SGX does JAK-2

2008 has been an interesting year. The drug-discovery industry has shrunk dramatically in market capitalization, as well as, I suspect, in the number of companies. One of the pioneers of biotech fragment-based drug discovery, SGX Pharmaceuticals, was acquired by Lilly for $64 million in August; hopefully the fragment-based know-how will percolate throughout Lilly. I thought I’d end this year by highlighting a recent communication from SGX published in the first 2009 issue of Bioorganic and Medicinal Chemistry Letters. The paper was accepted on 18 August, two days before the merger with Lilly was completed.



SGX relied heavily on crystallographic discovery of fragments, and their efforts towards inhibitors of JAK-2, a protein tyrosine kinase target for myloproliferative disorders, began by crystallizing the protein and performing fragment-soaking experiments. A bromoaminoindazole fragment with a mid-micromolar IC50 and high ligand efficiency was found to bind to the hinge region of the kinase. Examination of the crystal structure revealed a hydrophobic groove nearby, and replacement of the bromine by a phenyl group boosted the affinity by a factor of 25. Further elaboration of the phenyl group improved the IC50 to 78 nM, more than 500-fold better than the initial fragment. The molecule also exhibited respectable (38-fold) selectivity over JAK-3. Although ligand efficiency fell throughout the optimization process, it remained high; the final molecule remains relatively small and does not appear to violate Lipinski’s Rule of Five. There is no mention of cell activity or other pharmaceutical properties, but the authors do promise future publications.

In closing out this year, we would like to thank everyone for reading, and especially for commenting. Please pass along any fragment news or events and we will get the word out. May you all have a happy and productive 2009!

50% ain’t half-bad

In the world of fragment-based ligand discovery, researchers hope that two fragments, when linked together, will behave at least additively: the free energies of binding for each fragment will sum together, with a multiplicative effect on affinity. In ideal cases, linked fragments will behave synergistically (see for example the post from 18 August, below). But all too often, linking two fragments produces disruptive behavior, and the resulting molecule actually binds less tightly than would be predicted based on the binding energies of the individual fragments. This occurs not just when linking fragments, but in fragment merging and growing as well. Can such phenomena be modeled?

The mathematical groundwork was described more than forty years ago by Spencer Free and James Wilson at the old Smith Kline and French company, and came to be known as a Free-Wilson analysis. In a nice update of this work, Julen Oyarzabal and co-workers have applied this technique to the screening results of eight libraries consisting of several hundred compounds total. The molecules belong to five diverse chemical scaffolds (shown), and were tested against a variety of different targets, including a kinase, GPCRs, ion channels, and P450s.



For each library tested against each target, the authors asked whether the binding contribution due to a substituent Rx was additive, partially additive, or non-additive with the binding contribution of a substituent Ry. The mathematics get pretty intense, and the paper goes far beyond what I can summarize in a blog post, but the main conclusion is surprisingly encouraging: roughly half of all the data sets (10 of 19) show clear additive behavior, while another quarter (5 of 19) show partially additive effects. Only 4 data sets show non-additive behavior.

In many fields, a 50% success rate wouldn’t look too impressive, but in medicinal chemistry (in fact in much of chemistry in general), half-right sounds pretty good. The authors don’t further divide the non-additive data sets into sub-additive versus super-additive categories. In other words, the non-additive effects could well be due to synergy, the quality those of us pursuing FBLD ardently desire. But even if synergy is elusive, the paper suggests that you’ve got a better than even shot of producing a whole that is at least equal to the sum of its parts.

Smacks of SMAC

Maurizio Pellecchia’s lab at the Burnham Institute has been one of the most active academic groups using fragment-based ligand discovery, and their recent paper in J. Med. Chem. describes an NMR-based approach to discover small-molecule mimetics of SMAC. The four N-terminal amino acids of SMAC bind to the protein XIAP, thereby blocking its interaction with caspase-9 and allowing apoptosis to proceed. A small-molecule mimic of SMAC could thus be useful as a chemical probe to better understand the biology of apoptosis, and, ultimately, could be useful as a cancer therapeutic.

The researchers started with the alanine “fragment” of the tetrapeptide Ala-Val-Pro-Ile (AVPI) and generated a virtual library of nearly 1400 alanine-containing derivatives. Molecular modeling narrowed this down to 15 which were then actually synthesized and tested by NMR to assess their binding affinity to a domain of XIAP; BI-75A1 was found to be a weak binder. Molecular modeling suggested that this new fragment (with a molecular weight just under 300) could in turn be “grown” to improve affinity, and after roughly 900 compounds were docked, 28 were then synthesized and tested. Of these, the most potent turned out to be BI-75D2, with a low micromolar dissociation constant in both NMR and isothermal titration calorimetry assays.



BI-75D2 exhibited improved stability in human plasma and S9 fraction compared to the starting peptide AVPI, as well as increased permeability. BI-75D2 also showed modest (16 micromolar) activity in a cell-based apoptosis induction assay, in contrast to the (inactive) AVPI peptide. Further biological experiments support the hypothesis that the small molecule induces apoptosis by binding to XIAP.

From a drug perspective, BI-75D2 still has a long way to go: it is a relatively weak binder, has a molecular weight greater than 500 Daltons, and contains several structural features that make a medicinal chemist squirm and a toxicologist squeal. Moreover, BI-75D2 has a fairly low ligand efficiency (LE), and this actually got worse as the affinity was improved. Nonetheless, as a chemical probe it may have value. It is also a demonstration of how fragment-inspired techniques can be used to attain novel molecules in an academic setting.

Great Discussion

Those of who didn't read this link have missed a fabulous discussion buried in the comments.

Mekie started by asking (for a school paper) how widely FBDD is used.

Dan said widely. However, the current economic situation is seeing early technologies (those farthest from making money, like FBDD) getting axed. Exactly what Sunesis did :-(

Mekie followed up with the obvious question. Is it he cost of the biophysical techniques, such as X-ray/NMR/SPR that is the big problem. Would a cheaper technique be better

I jumped in with both feet and unafraid to piss anyone off by saying, "Nope. It's the chemist's hubris."

Then Dan, being the voice of reason, said it is more pragmatism over hubris. Too often weak hits ended up being complete crap and we are paying for that.

Tony G. joined in and said SPR may be the savior of FBDD (highly paraphrased. Go read his comments, quite cogent).

Then Pete joined the party and FBDD can negate the huge advantage in chemical space that Big Pharma has over small companies. He also agreed with Dan and expanded in that FBDD has not really been shown with membrane targets (which are only 50% of all the targets).
My comment about this is, the natural ligands are already fragments (Count the number of heavy atoms in serotonin.) He also says you need structural data and that is not forthcoming for membrane proteins. Hogwash says I. But we can debate that at a later time.

Finally NMR-soul pointed out that FBDD needs an early committment of resources when the chance of failure is the highest.

These were some excellent comments, well worth going in and reading. I think everyone would agree that FBDD practitioners (and can we come up with a cool name already) are also to blame for overselling what FBDD can deliver (I call this the NMR effect for obvious reasons.)

New FBDD Literature Resource

A blog devoted to tracking literature related to fragment-based drug discovery has recently been launched by Peter Kenny. It sorts papers into various categories (X-ray crystallography, NMR, FBDD theory, etc.) and should be a great resource. Coming soon: DOI links to all of the references!