03 December 2014

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. 


Peter Kenny said...

Hit 2 is more likely to exist predominantly as the 1,5-naphthyridone tautomer than as the hydroxy-1,5-naphthyridine. Protonation of hit 9 under assay conditions should be considered (I don't have a relevant pKa measurement to hand) and may not be as similar to its cluster-mates as inspection of the structures might suggest. I was struck by the number of compounds with aniline-like amino groups in the hit list. Were the des-amino analogs available or tested? Good to see 6 in the hit list (I may have even put this very same compound into a fragment screening library) and it's just the thing to get those tiresome anti-flat evangelists spitting feathers.

Dan Erlanson said...

I guess I have a more favorable impression of this paper than Teddy does. Sure, it's a low hit rate, but in the end you only need one good fragment (and a lot of luck). Also, new chemotypes are themselves valuable, and I applaud the researchers for both putting them in the public domain and for depositing the crystal structures in the protein data bank.

Dr. Teddy Z said...

The paper is fine, finding good chemical equity against a tough target is good science. My disappointment is that, once scooped, it made it into JMedChem without showing any of those compounds being advanced to at least the tool stage. Even without being scooped, how many papers without ZERO chemistry (or at least some analog by catalog) make it into JMedChem?

Douglas Kell said...

There is no need to have an actual synthesis to make a useful advance, e.g. in algorithms for selecting/combining/analysing fragments, that it might be of use for the Med Chem community to know about - related is http://link.springer.com/article/10.1007%2Fs11306-014-0733-z

Dr. Teddy Z said...

Dr. Kell, thank you for proving my point.

Peter Kenny said...

I would agree that measured affinity and crystal structures of protein-ligand complexes for a challenging and potentially valuable do represent a useful advance. JMC publishes articles which are essentially analyses of proprietary datasets sometimes also using proprietary methods (e.g. descriptors).

However, I don't see what the rule of 0.5 article has to do with the discussion since it was not published in JMC (it is highly unlikely that it would have got past the JMC reviewers if it had even been sent out for review). The 0.5 threshold appears to have been chosen for the purposes of defining a catchy drug-likeness metric and what medicinal chemists (and Rev Bayes) would really want to know is what fraction of compounds with >0.5 similarity to at least one metabolite would become drugs if they were taken into development.