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.”

First fragment-based drug approved

Today marks history with the first FDA approval of a drug to come out of fragment-based screening. The drug is branded as Zelboraf (vemurafenib), but readers of this blog are probably more familiar with its previous name of PLX4032. Although widely expected to be approved, the FDA acted more than two months ahead of schedule. The drug targets a mutant form of BRAF and has received widespread media coverage because of dramatic clinical results showing that it extends life for patients with a particularly deadly form of skin cancer. FiercePharma has an article with links to several others.

The drug was discovered at Plexxikon and developed in partnership with Roche; Plexxikon was acquired earlier this year by Daiichi Sankyo. The PLX4032 story is a case study in how rapidly fragments can enable a program: initiated in Februrary 2005, it took just six years to reach approval. It’s also an example of starting with a profoundly unselective fragment and winding up with a very selective drug (see here for early discovery and here for characterization of PLX4032).

Although I claim no prescience, I did state back in 2008 that it would be nice if a fragment-based drug would be approved by 2011. But more importantly, it is worth pausing to remember that this is a victory not just for the field of fragment-based drug discovery, but for those patients afflicted with metastatic melanoma. In the end, that’s what this is all about.

Fifth Annual Fragment-Based Drug Discovery

The first of two conferences in 2010 exclusively devoted to fragment-based drug discovery concluded in San Diego this week, and I thought I’d jot down some observations while my memories are still fresh.

The pre-conference short courses were quite successful (and I’m hopefully only slightly biased by the fact that Teddy and I were both instructors). Participants included folks with considerable experience in fragments, which allowed good discussion.

One talk from the conference that stands out in my mind was by Sandy Farmer of Boehringer Ingelheim. BI is a relative late-comer to fragment-based methods, really only starting in late 2004. Sandy described how fragment efforts are run in parallel with HTS. They use an intentionally modest fragment library of 2000 diverse compounds; increasing the size of this library tended to overwhelm downstream efforts. Fragments are confirmed using multiple assays, including SPR and size-exclusion chromatography coupled with mass spectrometry, with crystallography playing a pivotal role in determining which fragments to advance. Part of the challenge at BI has been getting chemists to accept FBS, or “faith-based synthesis,” particularly where initial fragments have low affinities. A focus on ligand efficiency helps, as do organizational strategies such as establishing a dedicated group of chemists focused on fragment projects.

Often at conferences you hear about success stories, but sometimes the continuing challenges are more instructive, as when Ravi Kurumbail at Pfizer discussed his efforts to discover drug-like inhibitors of the serine protease Factor XIa. One of the sobering findings was that, although a functional assay of 2500 fragments yielded a 6.5% hit rate, adding 0.01% detergent eliminated activity and revealed most ‘hits’ as false positives. Even one crystallographically characterized fragment with an apparent IC50 of 75 micromolar turned out to be an artifact after subsequent analysis – a reminder to always be vigilant at higher concentrations.

But back to success stories: Daniel Wyss gave an update on the BACE program from Merck (legacy Schering-Plough, which has run more than 30 fragment screens on various targets). We highlighted a couple publications resulting from this effort earlier this year. It turns out there are now three molecules from this program in early clinical trials – a clear indication of the importance of this target and the utility of fragment screening.

Finally, Rick Artis, formerly of Plexxikon (now Elan) gave an update on the PLX4032 Raf kinase program. This project demonstrates the potential for fragment-based efforts to move quickly: it was started in February 2005, the clinical candidate was identified in January 2006, the IND was filed in September, and the first patient was dosed in November of that year. The molecule has continued to move at warp speed through the clinic: it is now partnered with Roche in Phase III testing for metastatic melanoma, and was recently profiled in the New York Times. This lengthy but excellent article is well worth reading for a bit of perspective when life in the lab gets you down.

These are just a few of many nice talks and breakout discussions. I know that at least some readers of this blog were there – what were your impressions?

Molecular Medicine Tri-Conference 2010

The first event on our 2010 calendar, the Molecular Medicine Tri-Conference 2010, was held in San Francisco earlier this week. There were fragment talks and a roundtable, as well as a number of vendors selling fragment libraries – we’ve recently noted how rapidly this area has expanded.

Michael Hennig presented a nice overview of the history and development of fragment-screening at F. Hoffmann-La Roche (Basel). Work done there back in the late 1990s relied on NMR and crystallographic screening of a library of 300 fragments, described in the seminal “needle-screening” publication in J. Med. Chem. Today that library has grown to 6000 compounds following a relaxed rule-of-3 (allowing in particular more hydrogen-bond acceptors and higher lipophilicity) and requiring at least one hydrogen bond donor or acceptor and at least one ring. Also, the primary screening technique is now surface-plasmon resonance (SPR), with crystallographic follow-up; the entire collection can be screened on a single Biacore instrument in four weeks.

Hennig shared two case studies, one on BACE-1, the other on chymase. In the second case, a dozen fragments were successfully co-crystallized with the enzyme, and all but one of these bound in the S1 pocket, revealing the importance of this site for binding. In response to a question about how widely FBDD is used at Roche, Hennig said that it is applied to all targets that are technically feasible.

In another talk, James Madden described fragment-based discovery at Evotec. An increasingly stringent series of assays (from high-throughput high-concentration functional assays, through SPR and/or ligand-detected NMR, and finally crystallography and/or protein-detected NMR) helps keep the number of compounds manageable at each step. Madden also presented two cases studies, BACE-1 (clearly a popular target for FBDD, perhaps due to its intractability to many other approaches) and PDE10a.

A fun talk with relevance beyond FBDD was “Examples of X-ray Bloopers”, by Edward Kesicki of the Infectious Disease Research Institute (IDRI) in Seattle, WA. He described several cautionary tales from his own experience. In one case, a chemist provided the structure of the wrong enantiomer to a crystallographer, who duly refined the data, resulting in weeks of confusion and time-consuming follow-up experiments. In two others, crystallographers inadvertently omitted methylene units in fitting electron density. We’ve previously commented on the dangers of taking crystallographic data at face value, and Kesicki also mentioned an effort by Stephen Warren of Gonzaga University to comb through and correct structures in the protein data bank. He has a lot of work to do: of the 1000 structures examined thus far, roughly 20% have problems with the ligands.

Finally, in a panel discussion on “medicinal chemistry drivers,” someone asked about the role of fragment-based drug discovery. Consistent with the idea that fragment approaches are becoming increasingly integrated with other lead-finding activities, Hing Sham of Elan said that he was neither pro-fragment nor anti-fragment – “it’s just another tool in the toolbox.”

Genentech’s affinity for Graffinity

Heidelberg-based Graffinity today announced that they would be collaborating with the Genentech division of Roche. Graffinity will apply its surface plasmon resonance (SPR) fragment-based technology to several Genentech targets. Financial details and specific targets have not been released, though Graffinity CEO Kristina Schmidt is quoted as saying that they plan “to explore drug targets that would remain white spaces on the map of drug discovery” with conventional high-throughput screening.

SPR is rapidly becoming a workhorse in the stable of FBDD techniques. Although it provides less information than NMR or X-ray approaches, SPR is faster, and can rapidly distinguish true hits from bad-acting artifacts. Typically a protein is immobilized on a gold surface, and fragments are allowed to flow past to detect those that bind. Graffinity reverses this process: they have a collection of about 110,000 small molecules, just over a fifth of which are fragments, immobilized in microarrays which can be screened against proteins (see here for full description).

Genentech is no stranger to SPR; one of the highlights of the recent FBLD 2009 meeting was a talk by Tony Giannetti on the use of this technology at Genentech against roughly 40 target proteins. The collaboration further validates the use of SPR for FBDD, and suggests that Graffinity has an interesting – and useful – angle.