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?
Posts
2 comments:
Dan. Thanks for covering this RT fragments story that has emerged from Eddy's lab. I'd like to point readers to an interesting paper from Skolnik and Gao recently published in PNAS.
http://www.pnas.org/content/early/2013/05/17/1300011110.abstract
They point out that an anlysis of 1,284,577 entries extracted from the ChEMBL15 and BindingDB "more than 1,400 ligands, each binds to 40 or more nonhomologous proteins.
See also the post on this at In the Pipeline.
http://pipeline.corante.com/archives/2013/05/22/how_many_binding_pockets_are_there.php
I tend to think that most protein interactions are with proteins. In a folded protein, most residue interactions are with other residues. Thinking of it this way means that small molecules have to "mimic" protein residues / functional groups in order to "trick" their way into binding proteins.
By this logic, 4-bromopyrazole is "Crafty" "Tricky" Think of it like Run DMC would describe this molecule ...."its tricky to rock a rhyme, its tricky tricky ttttricky"
Just look at 4-bromopyrazole structure. Per unit area its chemical structure packs a tremendous number of possible types of interactions with proteins. I used to think Tyrosine was really "tricky", capable of H-bond donor acceptor, Pi stack, cation, edge binding. But 4-bromopyrazole is looking pretty darn tricky too now.
So, I have been mulling this over...it comes down to your definition. Something that binds is an active, it is is active in two or more orthogonal assays, it becomes a hit. Hits are what should be discussed, actives are of no interest. The "some" fragments that showed enzymatic inhibition are the interesting ones, everything else is promiscuous until proven otherwise.
Post a Comment