Review of 2013 reviews

The year is coming to an end, and as we did last year, Practical Fragments is looking back at notable events as well as reviews that we haven’t previously highlighted.

The fragment calendar started in March in Oxfordshire, at the RSC Fragments 2013 conference, closely followed in April by CHI’s FBDD meeting in San Diego (here and here). Closing out the year for conferences that Teddy or I attended was the Novalix conference on Biophysics in Drug Discovery in Strasbourg (here, here, and here).

There weren’t any new books published (though the special issue of Aus. J. Chem. practically counts as one), but there were several notable reviews.

Stephen Fesik and colleagues at Vanderbilt University published “Fragment-based drug discovery using NMR spectroscopy” in J. Biomol. NMR. This is an excellent overview that covers library design, NMR screening methodologies, and compound optimization. The researchers make an interesting case for including multiple similar compounds and allowing for larger, more lipophilic fragments, while always being careful to avoid “bad actors”. They also do a good job of summarizing the various NMR techniques, including their strengths and limitations, in language accessible to a non-spectroscopist. Finally, the section on fragment linking discusses the theoretical gains in affinity, the practical challenges to achieving these, and strategies to overcome them.

Turning to the other high-resolution structural technique, Rocco Caliandro and colleagues at the CNR-Istituto di Cristallografia in Italy published “Protein crystallography and fragment-based drug design” in Future Med. Chem. This provides a fairly technical description of X-ray crystallography and its role in FBDD, along with a table summarizing around 30 examples, five of which are discussed in some detail.

Of course, it’s always best to use multiple techniques for finding fragments, so it’s well worth perusing “A three-stage biophysical screening cascade for fragment-based drug discovery,” published in Nature Protocols by Chris Abell and colleagues at the University of Cambridge. This expands on a gauntlet of biophysical assays (involving differential scanning fluorimetry (DSF), NMR, crystallography, and isothermal titration calorimetry (ITC)) that we discussed earlier this year. Nature Protocols are highly detailed, with lots of troubleshooting tips, so this is a great resource if you’re exploring any of these techniques.

Finally, Christopher Wilson and Michelle Arkin at the University of California San Francisco published “Probing structural adaptivity at PPI interfaces with small molecules” in Drug Discovery Today: Technologies. Protein-protein interactions are frequent targets for FBLD: see for example here, here, here, here, and here – and that’s just for 2013! The current review gives a nice overview of the technology called Tethering, focusing on the cytokine IL2 and an allosteric site on the kinase PDK1.

And with that, Practical Fragments thanks you for reading and says goodbye to 2013. May your 2014 be happy and fulfilling!

Fragments in Australia

Last year we highlighted the first FBDD conference held in Australia. That meeting has now led to a dozen papers in the December issue of Aus. J. Chem. Many of the papers use the same fragment libraries, so this is a good opportunity to survey a variety of outcomes from different techniques and targets.

The collection of papers (essentially a symposium in print) starts with a clear, concise overview of fragment-based lead discovery by Ray Norton of Monash University. Ray also outlines the rest of the articles in the issue.

A well-designed fragment library is key to getting good hits, and the next two papers address this issue. Jamie Simpson, Martin Scanlon, and colleagues at Monash University discuss the design and construction of a library built for NMR screening. Compounds were selected using slightly relaxed rule-of-three criteria, and special care was taken to ensure that at least 10 analogs of each were commercially available to facilitate follow-up studies. Remarkably, of 1592 compounds purchased, only 1192 passed quality control and were soluble at 1 mM in phosphate buffer. The properties of the final library are compared with nearly two dozen other libraries reported in the literature; this is the most extensive summary I’ve seen on published fragment libraries. The paper also analyzes the results of 14 screens on various targets using saturation transfer difference (STD) NMR. As the researchers note, this technique is prone to false positives, and indeed the average hit rate of 22.5% is high, with only about 50% confirming in secondary assays. There is also a nice analysis of what features are common to hits, along with a list of the 24 compounds that hit in more than 90% of screens.

The other paper on library design, by Tom Peat, Jack Ryan and others (including Pete Kenny), discusses library design at CSIRO. The researchers started with 500 fragments commercially available from Maybridge and supplemented these with roughly the same number of fragments from a collection of small heterocycles that had been synthesized internally; additional “three-dimensional” fragments are also being constructed. At CSIRO the primary screening method appears to be surface plasmon resonance (SPR), in particular the ProteOn instrument that allows simultaneous analysis of six fragments against six targets. Eight of about ten targets have yielded confirmed hits. The researchers show examples of specific (good), nonspecific (probably bad) and ill-behaved (ugly) fragments.

Next up is an excellent discussion of PAINS by Jonathan Baell (at Monash) and collaborators. Although Practical Fragments has covered this topic repeatedly (here, here, here, here, here, and here) it is a sad fact that more examples appear in the literature every day, so there is always something new to write about.

Fragment-finding methods make up the next several papers, starting with a nice overview of native mass spectrometry by Sally-Ann Poulsen at Griffith University. This paper covers theory, practical issues, and recent examples. Roisin McMahon and Jennifer Martin at University of Queensland, along with Martin Scanlon, describe thermal shift assays. In addition to highlighting a number of published examples, the paper also delves into some of the technical challenges and issues with false positives and false negatives, concluding with a nuanced discussion of how to deal with conflicting data.

The subject of conflicting data is central to the work of Olan Dolezal and Tom Peat, both of CSIRO, and their collaborators. They screened the protein trypsin against 500 Maybridge fragments using SPR. Unfortunately they couldn’t go higher than 100 micromolar without running into problems of solubility and aggregation, but even at this relatively low concentration they found 18 hits. X-ray crystallography validated 9 of them, and isothermal titration calorimetry (ITC) also validated 9, with 7 confirmed by all three techniques. (Incidentally, there are lots of great experimental details here.) Four of the SPR hits could not be confirmed by either ITC or X-ray, and 3 turned out to be false positives when repurchased and tested; in one case this appeared to be due to cross-contamination with a more potent compound. In general, the more potent compounds tended to be the ones that reproduced best, and solubility seemed to be a limiting factor for ITC. Despite the imperfect agreement of biophysical techniques, these were still superior to computational approaches on the same target with the same library. As they conclude:

It is gratifying to know (at least for these authors) that experimental data are still of enormous value in the area of fragment-based ligand design and that the modelling community still has a way to go before the experimentalists are put out to pasture.

But experimentalists should not get too cocky: the next paper, by Jamie Simpson and collaborators at Monash University, describes some of the things that can go wrong. An STD NMR screen of the antimicrobial target ketopantoate reductase (KPR) using the same Maybridge library of 500 compounds revealed 196 hits! The 47 with the strongest STD signals were then tested in a ¹H/¹⁵N-HSQC NMR assay, leading to 14 hits, of which 4 gave measurable IC₅₀ values in an enzymatic assay. Unfortunately, follow-up SAR was disappointing, and subsequent experiments revealed that aggregation was to blame: when the biochemical experiments were rerun in the presence of 0.01% Tween-20, only a single fragment gave a measurable IC₅₀ value. The researchers redid their STD-NMR screen in the presence of detergent, resulting in 71 hits, all of which were tested in the biochemical screen. This led to the identification of a new (and fairly potent) hit that had previously been missed. This nicely illustrates the fact that false positives are not just a problem in terms of wasted resources, they can also overwhelm the signal from true positives. The moral? Always use detergent in your assay!

The question of whether structure is needed to prosecute fragments has come up before, and the next paper, by Stephen Headey, Steve Bottomley, and collaborators at Monash University, addresses this question directly. The target protein, a mutant form of α1-antitrypsin called Z-AAT, unfolds and polymerizes in vivo, causing a genetic disease. The researchers used an STD NMR fragment screen of 1137 fragments to identify several hundred hits, and focused on those that bound to the mutant form of the protein rather than the wild-type. They then used a technique called Carr-Purcell-Meiboom-Gill (CPMG) NMR (which relies on line broadening when fragments bind to a protein) to confirm 80 hits, the best of which had a dissociation constant of 330 micromolar. If you’ve stuck through this post thus far you’ll recall that the Monash library was designed for “SAR by catalog”, and 100 analogs of this fragment were purchased and tested, leading to several new hits, one with a dissociation constant of 49 micromolar. Although there is still a long way to go, metastable proteins are tough targets, so this is a nice start.

The next paper, by Ray Norton at Monash University and collaborators, describes a fragment screening cascade against the antimalarial target apical membrane antigen 1 (AMA1). An initial STD NMR assay of 1140 fragments produced 208 hits, but competition experiments with a peptide ligand whittled this number down to 57 that confirmed in both STD and CPMG NMR assays. Of these, 46 confirmed in an SPR assay, and although most are fairly weak, some SAR is starting to emerge as new analogs are synthesized.

Another antimicrobial target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), is the subject of a paper by James Swarbrick at Monash and collaborators. An initial STD NMR screen gave an unnervingly high hit rate (notice any themes emerging?), so 2D ¹⁵N-HMQC experiments were performed on 750 Maybridge fragments, yielding 16 hits. Competition experiments using CPMG NMR and close analyses of the chemical shifts suggested that these fragments bind in the substrate binding site, and SPR confirmed binding for some of the fragments.

Finally, Martin Drysdale of the Beatson Institute highlights some of the success stories of FBDD, including clinical compounds, and ends with a call for shapelier fragments.

All in all this is a great collection of papers, particularly for those relatively new to the field. It will be fun to revisit some of these projects in a few years to see how they’ve progressed.

Undruggable? Pshaw, Fragments can do it.

Bromodomains, as well as other epigenetic targets, are hot right now.  This paper adds to the growing library of fragment success against bromodomains.  As in any nascent field, some of the targets do not have a known biological role.  BAZ2B (bromodomain adjacent to zinc finger domain protein 2B) is one of these.  BAZ2B is interesting compared to the 41 other bromodomains where there is structural information.  Its KAc binding pocket is smaller than other bromodomains (92-105 Angstrom vs 130-220 Angstrom for the other bromodomains and lacks features of BET bromodomains, like the ZA channel and the hydrophobic groove adjacent to the WPF motif.  

BAZ2B (Left), BRD2-BD1 (Right)

Due to these structural differences, the strategies that have been applied successfully to other bromodomains will not transfer to BAZ2B; thus, it considered one of the least druggable bromdomains.  Hence, fragments to the rescue!  

They screened 1300 commercially available (and thus Voldemort Rule compliant) with an alpha screen with hits being defined as 50% activity at 1mM.  10 compounds were identified and confirmed using STD, Waterlogsy, and CPMG NMR experiments (0.8% hit rate).  

(The same library was screened against BRD2-BD1 and CREBBP and had hits rates of 1.8% and 6.1% respectively.)  The ten fragments were then soaked into BAZ2B crystals yielding structures for 1,3,6 and KAc. 

a) KAc, b) 1, c) 3, d) 6

Fragment 6 had poor solubility, so direct ITC was not possible, instead a competition ITC study was used and yielded a 65 uM Kd.  They attempted to optimize this fragment from the 1 position of THgammaC.  All of these modifications resulted in worse affinity.  They then attempted to replace the Chlorine with aryl substituents (based on modeling results).  These compounds showed some improvement in solubility, but no significant improvements in affinity.  They then tried to change the electronic properties of the aromatic substituent.  EWG showed the expected reduction in affinity, but EDG did not show an increase in affinity.  

The most ligand efficient fragments (7, 8, and 10) all contained thioamides, so they synthesized thioamide and thiourea analogs of fragment 6.  Both of these molecules did not bind to the protein. Finally, they attempted to merge 3 and 6 putting the KAc mimetic on the scaffold of 6 . 

This urea containing compound (40) showed improved solubility, and 8-fold reduction in Kd, and a corresponding increase in ligand efficiency. 
This paper is a nice story of using fragments, structural biology, and modeling to generate useful compounds as tools.  I think it also points to "druggability" being a useless term when it comes to fragments.  Archimedes may have wanted a lever long enough, but for me, I just want fragments diverse enough.

Fragment linking on RPA: another protein-protein interaction inhibitor

Protein-protein interactions have often been targeted by fragment efforts, partly I think out of desperation when all else fails. That said, there have been notable successes. Earlier this year we highlighted one example from Stephen Fesik’s group at Vanderbilt University. In a recent paper in J. Med. Chem., the same lab now reports progress on a different target.

Replication protein A (RPA) is important for DNA replication and repair, and is thus an intriguing anti-cancer target. RPA binds to single-stranded DNA as well as to various other proteins involved in the DNA-damage response, such as ATRIP. The site targeted here is the “basic cleft” of RPA that binds ATRIP.

The researchers used the venerable SAR by NMR approach, screening a library of 14,976 fragments against ¹⁵N-labeled protein using HMQC and looking for changes in chemical shifts. A total of 149 fragments produced significant and specific chemical shift differences at 0.8 mM concentration. One of the nice features of SAR by NMR is that not only do you get hits, you find out where they bind. In this case, most of the hits bind in the basic cleft. This region has two sub-sites; some fragments bind to one or the other, while many bind to both. Not surprisingly for a basic binding site, most of the fragments identified are negatively charged.

Although all the hits are relatively weak (the best have dissociation constants around 0.5 mM), some could be improved through various strategies to low micromolar inhibitors, the subject of a paper earlier this year.

In the current paper, the researchers used crystallography to further define the binding modes of select fragments. They found that fragment 2 and fragment 4 could bind to both subsites of the basic cleft, but that when co-crystallized together fragment 2 binds to one subsite while fragment 4 binds to the other. The two fragments come within a few Ångstroms of one another, suggesting that they could be linked.

Fragment linking doesn't always work as well as one might hope, and although the initial linked compound 7 is nearly 30-fold more potent than fragment 4, its ligand efficiency drops considerably. However, structure-based optimization to compound 8 was able to improve the affinity by another two orders of magnitude.

Teddy has argued that SAR by NMR is dangerous because of its reliance on labeled protein and because the initial application involved fragment linking, leading people to believe that these are necessary requirements for successful prosecution of fragments. There are now plenty of examples of using other methods to find and advance fragments, but this paper illustrates that SAR by NMR can still be incredibly powerful.

Of course, the final molecule reported here has warts, notably a thioamide, two carboxylic acids, a molecular weight over 600, and a ClogP>7. Indeed, the absence of reported cellular data is perhaps telling. And yet, Bcl inhibitors are also superficially unattractive but are in the clinic. Clearly more medicinal chemistry needs to be done on these molecules, if nothing else to improve potency, but that’s not to say there isn’t a path forward. It will be fun to watch this story progress.

Upon Request

Dan and I blog here because we love it; we don't get paid, it takes a lot of time, and has very little reward.  I love it when I meet someone new and they say, "Oh, I read your blog."  However, this allows us to have freedom to review what we want, when we want, and how we want.  We don't sell advertising, we don't generate revenue, and so on.  Sometimes people agree with us, sometimes they don't.  These posts are our opinions and like bellybuttons, everyone has one.  Sometimes, we get pinged by somebody who just published a paper and would really like to see us blog about it.  Sometimes we do, sometimes we already have and they missed it, and sometimes we don't.

I received a polite email recently, pointing out this paper.  It was already on my radar to blog about, so I bumped it up in the queue.  This paper caught my attention because it is a fragment screen against a DNA-target, specifically the G-quadruplex from c-MYC.  G-quadruplexes are found in the promoters to many oncogenes and the supposition is that by stabilizing them you can reduce their transcription.  It is an intriguing idea which has already been investigated with a number of compounds to date.  These authors decided to use fragments against the G-quadruplex without knowing if fragments would bind to a nucleic acid target with sufficient affinity and selectivity.  Their primary screen was an Intercalator Displacement Assay (IDA) which has been used previously to find G-quadruplex binding ligands.  A 1377 fragment library (@5mM) (previously used against riboswitches) was used and it obeyed the Voldemort Rule, had >95% purity, and 1mM aqueous solubility. The top 10 hits from this screen could be placed in three groups.

Then, in order to confirm their biochemical assay results they decided to dock them these top 10 fragments.  WHHAAAAT you say?  That was my initial reaction.  Why oh why doth they vex me so?  They then go into EXCRUCIATING detail about the docking results, even concluding from the results some SAR hypotheses.  I kid you not.  They also evaluated these top 10 fragments in a cellular assay (125um and 250uM) using a Western blot readout.  These concentrations were chosen in order to not show short or long-term toxicity, but Mirabile dictu, Data Not Shown.  All fragments, except two (7A3 and 2G5), showed significant changes in c-Myc expression levels. Interestingly, "no significant changes" still gives a 20% reduction in c-Myc levels. 

Four fragments were able to reproduce this effect, of which 11D6 was the best.  The four best were then run pair-wise to and every combination induced a significant reduction of c-Myc.  

So what does this tell us?  Well, I think they have found fragments which bind to the c-MYC promoter G-quadruplex.  It may be exhibiting this binding in the cells.  There are a few experiments that I would like to see (and would have asked for if I had reviewed this paper): a binding assay (SPR, ITC, NMR, whatevs) being he primary one.  We also continue to know that docking really does not add anything to the discovery process. 

Docking vs TINS on a GPCR

Practical Fragments has featured a number of posts comparing various fragment-finding methods. In some cases there is good agreement, while in others – not so much. Computational methods can in theory sample the greatest swath of diversity space: a virtual library can be orders of magnitude larger than any physical library. In a recent paper in J. Chem. Inf. Model. Gregg Siegal at ZoBio and Leiden University and Jens Carlsson at Stockhom University and their colleagues compare the performance of virtual screening with a biophysical method.

The target they chose, the A_2A adenosine receptor (A_2AAR) is a GPCR implicated in a variety of diseases. It also has the advantage of multiple published co-crystal structures with either agonists or antagonists bound, making it a good candidate for computational screening.

The researchers began by conducting a computational screen of 500 fragments using DOCK 3.6 against the crystal structure of an antagonist-bound A_2AAR and ranked these according to how well they scored. Next, the researchers physically screened the same library of 500 fragments against A_2AAR using an NMR-based screening method called TINS (see also here). This resulted in a whopping 94 primary hits, which were followed up in a radioligand displacement assay to yield 5 confirmed hits with K_i values ranging from 14-600 micromolar. Happily, 4 of the 5 hits from the TINS screen were within the top 5% scoring hits identified in silico.

This is satisfying at first glance, but what does it say about the other top-scoring computational hits? Computational screening virtually docks fragments in many possible positions, or poses, which are automatically evaluated. Manual inspection of the top 50 in silico hits showed that, in some cases, the best poses had desolvated polar groups, which would presumably be energetically unfavorable. Indeed, identifying the “correct” pose seems to be a general problem with docking fragments.

But some of the top-scoring fragments looked fine by visual inspection, so 5 of these were tested in a radioligand displacement assay. Surprisingly, 3 of these were active, with K_i values ranging from 18-128 micromolar. In other words, these were false negatives in the primary TINS assay.

Having found hits that had been missed using a biophysical screen, the researchers then docked 328,000 commercially available fragments against the target – an exercise that took only seven hours on a computer cluster. Of the top hits, 22 were purchased and tested in the radioligand displacement assay, and a remarkable 14 of these were active, with K_i values ranging from 2-240 micromolar. (I do wonder how much chemical intuition played a role in choosing hits to purchase.)

Interestingly, all of the 14 hits from docking had respectable ligand efficiencies (LE > 0.3 kcal/mol/atom, with a single exception). This is consistent with previous fragment docking studies that show that the best results are obtained with the most ligand-efficient fragments. It’s also a nice feature; after all, these are exactly the kind of hits you would hope to find, though of course you want to first filter out any garbage from your virtual library.

This paper provides more evidence that computational approaches can find fragment hits for GPCRs, at least relatively “druggable” ones with good structural characterization. It is also a useful reminder of the importance of using multiple methods, to avoid both false positives and false negatives.

Finally, if you haven't already voted on your fragment-finding methods, please do so on the right side of the page!

Fragments vs hematopoietic prostaglandin D2 synthase

Prostaglandins are modified fatty acids involved in myriad biological processes. The enzyme hematopoietic prostaglandin D2 synthase (H-PGDS) converts prostaglandin H2 to prostaglandin D2 and is a potential target for inflammatory disorders. In an article just published online in MedChemComm, Gordon Saxty and co-workers at Astex and collaborators at GlaxoSmithKline describe how they used fragment-based methods to develop orally available inhibitors of this enzyme.

The researchers started by screening their fragment library against crystals of H-PGDS, resulting in 76 fragment hits, some of which were quite potent (sub-micromolar). Two are described in the paper, with most of the focus being on Fragment 6, which wasn’t the most potent but did produce an interesting conformational shift in the protein. Also, although H-PGDS typically binds lipophilic molecules, the researchers were intrigued to observe that the polar pyrazole moiety made two hydrogen bonds to the protein.

Structure-guided optimization of Fragment 6 led to Fragment 8 with a modest improvement in affinity, and fragment growing led to Compound 9, with a satisfying 400-fold boost in affinity. In one of those “nice to have” problems, this compound was actually too hydrophilic (ClogP < 1), but increasing the lipophilicity slightly led to Compound 10 (AT24111 / GSK2696124A), which has low nanomolar potency and oral bioavailability in both mice and rats. The compound also blocked prostaglandin D2 production in mice when dosed orally.

This is a concise but elegant paper, and it is impressive that the researchers managed to maintain or improve ligand efficiency throughout optimization. Of course, all the molecules contain a pyrazole moiety, which is a privileged pharmacophore for kinases, so it will be important to carefully assess selectivity.

Finally, you may recall that Astex was acquired by Otsuka earlier this year. Whenever an acquisition happens there is always the worry that the acquired company will be decimated or shuttered entirely. Happily, this doesn’t seem to be the case here. In fact, Astex actually seems to be expanding: I recently saw a full page job advertisement from them in Nature, and as of this morning their website lists 9 openings, with several in research. Hopefully the honeymoon lasts a long time!