Anna Kohlmann and colleagues at Ariad started with a fairly small library, just 735 fragments from Maybridge. These were screened using STD-NMR at 2-3 mM per fragment, resulting in 38 hits, about half of which contained carboxylic acids – not surprising given that the substrate and cofactor are both negatively charged. Most of the fragments could be competed by the cofactor NADH, and although they bound too weakly to show any inhibition in an enzymatic assay, they did show binding by SPR. Crystal soaking led to a co-crystal structure of compound 1, which binds in the substrate and part of the cofactor site (where the nictotinamide moiety of NADH normally binds).
Fragment growing led to compounds 2 and 5, both with enhanced affinity. Interestingly, crystallography revealed that compound 5 binds in a distant part of the cofactor binding site, where the adenosine moiety of NADH normally binds. Elaboration of this molecule didn’t do much for affinity but did suggest a linking strategy, resulting in molecules such as compound 9, with nanomolar potency and detectable cell-based activity.
Apropos to Darwin Day, this is an interesting example of convergent evolution: two companies applying fragment-linking to discover molecules that bear some similarity to one another (Ariad compound 8 in blue, AstraZeneca compound 26 in red).
Near the end of the paper, the researchers also carefully investigated some of the other previously reported “inhibitors” of LDHA and found that they are in fact aggregators. This is not surprising given their structures, which look like something that might appear in an April Fool’s post. Unfortunately these molecules were reported in prominent journals such as Chem. Biol. and Proc. Nat. Acad. Sci. USA; the later, published in 2010, has already been cited at least 100 times. Publicly revealing them to be artifacts is a beautiful example of the self-correcting nature of science. I hope we’ll see more of it.