ATAD2 Again...Now with a good tool.

Epigenetics is big.  We keep on beating that drum.  Just to prove it, today's paper is on a target we have talked about before: ATAD2.  That previous paper was unsatisfying: leading to my summary: "if you throw enough fragments at a target you can find a few that bind."  Today's entry  from GSK has produced the first micromolar inhibitors of ATAD2.  

As noted previously, ATAD2 is "undruggable" or at least VERY difficult to find chemical matter against.  To add to the difficulty,  the BET activity needs to be minimized.  With that in mind, they set a high threshold of activity (pIC50 greater than7) and 100 fold selectivity against BRD4 (a representative BET domain).  The ATAD2 site is more polar and flexible than BET.  The authors felt that this would be exploitable to create selective molecules.  To address ATAD2 they started with Ac-K mimics from previous BET work.  They supplemented this with diverse cores not represented.  One such array (which I read as libraries, somebody correct me if I am wrong) was based on the cpd 1,

Cpd 1. 

which is similar to the chemotypes discussed last year.   A crystal structure of 1 was solved, confirming that it bound as expected.  

Additional arrays were made around this core and tested in a TR-FRET assay.  30,000 compounds gave a 0.25% hit rate.  Confirmation was performed by HSQC NMR.  A subset of compounds interacted at the Ac-K site based upon comparison to compounds with known binding modes.  In this case, the peak that shifted upon binding were the same.  I would like to know if this was by visual inspection of spectra or if it was accomplished using PCA, or similar method.  It probably doesn't matter, but intrigues the NMR jock in me.

In rounds of medchem and X-ray confirmation, they were able to drive the potency against ATAD2 to the single digit micromolar.  The ligand efficiencies were maintained right around 0.30. Compound 57 (R=4-Me) and 60 (R=4-OMe) had the "best balance of ATAD2 and BET activity".  These compounds were also active in a cell-based assay known to be sensitive to BET inhibitors.  However, there is no selectivity.  ATAD2/BET pIC50 for 57 was 1.1 and 60 was 1.0. So, despite the selectivity threshold they developed, these compounds are not selective.  Despite that, I think this paper shows that the aphorism Undruggable =Undone is true.

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.