Review of 2012 reviews

2012 has been a bumper year for fragment conferences and reviews. Starting with the Molecular Medicine Tri-Con in San Francisco, moving south to the CHI FBDD meeting in San Diego, east to the ACS Fall Meeting in Philadelphia, back to FBLD 2012 in San Francisco, and ending with FBDD Down Under in Melbourne, there have been plenty of opportunities to learn about the latest work in the field. Two new books were also published, one focused particularly on crystallography and the other focused heavily on computational methods.

Practical Fragments has highlighted one review paper, and I thought I’d mention a few others that came out over the past year.

Chris Abell and colleagues at the University of Cambridge published “Fragment-based approaches in drug discovery and chemical biology” in Biochemistry. This is an excellent and wide-ranging general review, covering theory, library design, screening methods, fragment advancement, applications, limitations, and future trends. If you’re new to the field or want a good refresher, this is the place to go.

Tom Blundell and coworkers, also at the University of Cambridge, published “Biophysical and computational fragment-based approaches to targeting protein-protein interactions: applications in structure-guided drug discovery” in Quarterly Review of Biophysics. As the title suggests, the focus is on protein-protein interactions, but there is plenty of general interest, including lots of unpublished data and practical suggestions.

Aaron Oakley and colleagues at the University of Wollongong, Australia, published “Fragment-based screening by protein crystallography: successes and pitfalls” in Int. J. Mol. Sci. This covers the entire process of crystallographic screening, from library assembly through model building, with a nice table of recent examples and several in-depth case studies. It also touches on potential pitfalls and complementary fragment-finding methods.

Finally, Chungquan Sheng and Wannian Zhang of the Second Military Medical University in Shanghai published “Fragment informatics and computational fragment-based drug design: an overview and update” in Medicinal Research Reviews. With 267 references, this is a great compilation of computational methods that touch on numerous aspects of fragment-based lead discovery.

And with that, Practical Fragments thanks all our readers and says goodbye to 2012. Please keep your comments coming, and may 2013 be a splendid year!

GDB-17: 166 billion fragments and counting

How many possible fragments are there? Jean-Louis Reymond and colleagues at the University of Berne have been trying to answer this question computationally by enumerating all stable molecules from first principles. In their previous effort they found nearly a billion molecules with up to 13 atoms. In a new paper published in J. Chem. Inf. Model. they have now extended this analysis to molecules containing up to 17 carbon, oxygen, nitrogen, sulfur, and halogen atoms. There are 166,443,860,262 of them.

What do they look like? Before addressing that question, it is worth noting that this set of molecules—dubbed the GDB-17—is not exhaustive. The researchers intentionally excluded many potentially unstable moieties. Most of these are probably best ignored, though doing so does leave out functionalities found in some drugs, such as hemiaminal ethers (acyclovir), sulfoxides (omeprazole), and some non-aromatic double bonds (cyclosporine). In fact, more than 40% of similarly-sized molecules in PubChem (ie, they’ve actually been synthesized) are not represented in GDB-17.

But even looking at the PubChem molecules that do show up in GDB-17, there are dramatic differences between existing molecules and enumerated possibilities. For example, a huge fraction of the GDB-17 set contains 3- or 4-membered rings. Aromatic rings are surprisingly rare, at only 0.8%, compared with roughly a third of similar-sized molecules in PubChem. On the other hand, 57% of the GDB-17 molecules contain nonaromatic heterocycles, compared with just 12% in PubChem; these may be particularly attractive in terms of drug-like properties.

Consistent with their reduced aromaticity, the GDB-17 molecules also contain many more stereocenters: on average more than 6 per molecule compared with just 2 for PubChem. With all these stereocenters, it’s inevitable that the molecules are more three-dimensional than those in PubChem. By the same token, though, synthetic accessibility is likely to be a challenge.

In general the GDB-17 molecules are also considerably more polar than known molecules of similar size, with more than half of them having ClogP ≤ 0. This could in part be due to the fact that it’s often tough to purify molecules that are too soluble in water.

There’s a lot of other fun stuff here: for example, almost half a billion isomers of procaine! And for the synthetic chemists in the audience, this work illuminates vast fields of uncharted chemical territory, just waiting to be explored.

Drugs Against Bugs

This paper, from a consortium  of academics in Holland, Belgium, and Switzerland and a UK pharmaceutical company, reports on inhibitors for parasite specific phosphodiesterases (PDE). Trypanosomal disease is a major health obstacle in Africa; if the diseases enters into stage 2 (CNS penetration by the parasite) the patient dies. Trypanosoma brucei has two subspecies: T.b. gambiense and T.b. rhodesiense which makes the search for a pan-anti-trypanosomal agent even harder.  The current treatment options: 

...are limited and suffer from suboptimal dosage regimens and/or severe toxicity. During the second stage of the disease, the only choices are the arsenic-containing drug melarsoprol and the ornithine decarboxylase inhibitor eflornithine, used as monotherapy or in combination with nifurtimox (NECT).  However, eflornithine is not effective against T. b. rhodesiense.

Mapping of the T. brucei genome lead to the identification of trypanosomal PDEs.  Two of these PDEB1 and B2 play a pivotal role in proliferation.  Knocking down one or both TbrPDEB1 andTbrPDEB2 simultaneously leads to an arrest of parasite cell division, lysis of the parasites, and elimination of the infection in vivo (in an infected mouse model). Of supreme interest, the "P-pocket" (found in the crystal structure of  Leishmania major crystal structure) was conserved in their homology model. 

The HTS found compound 1 and in their follow up to chemotypes related to this, the came up with compound 6b.  Rolipram itself was inactive (>100uM) despite fitting nicely in the pocket (according to modeling).  However, the catechol moiety was shown to be the key for activity.  Replacing it with a less "worrisome" moiety led to a significant decrease in potency.  Compound 8d (R1, Obenzyl, R2=cycloheptyl) had 500 nM potency that maintained the same ligand efficiency, which indicates the atoms added were reliably efficient, but not super-efficient.  It also had anti-proliferative effects without cytotoxicity; the authors guess that this is due differences in cell-permeability compared to the cyclopentyl compounds. 

The benzylcatechol chemotype was then chosen for further optimization.  The docking shows that the benzyl moiety is at the entrance of the P-pocket (figure 3a) which should afford access to it through growing.  Table 2 shows the results of their efforts here with both the cycloheptyl and isopropyl (which were more ligand efficient).  The docking suggested that they should be able to grow into the p-pocket from the 4 position (Figure 3a).  Compound 20b (49nM) was the best inhibitor and as shown in the docking (Figure 3b) this is most likely due to interactions in the P-pocket.  Interestingly, they also propose that it may be displacing water from this pocket causing some sort of increase in affinity.  

The cLogP of the cycloheptyl compounds was a real issue affecting solubility and may have affected the antitrypanosomal activity.  20b, with a solubilizing tetrazole group, concentration-dependently worked in the anti-trypanosomal assay with a IC50 of 520nM.  It was also found to be very potent against PDE4A-D enzymes.  It should also be noted that 20b is 40 heavy atoms, which is 0.18LEAN. However, 20b was a good inhibitor of  T. b. rhodesiense (60nM).  It was tolerated by a human fibroblast cell line (250 fold sensitivity index)  Lastly, they were able to show that cAMP accumulated in the cells, indicating the target specific effects.

Often, I am very harsh on academic drug discovery, but this paper is an excellent example of what can be accomplished when it is done right.

 

Entropy-enthalpy transduction: time to throw up our hands?

Thermodynamics has come up several times on Practical Fragments. The binding of a ligand to a protein can be dissected into enthalpic and entropic components. Very simplistically, enthalpy underpins directed, often polar interactions, while entropy plays the dominant role in non-directed, hydrophobic interactions. Ligands that bind primarily through enthalpic interactions (such as hydrogen bonds) have been suggested to be more selective and “best in class”. Historically, a key theoretical advantage of FBLD is the notion that linking two fragments can provide an entropic advantage to the combined molecule compared with the isolated fragments. However, as discussed recently, reality sometimes cocks a snook at theory.

One stumbling block in trying to apply thermodynamics rationally is enthalpy-entropy compensation, a perverse trick of the universe in which, when you improve the enthalpy of an interaction, you may worsen entropy, and vice versa. For example, if you introduce a hydrogen bond into a protein-ligand interaction, the precise positioning required may cause increased rigidity, at an entropic cost.

Now a new paper in Proc. Nat. Acad. Sci. USA from Michael Gilson and colleagues at UCSD suggests that things may be even more complicated. They analyze a previously published 1 millisecond molecular dynamics simulation of the small (58 residue) protein BPTI. There are three main conformational states (or clusters), each with similar overall energies. However, the researchers find that the different conformational states have very different global enthalpies and entropies. Worse, very tiny perturbations, such as the distance between two side chain atoms, can cause one state to shift to another, in turn dramatically changing the overall thermodynamic signature.

In practice, this means that when you measure the thermodynamics of a ligand binding to a protein, the enthalpic and entropic changes observed could have more to do with subtle changes in the global conformation of the protein, or even changes in solvent binding, than to the ligand-protein interaction itself.

The researchers call this phenomenon entropy-enthalpy transduction (EET):

The thermodynamic character of a local perturbation, such as enthalpic binding of a small molecule, is camouflaged by the thermodynamics of a global conformational change induced by the perturbation, such as a switch into a high-entropy conformational state.

The researchers argue that EET could occur in many protein systems, so experimentally determined values of entropy and enthalpy for ligand binding are actually unreliable indicators of the local thermodynamic driving forces we normally try to influence.

Although the researchers develop a sophisticated mathematical framework to describe EET, at the end of the article I’m left wondering, is there any hope of using thermodynamics for practical drug discovery?

Fragments vs PI3K – AstraZeneca’s turn

Last year we highlighted a paper from AstraZeneca in which they used virtual screening to identify fragments that inhibit the p110β isoform of phosphoinositide 3-kinase (PI3K), a potential anti-cancer and antithrombotic target. In two recent papers in Bioorg. Med. Chem. Lett., Fabrizio Giordanetto and colleagues describe the optimization of one of these fragments to a potent, selective molecule with in vivo efficacy.

The first paper describes the initial fragment-to-lead work. A variety of changes to fragment 1 were explored, including adding a lipophilic phenyl group to increase potency (compound 2). At the same time, modifications were explored in the central pyrimidinone ring. Although compound 3 was less active than compound 2, it also had a considerably lower logD. Many additional changes were explored, and ultimately one of the most potent compounds was compound 16. The isomeric compound 22 was less potent, but had significantly better solublility and stability in a microsomal assay.

The second paper describes subsequent optimization, ultimately yielding compound (S)-21. There’s a lot of good medicinal chemistry that I can’t do justice to here, so definitely check out the two papers themselves. Compound (S)-21 is potent, selective against the related kinase p110α, and shows good activity in a dog model of platelet aggregation without causing an increase in bleeding time.

One of the nice things about this work is the fact that the researchers used a fragment-hopping approach and were not focused on potency to the exclusion of all other properties. Although one could argue that this is simply good medicinal chemistry, it can sometimes fall into the category of what Mike Hann has memorably christened “unknown knowns,” a trap this team avoided.

MK-8931, BACE1 inhibitor, enters Phase 2/3 for Alzehimer’s

Perhaps no other single target so successfully demonstrates the potential of fragment-based approaches as BACE1, a challenging aspartyl protease implicated in Alzheimer’s Disease. Practical Fragments has previously written about efforts from Merck, Lilly, Pfizer, Evotec, and Amgen (there’s also the Astex-AstraZeneca collaboration).

Today Merck announced that their MK-8931 has entered a Phase 2/3 clinical trial, a randomized placebo-controlled study which will run for 78 weeks and enroll up to 1700 patients in the phase 3 portion.

There have been concerns lately as to whether or not BACE1 is a viable target for Alzheimers; it will take large studies like this to answer that question. Practical Fragments wishes them luck.

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.