The researchers assembled a library of 775 fragments, 500 from Maybridge and most of the rest from Sigma-Aldrich and Acros. These were combined into 143 pools of 4 to 8 fragments, each at 100 mM in DMSO. Crystals of RT grown with the drug rilpivirine were soaked with each of the pools; rilpivirine stabilizes the protein and yields crystals that diffract to high resolution. The researchers also added 80 mM arginine and 6% trimethylamine N-oxide (TMAO) to the soaking solutions; arginine helped solublize some of the more hydrophopic fragments and improved electron density, while TMAO improved diffraction.
Overall, the researchers found 34 fragments that bound to HIV RT, a hit rate just over 4%. Interestingly, halogenated fragments seemed to give a much higher hit rate: 7 of 29 fluorine-containing fragments produced structures, as did 4 of the 17 brominated fragments and one of the two chlorinated fragments. I don’t recall seeing halogens previously over-represented among fragment hits, though last year we did write about halogen-enriched fragment libraries. The sample sizes reported here are small, but if the findings hold up in other studies, fluorine fetishism may be further justified.
But just as interesting as the composition of the fragment hits is the number of binding sites in the protein: 16, with names ranging from the descriptive (“NNRTI Adjacent” and “Incoming Nucleotide Binding”) to the concise (“399”) to the downright thuggish (“Knuckles”). In the case of three of these sites, some of the fragments also inhibited enzymatic activity.
There is a lot of nice information here, and eight co-crystal structures have been deposited in the protein data bank. Still, I am left a bit dizzy at the sheer number of sites. In fact, one fragment (4-bromopyrazole) bound to all of the 16 sites! What are we to make of this – is this a privileged fragment or a promiscuous binder? And as for the sites with no known functional activity, are these useful? What do you think?