One of the first fragment screens I was ever involved with had RNA as the target (this is back when people did anti-bacterial research). Because of that, I always try to write about targets, not proteins when referring to generic things we wish to find ligands for. Nucleic acids have secondary and tertiary structure, just like proteins, and thus have ligandable pockets. We have covered RNA as a target previously. Well, we get to add another paper to the list.
In this paper from researchers at Goethe-Universität Frankfurt am Main present their results on HIV Tat-TAR. This target was discussed over three years ago. I am not particularly impressed with the compounds or the work (even though it included RNA NMR). I was more impressed with comment made in the paper which hint at the kind of compound understanding we often cite as lacking from academic papers.
First, let's get to the guts of the paper. They have been trying to identify ligands to this target for years, yielding nanomolar affinities but limited specificity. Moving to fragments when all else fails, they decided to utilize very simple fragments: benzenes and amines, amidines, or guanidines able to be protonated at physiological pH. Their fragments were screened in a fluorescent Tat-TAR-peptide assay. Figure 1 shows the compounds tested.
|Figure 1. Compounds tested. IC50s shown in parentheses.|
Cpds 1-6 were inactive, but cpd 7 looked promising...at first glance. As the authors state:
"However, the IC50 value of this compound improves steadily when aqueous solutions are kept under air. This effect was also found with other compounds, for example tetraaminoquinazoline 23. The electron rich heterocycles in particular have the tendency to produce false positive results, presumably by forming positively charged oligomers."
I presume that they ran the assay and a few days later went back to re-test the compounds and saw anomolous results. What really strikes me is that with a 7 mM IC50 upon retesting they saw a number sufficiently different (and that they trusted) to flag it. They also note that such compounds must be carefully recrystallized and fresh powders ONLY used.
The rest of the story is not nearly as interesting. They performed 1H NMR titrations and 2D NOESY to confirm that these compounds are binding to the RNA. They do some SAR and re-discover cpd 22, a known Tat-TAR inhibitor, that has already been patented.
So, what do we learn here? Understanding your fragments and their potential liabilities in your assay is crucial.