts than other biophysical methods. In particular, had the researchers not been able to do competition experiments (if, for example, they did not have another small molecule inhibitor available) they would have had a harder time sorting things out. Also, the researchers actually had crystal structures of both compounds 1 and 2 bound to PtS, so it is not clear how valuable the ILOE data really were for linking. Still, the potential advantages of an NMR-based method that doesn’t require structure are appealing. Hopefully we will see more applications of SAR-by-ILOE, now that people are more aware of the dangers.

To grow or to link: why not both?

What can you do with fragments? The idea of linking a couple together, while successfully demonstrated in the first SAR by NMR paper, generally seems to be more difficult than gradually growing one fragment. Now a new paper in Angewandte Chemie from Chris Abell and colleagues at the University of Cambridge presents a lovely comparison of these two strategies applied to a single target.

The researchers were interested in the M. tuberculosis enzyme pantothenate synthetase (PS) as a potential therapy for TB. Using a number of biophysical techniques including thermal shifts, NMR, and isothermal titration calorimetry, Abell and colleagues identified indole fragment 1 as a low-affinity binder from a library of about 1300 fragments (see figure below). X-ray crystallography revealed that the fragment binds in the ATP-binding site. An attempt to partially mimic the triphosphate by introducing negatively charged moieties led to modest improvements in potency (compounds 1a, 1b, and 2). Compound 2 bound in a similar position as compound 1, with the advantage that the methyl group off the sulfonamide is nicely positioned for further growing the molecule. Replacing this methyl group with a methylpyridine produced compound 4, increasing the affinity by about two orders of magnitude while maintaining ligand efficiency, and crystallography revealed that this moiety binds in the P2 pocket. Thus, the fragment growing approach began with an indole of low millimolar affinity and produced a molecule with low micromolar affinity after several iterations.



At the same time, the researchers also identified benzofuran fragment 5 (see figure below) and discovered that it binds in the P1 pocket some distance from the indole fragment 1, suggesting the two could be linked. In fact, a crystal structure revealed that the two fragments are able to bind to PS simultaneously. Linking these together through the acylsulfonamide linker employed above led to compound 8, with a potency similar to that obtained from fragment growing. Compounds 4 and 8 structurally resemble each other, but although the indole fragment of each binds in the same location, the terminal fragments (the methylpyridine in compound 4 and the benzofuran fragment in compound 8) bind in different locations, the former in the P2 pocket with the later in the P1 pocket. However, the benzofuran is somewhat twisted relative to the binding mode it adopts as a free fragment.



As the researchers observe, the ligand efficiency of compound 8 derived from fragment linking is lower than those derived from fragment growing, though even the molecules developed from growing have lower ligand efficiencies than the initial fragments.

The researchers conclude:

The two strategies resulted in similar compounds with similar potencies. This outcome obscures the fact that although the linking strategy appears more elegant, the limited repertoire of linkers is likely to compromise the binding of the original fragments. In comparison, the fragment-growing strategy provides more freedom for development at each stage and allows more room for further optimization.

True. But, the fragment linking strategy does provide a clear starting point for further optimization. The researchers did not describe how they selected the methylpyridyl fragment in compound 4 or how many other moieties they tested; 5-methylpyridine-2-sulfonamide does not seem like the first reagent one would grab from the shelf. However, the methylpyridine fragment is not dissimilar to the benzofuran fragment: swap the (hydrogen-bond accepting) oxygen for the (hydrogen-bond accepting) nitrogen, and the methyl would sit in a similar position as the phenyl ring (see figure above). In other words, medicinal chemistry on compound 8 could lead quite naturally to compound 4.