Huge Library + Tiny Hit Rate = Novel Chemotype

As Dan recently pointed out that I pointed out, epigenetics is big.  Bromodomains get a lot of play on this blog.  One bromodomain that is not mentioned a lot in the literature is ATAD2 (because everyone is actually working on it?).  It is promising because of the diverse cellular activities it is involved in.  However, its bromodomain is quite dissimilar from to "druggable" bromodomains.  [Just an aside, can't we get away from druggable already?] Only 3 of seven residues lining the KAc pocket that interact with peptide are similar (compared to Brd4) (Figure 1)

Figure 1.  View of residues within the KAc binding site of BRD4 that interact with diacetylated residues.  Residues from the peptide are shown in teal.

So, in this paper from the Fesik lab at Vanderbilt, the use fragments to discover chemical matter against this tough target.  They utilized 15N-HSQC, like the previous post on bromodomains, because it can detect millimolar binders and (with resonance assignments) determine where on the protein it is binding.  They screened 13800 fragments (NOT a typo!) as mixtures of 12, or 1150 individual experiments.  Using the SO-FAST pulse sequence allows each experiment to be acquired in 7 minutes (6 days of acquisition).  This required more than 2 grams of labeled material.  Hits were then deconvoluted as singletons, resulting in 65 actives with Kds from 350uM to more than 2 mM (determined by HSQC titration).  12 had affinities of less than 1 mM.  This hit rate of 0.1% is low, especially for a fragment based screen, even against a PPI.  While it may ligandable, a hit rate this low still indicates this will be a very tough nut to crack. 

The assignments of ATAD2 are NOT known, but they observed a consistent cluster of resonances being perturbed (Figure 2).

Figure 2.  A. Fragment 1, B, Fragment 5, C Fragment 12.  Green Circles represent resonances which may report on ligand binding.

They discovered several novel chemotypes, never seen against bromodomains, albeit with a very low hit rate, that could be put in three clusters (Figure 3).  Cluster 1 represents known bromodomain inhibitors, while cluster 2 and cluster 3 are unique to ATAD2.  Interestingly, the Kds only differ by 2-fold, but are still more potent than other recently published ATAD2 compounds.

Figure 3.

One representative from each cluster was crystallized (1, 5, and 12).  All three fragments occupy the same pocket and make a critical contact to the conserved N1064.  They also compare their fragments to work from the SGC that scooped them. 

Figure 4. A. Fragment 1, B, Fragment 5, C Fragment 12.

In the end, this is an unsatisfying paper.  There is speculation as to how these fragments can be progressed and made more potent.  But, this entire paper is about the novel chemotypes for ATAD2.  There is no chemistry in a journal that has Chemistry in its title.  I expect more from this journal and this group.  To summarize, if you throw enough fragments at a target you can find a few that bind.