Is this a Fragment?

Fierce Biotech has a listing of the top 15 (potential) late stage blockbusters in development.  I think these lists Fierce put out are very interesting.  The last one on the list is alpharadin.  Its a potential treatment for bone metastases and Bayer just came up with data that it could also be a broad spectrum treatment for prostate cancer.  We typically don't discuss late stage development; so few of us have actually been there its like Oz.  What makes this so interesting to me is the chemical structure of alpharadin: radium-223 dichloride.  RaCl2.  Three atoms or 297 g/mol. 

I realize this is not a fragment in the true sense of what most of us do.  I found it fascinating nonetheless.

FBDD Down Under

Fragment-Based Drug Design Down Under was held at Monash University in Melbourne, Australia earlier this month. The first dedicated FBDD conference in this country was full of enthusiasm: I had the impression many of the 100 or so participants, most of them Australian, were happily surprised to meet so many other fragment aficionados. With 18 oral presentations, nearly as many posters, and a lively panel discussion I can only touch on some of the broader themes here, so please weigh in with your own observations.

Fragment library design received considerable attention, which was nice as this is an area that is all too often ignored in conferences. Pete Kenny's name came up a couple times in helping to put together the CSIRO fragment library. Craig Morton gave an excellent overview of the SVIMR fragment library and some of the challenges constructing it: of roughly 1600 fragments purchased, 450 were either not sufficiently soluble in water or DMSO or not sufficiently pure to be included. David Chalmers of MIPS presented an analysis of the physicochemical properties of approved drugs, noting that roughly three quarters are ionizable, with potential implications for library design.

Three dimensional fragments have been much discussed lately, and Martin Drysdale of the Beatson Institute described a UK consortium, 3Dfrag.org, to put together a library of 3-dimensional fragments, as defined by having a principal moment of inertia closer to a sphere (think adamantane) as opposed to a plane (benzene) or a rod (2-butyne). The project is still in its early stages, with about 200 fragments acquired thus far. Martin also described an interesting collaboration with the Broad Institute to use existing DOS-derived fragment-sized molecules for screening.

There were several talks on fragment screening methods, especially NMR and SPR. In an intriguing comparison, Jerome Wielens of SVIMR described parallel efforts on HIV integrase, both using essentially the same (Maybridge) library. STD NMR screening produced more than 50 hits, ultimately yielding 15 co-crystal structures, while SPR screening (also discussed later by Tom Peat of CSIRO) produced 16 hits and ultimately 6 crystal structures, yet few of the hits were in common. There were differences in the protein constructs and pH, and some of the NMR hits may have been artifactual, while the use of a reference protein in SPR may have weeded out some true positives. All of which underlines the fact that using multiple biophysical methods is ideal.

STD NMR came under scrutiny from others as well: San Lim of MIPS described compounds that showed a signal when screened in mixtures but not when tested individually, and Martin Drysdale discussed one target that gave a 36% hit rate using the technique, leading him to pick SPR as a primary screening method. Still, there are some interesting possibilities: Thomas Haselhorst of Griffith University discussed using STD NMR not just for screening membrane proteins but for screening viruses, cells, and even fungal spores!

Markku Hämäläinen of GE Healthcare discussed the use of both SPR (specifically Biacore) and ITC. In the case of SPR, he termed one class of problematic compounds “selective promiscuous binders”: for example, a positively charged protein may cause negatively charged fragments to aggregate around it, giving anomalously high signals. Using a positive control and setting a maximum Rmax in fitting the data can help weed these out and provide more accurate dissociation constants. In a collaboration with Merck Serono on a kinase target, 105 hits from a 1920-fragment library gave an 80% confirmation rate when tested in ITC, and 41 of 48 produced co-crystal structures.

But as we are increasingly seeing, Biacore is no longer the only name in the SPR game: Olan Dolezal of CSIRO described Bio-Rad’s ProteOn instrument and found that, while it was less sensitive than Biacore, its higher throughput made it an attractive primary screening instrument.

There were also a couple interesting talks on in-situ methods for fragment assembly, including MS-based methods described by Sally-Ann Poulsen of Griffith and click-based methods discussed by William Tieu at the University of Adelaide. One of the problems with assembling a high-affinity molecule in situ is product release: a molecule made in situ might bind so tightly it never leaves the protein, which essentially stops production once a stoichiometric amount of the inhibitor is made. In Tieu’s case, the problem was cleverly overcome by introducing a mutation to lower the affinity.

Finally, Jonathan Baell of MIPS gave an excellent (though disturbing) talk on PAINS – a topic which is still unfortunately not sufficiently appreciated. In one illuminating example, he found that an in-house screen of a histone acetyltransferase produced only a single legitimate hit, along with a plethora of PAINS.

One common theme both in the presentations and offline discussions was the relative lack of chemistry support; definitely a pity, since there are certainly plenty of chemists looking for new opportunities. Of course, funding chemistry is a problem not unique to the Southern Hemisphere.

Australia is clearly a new world for fragments, and it will be fun to see how the field develops there. And on a personal note, I found Aussies to be some of the warmest, most genuine people I have met in any country. I definitely look forward to finding an excuse to return.

The End of the Trilogy

If you haven't had enough computational fragment papers, here is one more.  In this paper, Zhao et al. set out to find potent inhibitors of EphB4, a receptor tyrosine kinase.  This is not novel target space; the inhibitor dasatinib is already on the market.  This paper is an extension of the groups previous computational method, ALTA (Anchor-based Library Tailoring).  In ALTA, 1. small, mainly rigid fragments are docked. 2. Compounds with the most favorable binding energy are used to select  compounds which contain that fragment.  3.  Fragment is then flexibly docked.  In this paper, they add explicit solvent molecular dynamics to this process. 

The figure below shows the approach to selecting fragments for screening.  Most of the 563,000 fragments are 150-300 Da, have fewer than 5 rotatable bonds, and no formal charge.  They then selected a kinase focused collection by retaining only those with molecular weight smaller than 300 Da, a maximum of three rotatable bonds, more than one ring [Emphasis mine], and the capability to form two hydrogen bonds with the backbone polar groups of the so-called hinge region. For the latter criterion acidic CH groups (e.g., in aromatic rings) were also considered as donors.  The one ring criterion is because single ring anchors don't give enough energy of binding AND to open up IP space.

I find it strange that they consider an aromatic hydrogen capable of H-bonding for the purpose of calculating free energies.  I would like to know what impact this additional factor had in picking the compounds; it is not explained in the paper, nor is it explained in the SI.  

This led to the three active compounds shown in the table below.  Previous work by them showed that the hydroxy at position 5 of compound 1 (Compound 7) would generate a significant increase in binding energy, through two additional hydrogen bonds.  [And excuse my pedantry here, but there is no position 5, is there?  Aren't position 3 and 5 here the same and indistinguishable?  I am not the worlds best chemist, but I do know an equivalent position when I see one.]  

 Modeling confirmed this (prior to the initiation of chemistry).  This was confirmed by compound 7 being 50x more potent than the parent compound 1.  It is nice to see that this potency correlates with 2.5 kcal/mol or the addition of 2 additional hydrogen bonds, as was predicted.  They then co-crystallized the compound with the target EphA3, despite EphB4 being the actual target.  32 of 36 residues in the actives site are the same, including the 100% identity for those involved in binding 7. The 1.7A structure confirmed the predicted mode of binding.

 

It was then tested against related Y-kinases: 0.338 μM for Src, 0.864 μM for Abl1, 1.38 μM for Lck, 1.62μM for EGFR, while no inhibition was observed for IGF1R.Thus compound 7 has higher affinity for EphB4 than for these five tyrosine kinases. It also showed cellular activity.  

To sum up the state of the art of computational FBDD:

You can find fragments that fit in an active site, even if you have to model the active site.

Honesty is the Best Policy

Continuing our theme of computational FBDD, in this paper Brožič et al. present their results of virtual screening using fragments. Aldo-ketoreductases control the activity of androgens, estrogens, and progesterone modulating the occupancy and transactivation of their receptors.  Selectivity is key because the different AKR1C isoforms (1-4) have different physiological outcomes.  Specifically, you want to  inhibit 1 and 3, while having no activity at 2 and 4.  Crystal structures exist for all four isoforms and there is a good history of drug discovery against these targets.  Salicylates are selective for isoform 1, while phenylanthranilates are potent but unselective for all isoforms. 3-bromo-5-phenylsalicylic acid is 4.1 nM inhibitor against 1 and 20x selective against 2 and 100x for 3 and 4.  Phenylaminobenzoates are potent and selective for 3.  

A virtual screen followed by biochemical evaluation.  Compounds from Asinex, Chembridge, Maybridge, and NCI were pulled from ZINC, yielding 1.9M cpds.  After applying filters (fragment like properties, reactive groups, problematic groups, and predicted and known aggregators) they were left with 143000 cpds.  Active site was defined as portion of the enzyme within 6 A of crystallized ligands.  Docking (using FlexX) of these compounds in the active was performed with the mandated interaction that an H- bond acceptor no more than 3A from Y55 was present.  37 cpds for AKR1C1 and 33 for AKR1C3 were found. Of these 70 compounds, 11 were insoluble.  Single point inhibition at 400 uM was performed on both enzymes, 1 and 3.  Compounds with >55% Inh had the IC50 determined, as well as selectivity vs. 2.  25 actives were found against isoform 1 or 3, 11 of which are salicylates or aminobenzoates (known scaffolds) .  Compounds 1-15 represent new chemical space (based upon similarity calculations) with known scaffolds against this slate of targets. 

 Compounds 16-28 represent structures from new chemical classes. 16-21 show no selectivity, 22 and 23 (ketone and aldehyde) show no selectivity and selectivity for Isoform 3 respectively.  Compound 23 brings up the question, if you filtered to eliminate reactive groups how did an aldehyde make it through?

Compound 25 is selective for isoform 1, while 25-28 are selective for isoform 3.  Compound 26 was the most interesting, as it was potent and selective for isoform 3 (based upon a fluorescence assay). 

However, this selectivity could not be explained by docking (isoform 1 and 3 are 88% identical), which predicted the inhibition of isoform 1, but completely missed isoform 3 (docking rank 21509).  The authors then did MD studies (10ns only) to see what could possibly happen.  What follows is honest to goodness handwaving.  What they conclude is that binding of 26 could induce conformational changes in both inhibitor and enzyme to make it bind really well.  Isoform 3 has a larger SP1subpocket. 

In a rare case of brutal (if unintended honesty), the authors state: 

"It is obvious from these results that our virtual screening protocol is capable of finding potent AKR1C inhibitors but is unable to predict the isoform selectivity."

Atoms are like apples...

Many, many moons ago Mike Hann at GSK published a paper that is either the most cited paper in FBDD, or should be (Hann et al.  J. Chem. Inf. Comput. Sci. (2001) 41: 856).  In it, he presented a simple, but quite useful, model of binding.  

The whole point of this model is it illustrates how less complex ligands (fragments)have a better probability of binding productively, but more importantly have less chance of having a detrimental binding event (the dreaded +/+ or -/- in this model).So, in this case, atoms are like apples, one bad one ruins the whole bunch.  

As has been pointed out previously on this blog, the magic methyl is fleeting and may not even exist.  But it has unitary power that makes everyone seek it, like the One Ring.  So, in my neverending quest to generate new terminology (e.g. Fragonomics) and be entertaining, if not informational, I would like to coin the term "Sauron atom" for that one bad atom that can cause a fragment not to bind.