FBLD 2014

FBLD 2014, the fifth in an illustrious series of conferences, took place in Basel, Switzerland last week. Organizers Wolfgang Jahnke (Novartis), Michael Hennig (Roche), and Rod Hubbard (University of York & Vernalis) put together a fantastic event. With 35 talks, 45 posters, and more than 200 delegates, I won’t attempt to give more than a few impressions here. In addition to Teddy’s (and others’) Tweets, Derek Lowe put up several posts (see here, here, and here); please share your thoughts below.

Harren Jhoti delivered a lively and wide-ranging opening keynote summarizing the past 15 years of FBLD as viewed from Astex. Among many other innovations, researchers there are responsible for the Rule of 3, which has been the subject of some debate. Harren emphasized that the “Voldemort Rule” should not be a strait-jacket. Like the Kobayashi Maru, rules are there to be broken, though you need to be something of a James T. Kirk to do so effectively.

Astex has produced what is likely the largest collection of protein-fragment crystal structures, and Harren noted that many proteins appear to have fragment binding sites outside the active site: across 25 different proteins, the average number of total sites is slightly greater than 2. Astex is increasingly targeting these sites for allosteric lead discovery.

The theme of crystallography carried through the conference. As Armin Ruf (Roche) exhorted, “more crystals, more structures.” One challenge is that not all crystal forms are suitable for fragments, and it is not always clear from the outset which forms will work. Armin described their chymase project in which an initial crystal form gave 8 fragment structures, but additional crystal forms allowed them to obtain 6 more. Without the different crystal forms these later fragments would have been crystallographic false negatives, yet the potential of different crystal forms to reveal more hits is under-appreciated: Armin noted that the majority of recent fragment papers reported using only a single crystal form.

The importance of crystallography was emphasized again by Nick Skelton (Genentech), who discussed their NAMPT program (which we covered here). In this project, which utilized dozens of crystal structures, a single atom change to a fragment could completely and unpredictably alter the binding mode.

Obtaining a good crystal is not necessarily easy, though. Andreas Lingel (Novartis) described their efforts to produce a form of B-RAF that would diffract to higher resolution and allow fragment soaks (as opposed to co-crystallization). They tried reducing the “surface entropy” by mutating glutamate and lysine residues to alanine, but only one of a dozen or so mutants expressed well and gave superior crystals. Although this turned out to be useful, the team is still at a loss to explain why the specific mutants are effective.

Continuing the theme of crystallography, Matt Clifton (Beryllium) described what looks to be a significant advance for the protein MCL-1. (This is a collaboration with the Broad Institute, and we previously noted some of their progress here.) The researchers have developed a maltose-binding protein (MBP) fusion of this oncology target that diffracts to 1.9 Å in the absence of any ligand. (MBP fusions are used to help crystallize challenging proteins.) Since they developed this construct in May of this year, the researchers have already solved more crystal structures than had been reported publicly to date, and uncovered some interesting findings. For example, the initial fragment that Steve Fesik’s group found binds in a similar manner to one of his more potent later leads, as does one of the AbbVie fragments; however another AbbVie fragment binds in a somewhat different fashion than the elaborated lead.

The subject of how to effectively sample chemical space was another theme, and to this end Alison Woolford (Astex) proposed the concept of a “minimal pharmacophore”: the minimal interactions necessary to drive fragment binding. Researchers at Astex have systematically cataloged several dozen of these, which include such simple entities as amines, acids, aromatic chlorides, and more abstract concepts such as a 1-bond donor-acceptor (think pyrazole). Alison showed an interesting graph with targets on the y-axis and minimal pharmacophores on the x-axis which revealed some obvious patterns such as the preference of donor-acceptor minimal pharmacophores by kinases, but there were unexpected features as well. In a sense, this is an empirical realization of early docking studies, but it also has interesting implications for library design. For example, Alison suggested avoiding fragments with more than one minimal pharmacophore, as these will not be able to effectively sample as many different sites on a protein: with two pharmacophores, a fragment would be limited to binding sites having matching recognition elements to both rather than just one. This idea ties in with the concept of molecular complexity, but from a more chemocentric point of view.

On the subject of chemistry, Dalia Hammoudeh (St Jude’s Hospital) gave a lovely talk on her experiences developing allosteric inhibitors of DHPS, an antibiotic target. Among other fragment hits from the Maybridge library, one was ostensibly 4-trifluoromethylbenzylamine, but turned out to actually be the Schiff base of this with the corresponding aldehyde. Yet another reminder to always carefully check what you think you have.

Practical Fragments has previously discussed the Genentech MAP4K4 program (here and here), and Terry Crawford gave a nice summary. One of the challenges they faced was that their initial leads had excellent brain penetration, leading to animal toxicity. This forced them to increase size and polar surface area. Although it was problematic in this case, this emphasizes how small and drug-like fragment-derived leads can be. Indeed Vicki Nienaber, who was a prime mover behind the original FBLD 2008 meeting, has devoted much of her efforts at Zenobia to CNS targets.

Finally, Derek Lowe (Vertex) gave a rollicking history of the drug industry, ending with his view of where fragments fit in. He noted that chemists – Valinor not withstanding – play a central role, and in that sense the field is a departure from the general trend of the past decade or so. It still remains to be seen how many of the 30+molecules FBLD has delivered to the clinic will come out the other side, but at least for now the field is thriving. As Chris Lipinski stated last year, “if I had to single out one technology that really took me by surprise and has been very successful, it has been fragment screening.”

PAINS in Nature

Practical Fragments has previously noted that many pan-assay interference compounds (PAINS) can be found in nature. Indeed, they’ve also found their way – unintentionally – into journals published by Nature Publishing Group. In an effort to educate the scientific community about these artifacts, Jonathan Baell (Monash University) and Mike Walters (University of Minnesota) have just published a Comment in Nature entitled “Chemical con artists foil drug discovery”. This is the clearest discussion I’ve yet seen of PAINS, and it deserves to be widely read.

Since the article is open-access I won’t go into depth here, other than to say that the researchers propose three steps to avoid PAINS.

1) Learn disreputable structures.

As a start, the paper provides a rogue’s gallery of some of the worst molecules, along with memorable interpretations by award-winning New Yorker cartoonist Roz Chast. It would be nice to see this posted in every academic screening center.

2) Check the literature.

This is even easier than having to learn structures, and should prevent people from embarrassing themselves by publishing research that is obviously flawed.

3) Assess assays.

Multiple orthogonal assays are useful for all science, not just FBLD!

Together with the recent C&ENstory and ACS symposium, this article ensures that PAINS are finally reaching the level of recognition such that scientists, reviewers, and editors will no longer be able to claim ignorance. Willful negligence may be another matter, but at least people will be able to recognize it as such.

Substrate activity screening revisited: substrates as inhibitors

Last year Practical Fragments highlighted substrate activity screening (SAS) as a means for identifying enzyme inhibitors. The idea is to make libraries consisting of potential substrates, modified to reveal interactions: for example, amides that release a fluorescent reporter group when cleaved by a protease. These libraries are then screened against a protein of interest, and any new substrates identified can be transformed into inhibitors by replacing the cleavable bond with some sort of warhead. At the end of that post, we asked why more people weren’t using SAS. In a new paper in ChemBioChem, Pieter Van der Veken and colleagues at the University of Antwerp have partially answered that question, and provided a solution.

The researchers were interested in the oncology target urokinase (uPA), a trypsin-like protease. They built potential substrates from an amino-methylcoumarin designed to fluoresce when cleaved. They used this to assemble a library of 137 molecules, each consisting of the amino-methylcoumarin coupled to a variable fragment. Of these, about 50 contained positively charged moieties likely to interact with the large S1 pocket of uPA, which has a predilection for cationic species. (The rest were diverse molecules.) The library was then screened against the enzyme to look for substrates, but the researchers ran into several difficulties.

First, since all the substrates are poor, the researchers had to use quite a bit of enzyme (about 2.5 micrograms per well) to get a good signal. Second, for the same reason, they had to run the assay for a long time (6 hours). Third, and somewhat unexpectedly, it turns out that SAS is susceptible to an interesting artifact: low levels of contaminating enzymes can cleave substrates, giving false positives. Indeed, the researchers found that commercial uPA isolated from human urine produced a number of hits that did not repeat with recombinant (and presumably purer) enzyme and could not be competed by addition of a potent uPA inhibitor.

Despite these challenges, the researchers identified 11 hits. However, notably absent were some of the fragments known to have affinity for the S1 pocket, such as several guanidines. This is not surprising: for a molecule to be processed as a substrate it needs to be able to fit in the S1 pocket as well as to position the cleavable bond near the catalytic machinery – subtle changes in geometry will prevent processing. This got the researchers thinking about alternative ways to use their library.

The approach they came up with, “modified substrate activity screening” (MSAS), starts by first looking for inhibitors rather than substrates, since a poor substrate can behave as an inhibitor. The idea is to incubate library members with the enzyme and a single potent substrate. This allowed the researchers to reduce the enzyme concentration by 10-fold and run a much shorter assay (10 minutes). It also reduces the risk that contaminating enzymes will be responsible for activity, though of course inhibition assays are susceptible to all sorts of other artifacts.

When the researchers applied MSAS to uPA, not only did they rediscover the 11 hits they had identified as substrates previously, they also identified 17 additional molecules, including all the guanidine-containing fragments.

The researchers propose a flowchart for MSAS in which compounds are first screened for inhibition. These hits are then followed up using SAS to determine whether some of these inhibitors are substrates too. Any substrates thus identified can be readily transformed into inhibitors by adding an appropriate warhead. Inhibitors identified in the first step that aren’t substrates can also be useful to provide structure-activity relationships and new fragments to take forward.

Of course, one could argue that if you are doing inhibition assays, there is no point in going to all the trouble of making custom libraries for MSAS. That said, if you’ve already got the substrate-based libraries, doing an initial inhibition screen is probably a good idea.

From fragment to lead: just remove (high energy) water

The proverb "well begun is half done" suggests that getting started comprises much of the work. Such is the case for fragments that bind to “hot spots,” sites on a protein that are particularly adept at binding small molecules and other proteins. Though fragment-to-lead efforts can give impressive improvements in potency, much of the binding energy of the final molecule resides in the initial fragment. In a new paper in ChemMedChem, Osamu Ichihara and colleagues at Schrödinger have asked why.

The researchers examined 23 published fragment-to-lead examples for which crystal structures and affinities of the fragment and lead were available and in which the fragment maintained its binding mode. They then used a computational tool called WaterMap to assess the water molecules displaced by both the initial fragment as well as the optimized molecule. They compared the calculated thermodynamic parameters (free energy, enthalpy, and entropy) of the water molecules displaced by the initial fragment (core hydration sites) or the bits added to it in the lead (auxiliary hydration sites).

When a protein is surrounded by water, water molecules bind just about everywhere. However, some of these water molecules may “prefer” to be in bulk solvent rather than, say, confined within a hydrophobic pocket on the protein. Perhaps not surprisingly, most of the water molecules displaced by ligands turned out to be of this “high-energy” or unstable variety. Also, the researchers consistently found that the core hydration sites were more unstable than the auxiliary hydration sites. In other words, fragments appear to displace the most unstable water molecules. Moreover, most of this higher energy was due to unfavorable entropy.

It is important to note that the focus here is on individual water molecules (or hydration sites) assessed computationally. The researchers are careful to stress that these may not correlate with thermodynamic parameters obtained by isothermal titration calorimetry (ITC). This is because ITC measures the entire system – protein, ligand, and all of the water – and factors such as protein flexibility can confound predictions.

The researchers summarize their findings as follows.

1) The presence of hydrogen bond motifs in a well-shaped small hydrophobic cavity is the typical feature of the hot spot surface  

2) Because of these unique surface features, the water molecules at hot spots are entropically destabilized to give high-energy hydration sites 

3) Fragments recognize hot spots by displacing these high-energy hydration sites

This provides a framework for understanding several phenomena. First, it describes the origin of hot spots. Second, it explains why much of the binding energy of an optimized molecule resides in the initial fragment; additional waters displaced are not as unstable as those displaced by the fragment, so they don’t give you as much bang for your atom. As a corollary, this might help explain the leveling off or decline in ligand efficiency often observed as molecules become larger.

The researchers go on to discuss specific examples of high-energy waters, noting that a water molecule involved in one or more hydrogen bonds may be particularly hard to replace because recapitulating the precise interaction(s) may be difficult. This is especially true for fragment-growing efforts (where one is likely to be limited in the choice of vector and distance) that aim to displace a high-energy water. Thus, the researchers suggest focusing on fragments that themselves displace high-energy waters, rather than trying to displace these later.

This seems like sound advice, but it likely reflects what folks already do. Since fragments that displace high-energy waters are likely to bind most effectively, won’t these be prioritized anyway? Regardless, this is an interesting and thought-provoking paper.

A COMT Tease...

S-adenosyl-methionine (SAM) is a hot molecule; you could probably make a good living selling it these days.  SAM-transferases of all types are "hot" targets, especially in epigenetics.  However, one current target is COMT, or catechol-O-methyl transferase.  COMT lives in a far different space than the epigenetics one, neurodegeneration.  There are several current Parkinson's Disease treatments based upon catechol, but as you would expect, there is toxicity associated with these.  

So, a team at Takeda decided to go after SAM-competitive molecules.  To this end, they screened 11,000 fragments using a enzymatic assay @100 uM.  52 hits (>15% inhibition) were found for a 0.15% hit rate.  They note this is a very low hit rate for what appears to be a very ligandable pocket. They then used LC-MS/MS and SPR to remove reactive moieties and non-SAM competitive molecules.  This led to compounds (4-6) and SAR by Corporate Collection (7). 

They followed up on these four compounds with DSF, STD-NMR, and X-ray.  They were able to co-crystallize 5 with mouse COMT.  This is the first (reported) structure of COMT with a SAM-competitive molecule. 

They mention that they took a "build up" approach, but I presume that is for for future papers. 

Whatcha Want? Whatcha Really Really Want?

There is a rule in our house: You cannot decorate your room for Christmas until November 1st.  Well, the countdown has begun as my son reminded me that is less than two months away.  So, to help everyone get into the Christmas (Hannukah, Diwali, and so on) spirit, I wanted to ask what cha want?  So what cha want?

Let's divide this in to two lists: Aspirational and Possible.  Below are some that Dan and I came up with.  But this is really your wish list.  Let us know in the comments.

Possible: Aqueous spectra for all commercially available fragments. Maybridge and Key Organics are here/getting here. 
Experimental solubility and 24 hour stability for commercial libraries.  

No PAINS in commercially available libraries.  I believe it is ~8% right now.  
No more rhodanines, anywhere and ever!

Aspirational: I know Peter Kenny wants people to stop using metrics that are arbitrarily defined.

Standard vocabulary for the field.  What's an active, hit, lead, etc.?

Fragments vs MAP4K4

Mitogen Activated Protein Kinase Kinase Kinase Kinase 4, or MAP4K4, is one of the 500+ human kinases that doesn’t get a whole lot of attention, in part perhaps because there aren’t many good tool compounds out there. A new paper from Genentech in Bioorg. Med. Chem. Lett. reports an attempt to change this.

The researchers started with a surface plasmon resonance (SPR) screen of 2361 fragments, yielding 225 confirmed hits with K_D values between 10 and 2010 µM, all with ligand efficiency (LE) values above 0.3 kcal/mol/atom. This seems like a good use of LE: with hundreds of hits to choose from, some sort of triage is necessary, and you might as well go with those with the highest LE.

Compound 1 had moderate potency and excellent LE, as well as a structure familiar from other kinase programs. Modeling suggested growing off the amine, and a small set of compounds were made including compound 7, which gave a 10-fold boost in potency, albeit with a loss in LE. Crystallography of a close analog of compound 7 revealed that it bound as expected, and also suggested a fragment growing approach.

A number of substituents were introduced, all with an eye towards keeping lipophilicity low (clogD < 3.5). Compound 16 was the most potent, though the solubility was poor, and adding polar substituents didn’t help much. Compound 25 had similar potency, and in this case adding a polar substituent (compound 44) improved solubility too. The PK profile in mice was also reasonable.

Unfortunately, when tested at 1 µM against 63 kinases, compound 44 inhibited 16 of them by at least 75%, suggesting that it will not make a useful tool compound. The team reported better selectivity earlier this year with a series of compounds derived from a different fragment hit identified in the same SPR screen. Yet despite the outcome, this is a nice case study in using ligand efficiency, calculated hydrophobicity, and structural information to guide fragment growing.

Fragment growing vs fragment linking

Results from our latest poll are in. As expected, fragment growing is both more successful and (thus) more popular than fragment linking, but there are a few surprises.

First, it was interesting to see that more than a third of the 69 respondents have not tried fragment linking. Actually, the fraction is probably higher since people could vote more than once (though unfortunately Polldaddy does not provide information as to how many did).

Second, despite its reputation for difficulty, roughly half of respondents who tried fragment linking reported that it worked “OK” or “well.” In fact, more people said that it worked well than that it failed outright. Again, these numbers should be taken cautiously since multiple people at the same organization may have voted on the same projects. And of course, one person’s definition of “OK” may be another’s definition of “marginal.” Still, if you have a tough target where fragment linking looks like a way forward, feel free to use these poll results to bolster your case. And please share your experiences in the comments, positive or negative.

PAINS in C&EN

Just a quick heads-up that Celia Arnaud has a nice story on pan-assay interference compounds (PAINS) in the latest issue of Chemical and Engineering News. Celia attended the PAINS symposium at the ACS meeting last month and spoke with several of the speakers for the piece.

As far as I know this is the first time C&EN has devoted an entire article to PAINS. It’s a fast read so I won’t summarize it here, other than to say that she does pick up on the concept of PAINS-shaming, which Teddy has also advocated. Although Practical Fragments has done some of this, most PAINS are not fragments, so it wouldn’t really be appropriate to do much of it here (though please visit HTSpains).

I do hope Celia’s article is widely read by practicing scientists, journal editors, and reviewers. The need for more PAINS recognition is amply illustrated by this article published in the most recent issue of J. Med. Chem. which reviews reported inhibitors of AP-1, many of them dubious. Let's hope that the C&EN piece cuts down on future pollution.