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.”

How effectively can fragments sample chemical space?

One of the key advantages of fragment-based drug discovery is that, since there are fewer fragments than lead-sized or drug-sized molecules, it is possible to sample chemical space far more efficiently with fragments than with larger molecules. At least, that’s the theory, but does is it hold true in the real world?

To put it another way, do fragments sample all of the space in which drugs are found? And what kinds of fragments are best for this sampling? In the most recent issue of J. Med. Chem., Stephen Roughley and Rod Hubbard of Vernalis address such questions.

The system they investigate, heat shock protein 90 (Hsp90), is an ideal model system: it is both a popular anti-cancer target as well as structurally tractable, and is thus arguably the most heavily explored single target in terms of fragment-based lead discovery. At least 8 antagonists have entered the clinic, of which at least 2 have come from fragments (see the posts on AT13387, NVP-BEP800/VER-82576, and posts on Evotec compounds discovered by fragment growing or linking.)

Vernalis has had a long-running fragment-based program targeting Hsp90, which has resulted in numerous fragments whose binding modes have been determined by X-ray crystallography. Roughley and Hubbard analyzed these fragments and compared them to published inhibitors. Just 5 distinct fragments can be mapped onto all of the clinical compounds: a handful of fragments effectively samples relevant chemical space. As the authors put it:

For Hsp90 at least, the fragments do cover an appropriate chemical space; what is then important is the imagination of the chemist in evolving the fragments into potent inhibitors.

The second point – about the imagination of the chemist – is critical. Mapping fragments onto elaborated molecules is easier to do retrospectively than prospectively; a cynic could argue that methane is a fragment of just about any drug out there. However, Roughly and Hubbard also point out that, particularly in cases such as this where there are many co-crystal structures, fragments can help identify bioisosteres, including cryptic ones that would not be obvious purely from studying functional SAR.

The paper also addresses the issue of optimal library design, in particular the dilemma of size. Although all five representative fragments were found in an initial set of just 719 fragments, subtle changes can dramatically change the binding mode, an issue we’ve touched on previously. It may not be practical to have multiple similar fragments present in a primary screening library, but testing close analogs after identifying initial fragment hits is likely to be worthwhile.

Finally, one of the concerns about fragment-based approaches is that, if everyone is buying the same set of fragments from the same suppliers and screening them against the same targets, they will end up in the same place – and stumbling over each others’ intellectual property. Reassuringly, this turns out not to be the case:

Even though the various companies discovered rather similar compounds from a fragment screen, exploiting similar binding motifs, there were no exact matches. [Also], the subsequent evolution of the fragments sometimes took very different paths and produced mostly very different chemical leads and candidates.

If this holds true for such heavily mined targets as Hsp90 (and kinases, as discussed previously) it should be even more true for newer classes of targets.

There is a wealth of information in this paper, and it is worth perusing, especially if you find yourself longing for some science over the long holiday weekend.

Pin1 revisited

Earlier this year we highlighted a paper from Vernalis that described the use of NMR methods to discover inhibitors of the anti-cancer target Pin 1. In a recent issue of Bioorg. Med. Chem. Lett. the same team now reports a second series of compounds that inhibit this protein, also discovered and advanced through FBLD. The two papers together provide some interesting lessons.

Rather than using NMR, the researchers identified the second series of compounds with an inhibition assay. After screening 900 fragments at 2 mM, they obtained 40 hits, including 3 compounds previously discovered by NMR. Disturbingly though, follow-up NMR experiments confirmed binding for only 2 of the 37 new hits, suggesting that the remaining compounds may act through pathological mechanisms (see also here). Still, two hits are better than none, and the binding mode of one of the fragments (compound 3 in figure) was determined by X-ray crystallography. Several analogs of this were purchased and tested, and compound 5 was found to have an improved potency and ligand efficiency.

At this point chemistry entered the picture. The researchers synthesized several analogs of compound 5, guided by crystallography and modeling. This led to compound 10e and eventually to compound 20, with sub-micromolar biochemical activity and measurable cell activity.

Ultimately though, as in the previous Pin1 series, even this modest cellular potency was gained at the cost of unacceptable increases in size and hydrophobicity. This brings up an interesting question: at what point do you declare a target undruggable? The authors note that “the nature of the Pin1 active site makes it difficult to optimise hits into drug-like molecules.”

Fragment-based approaches can sometimes deliver inhibitors to challenging targets where HTS has failed. However, if the inhibitors can’t be transformed into drugs, is finding them actually a good thing? Researchers are getting better at improving potency at the same time as ligand efficiency for some targets, but ultimately getting to clinical candidates for harder targets may come down to how many resources you are willing to throw at a project: molecules such as ABT-263, though far from rule-of-5 compliant, are doing well in the clinic, but only after the investment of dozens if not hundreds of people-years.

Pinning fragments on Pin1

Pin1, a potential anti-cancer target, catalyzes isomerization around phosphoserine-proline and phosphothreonine-proline bonds. Its binding site is relatively shallow, complicating efforts to discover small, non-peptidic inhibitors. In a recent paper in Bioorg. Med. Chem. Lett., Jonathan Moore and colleagues at Vernalis describe their fragment-based approach to tackling this problem.

The researchers used NMR-screening of roughly 1200 fragments to identify five that competed known inhibitors; Compound 4 (see figure) was the most potent, with an IC50 of 16 micromolar in a functional assay. NMR experiments showed a weaker binding interaction, on the order of 200 micromolar, and surface-plasmon resonance (SPR) experiments were even less conclusive: the compound showed super-stoichiometric binding, indicating that multiple molecules were interacting with the enzyme rather than sitting specifically in the binding site. However, the researchers were able to obtain a crystal structure showing that the molecule binds in a hydrophobic pocket at the active site. Although they don’t mention it in this paper, in public presentations the researchers have reported seeing additional molecules of Compound 4 pile on top of each other, essentially forming a stack on top of the protein. Perhaps, as the authors suggest in the supplementary material, this is an example of a particularly insidious aggregation phenomenon: a legitimate hit that can also form aggregates.


In order to access other parts of the protein, and reduce the propensity for aggregation, the researchers relied on analog screening and modeling to generate Compound 18a, which is a more “three-dimensional” molecule. Further elaboration led to a series of compounds such as 19e, with low nanomolar potency, as well as one-to-one binding in the SPR assay.

Despite their potency, these molecules were inactive in cell-based assays, likely due to high polar surface areas and their resulting low cell permeabilities. To fix this, the researchers replaced the benzimidazole fragment with a naphthyl group, which led to a decrease in biochemical potency but did lead to cell-active molecules such as Compound 23b. Moreover, a crystal structure revealed that this molecule binds in a similar manner to the original fragment 4.

This paper exemplifies another example of fragment-assisted lead discovery: the original fragment morphed from an indole to a benzimidazole to a napthyl group, yet the final molecule still owes a debt to the initial fragment.

Too many aromatics stink

A recent discussion centered on whether fragment libraries should be designed to include more “3-dimensional” molecules and reduce the number of flat, aromatic compounds. Two new papers suggest that doing so may improve pharmaceutical properties. What effect this would have on screening success is still unclear.

The first paper, published by Timothy Ritchie and Simon Macdonald of GlaxoSmithKline in this month’s Drug Discovery Today, correlates the number of aromatic rings with several metrics associated with success in drug development. For this analysis, each ring in a fused system is counted separately, so indole is counted as having two aromatic rings. The researchers conclude that more than three aromatic rings correlates with an increased risk of compound attrition during drug development:

The fewer the number of aromatic rings contained in an oral drug candidate, the more developable that candidate is likely to be.

This is not surprising, but with their access to a vast internal data set the researchers provide considerable supporting evidence. For example, the mean aromatic ring count declines from 3.3 to 2.3 as GSK compounds move from preclinical candidate selection to proof-of-concept in humans. Measured (kinetic) solubility decreases dramatically with increasing ring count: even two aromatic rings leads to many low solubility compounds, and with four aromatic rings the median solubility is only 0.012 mg/ml. Both c log P and log D increase with increasing ring count, as do serum albumin binding, P450 3A4 inhibition, and hERG inhibition – all factors one usually wants to decrease in drug development.

One caveat is that the authors do not control for size. As aromatic rings are added, molecular weight is likely to increase, and thus many of the properties could simply reflect the pharmaceutical liabilities of larger molecules. This is where the second paper comes in. In J. Med. Chem., Frank Lovering and colleagues at Pfizer (nee Wyeth) analyze the effect of aromaticity itself by defining a simple metric:

Fsp3 = number of sp3 hybridized carbons / total carbon count

The smaller the number, the more aromatic the compound; the larger the number, the less aromatic. Besides being a straightforward measure of saturation, the formula inherently controls for molecular size.

When the researchers examined published data sets, they found that the mean Fsp3 increases from 0.36 for 2.2 million molecules in discovery to 0.47 for 1179 approved drugs. They also investigated measured solubility and found a strong correlation: 104 molecules with a log S of -6 (quite insoluble) had an average Fsp3 of 0.31, while 194 molecules with a log S of 0 (very soluble) had an average Fsp3 of 0.56. The effect is even more striking with melting points, which negatively correlate with solubility: 1153 molecules with a melting point of 125 deg. C had an average Fsp3 of 0.31, while 375 molecules with a melting point of 275 deg. C had an average Fsp3 of 0.18.

OK, so let’s say we accept the premise that increasing aromatic character in a molecule leads to lower solubility and worse properties overall. The easiest solution might be to reduce the number of aromatics in a screening collection, but would this really be wise? Ritchie and MacDonald note that aromatics, with their rigid structures, are likely to have increased potency relative to unsaturated molecules. And particularly for fragment libraries, you want all the binding energy you can get.

An interesting study would be to correlate the hit rate for fragments with their aromatic character. Does the hit rate increase with decreasing Fsp3? These data must exist in companies that have been doing FBDD for years. Indeed, at FBLD 2009, Ijen Chen of Vernalis presented a nice analysis of hits against 12 targets, in which she noted that roughly 2/3 of the fragment library members didn’t hit any of the targets. I don’t think she mentioned aromaticity specifically, but she did note that the hits tended to be slightly more rigid and hydrophobic than the non-hits – just what you would expect for low-Fsp3 molecules.

So by all means avoid having too many aromatics, but don’t go to extremes: it’s finding the right balance of binding energy and pharmaceutical properties that makes drug discovery such a tricky business.