FBLD 2016

Last week the sixth FBLD meeting was held in Cambridge, MA. Like its predecessors in 2014, 2012, 2010, 2009, and 2008, this meeting was an enormous success, mixing more than 230 scientists with excellent (and liberal) food and drink. With 33 talks, more than 30 posters, and several vendor booths and workshops I won’t be able to do more than capture a few highlights.

The most striking feature for me was the number of success stories. This began with Steve Fesik’s keynote lecture, in which he discussed the MCL-1 inhibitors he and his team at Vanderbilt have discovered. When we highlighted his work last year he had reported low nanomolar inhibitors, but these did not have cell-based activity. His group has now optimized the molecules to low picomolar biochemical potency, low nanomolar cellular activity, and good activity in mouse xenograft models. This has not been easy: more than 2210 compounds were made, guided by 60 X-ray structures and dozens of pharmacokinetic experiments. It seems to be paying off though, and the researchers are developing biomarkers with the goal of advancing a compound into clinical testing.

Two other notable success stories about clinical candidates must be mentioned, though I’ll wait until publications come out before going into detail. Kathy Lee described how she and her colleagues at Pfizer chose a fragment that was less potent and ligand-efficient than other hits due to its interesting binding mode and were able to advance it to PF-06650833, an IRAK4 inhibitor with potential for inflammatory diseases. And Wolfgang Jahnke discussed how he and his colleagues at Novartis were able to discover and advance ABL001, an allosteric inhibitor of BCR-ABL, despite having the project halted twice – a reminder that persistence is essential.

Several other success stories have been covered at least in part on Practical Fragments, including inhibitors against PDE10A (presented by Izzat Raheem of Merck), Dengue RNA-dependent RNA polymerase (presented by Fumiaki Yokokawa of Novartis), lipoprotein-associated phospholipase A2 (presented by Phil Day of Astex), and BACE1 (presented by Doug Whittington of Amgen).

Crystallography was another theme, and several of the success stories relied on crystallographic fragment screening. Frank von Delft of the Structural Genomics Consortium described developments that allow screening 1000 crystals per week at Diamond’s Xchem facility in the UK, which include acoustic dispensing of compounds into crystallization drops – while carefully avoiding hitting the crystals head-on.

Several computational talks reported results that run contrary to conventional wisdom. Vickie Tsui of Genentech discussed their CBP bromodomain program (which we recently discussed here). Several water molecules form a highly ordered network in the protein, and a WaterMap analysis suggested that these were high-energy and that displacing them would lead to an enhancement in activity. Unfortunately this turned out not to be the case, though the researchers were able to get to low nanomolar inhibitors by growing towards a different region of the protein.

Li Xing mined the Pfizer database of 4000 kinase-ligand structures to extract 595 unique hinge binders. Not surprisingly, some of these – such as adenine and 7-azaindole – bound to multiple kinases, but 427 were complexed to just a single kinase. Hinge binders typically form 1 to 3 hydrogen bonds to the protein, and while there didn’t seem to be a correlation between the number of hydrogen bonds and potency, more hydrogen bonds did correlate – perhaps counterintuitively – with lower selectivity. To the extent that hydrogen bonds are thought of as enthalpic interactions, this further muddies the argument that enthalpy and entropy can be useful in drug design.

On a more positive note, Sandor Vajda (Boston University) suggested that, according to analyses done in FTMap, perhaps 60-70% of protein-protein interactions may be druggable – as long as we accept that this may require building larger molecules than commonly accepted. And Chris Radoux (Cambridge Crystallographic Data Centre) discussed the computational tool for characterizing hotspots that we previously covered here; a web server for easy search should be available soon.

Library design was also a key topic. Richard Taylor of UCB described his analysis of all FDA-approved drugs, which revealed >350 ring systems. Interestingly though, 72% of drugs discovered since 1983 rely exclusively on ring systems used prior to that date. Clearly there is plenty of untapped chemical real estate.

But getting there won’t necessarily be easy. David Rees stated that 33 fragments recently added to the Astex library required 13 different reaction types. Importantly, many of the fragment to lead successes at Astex have required growing the fragment from the carbon skeleton rather than from more synthetically tractable heteroatoms. Knowing in advance how to do this with every new member of a fragment library should make life much easier in the long run, though it is a serious challenge for chemists.

There is far more to write about, including a great discussion led by Rod Hubbard on how FBLD is integrated effectively into organizations and how it enables difficult targets, but in the interest of space I’ll stop here. If you were at FBLD 2016 (or even if you weren’t) please share your thoughts!

Tenth Annual Fragment-based Drug Discovery Meeting

Last week marked the tenth anniversary of CHI’s three-day Drug Discovery Chemistry conference in San Diego. The conference consists of six tracks, with three happening simultaneously. The FBDD track is the only one which dates all the way back to the beginning in 2006. In fact, this is the oldest recurring fragment conference, predating both the Royal Society Fragments meetings as well as the independent FBLD meetings.

It’s worth reflecting on how far fragments have come since 2006. Back then, as Rod Hubbard (Vernalis and University of York) noted, most of the talks were prospective and methodological. Even as late as 2010 there were talks describing how dedicated fragment groups needed to be shielded from the larger organization. Now fragments are mainstream: a large fraction of the talks in the protein-protein interaction track involved fragments, as did both plenary keynote addresses to the entire conference.

Harren Jhoti’s keynote focused on lessons learned at Astex over the past 15 years. There has been some debate in the literature over ligand efficiency (LE), and one slide that struck me was a summary of 782 dissociation constants (measured by ITC) against 20 projects. The vast majority of these compounds had LE > 0.3 kcal/mol/atom. Given that Astex has put multiple fragment-derived drugs into the clinic and was acquired by Otsuka in one of the largest M&A events of 2013, the metric appears to have some utility.

Still, it’s important not to be dogmatic, particularly for difficult targets. Harren described a program for XIAP/cIAP where they started with an extremely weak fragment with LE < 0.2, but its binding mode was sufficiently interesting that they were willing to work on it. This program also revealed the importance of biophysical measurements, as functional activity was uninterpretable and even misleading until higher affinity compounds were discovered.

One theme throughout the conference was the observation that fragments bind at multiple sites on proteins. Harren noted that Astex researchers have found fragments bound (crystallographically) to 54 sites on 25 targets – an average of 2.2 sites per target. Some targets are even more site-rich: Joe Patel (AstraZeneca) performed a crystallographic screen on a complex of Ras and SOS and found four binding sites, including one previously discussed here. In this effort, 1200 fragments were screened in pools of 4, and in one case two fragments from the same pool each bound only when they were both present at the same time – each fragment alone showed no binding by NMR or crystallography.

Troy Messick (Wistar) described his work against the EBNA1 protein from Epstein-Barr virus. An HTS screen of 600,000 compounds came up with at best marginal hits, but soaking 100 different Maybridge fragments into protein crystals led to 20 structures, with fragments bound to four different sites. Some of these fragments were then merged to give cell-active compounds with good oral bioavailability.

Rather than exploring different ligands binding at different sites, Ravi Kurumbail (Pfizer) described an interesting case of the same ligand binding at different sites. A screen against the kinase ITK identified a (large) fragment that could bind both in the adenine binding pocket as well as a nearby pocket, as determined crystallographically. Determining the affinities of the same fragment for the two sites necessitated some clever SPR and enzymology, but did lead to a highly selective series.

In terms of targets, BCL-family proteins were certainly well-represented, featuring heavily in talks by Chudi Ndubaku (Genentech, selective Bcl-x_L inhibitors), Mike Serrano-Wu (Broad Institute, MCL-1 inhibitors), Zaneta Nikolovska-Coleska (University of Michigan, MCL-1), Roman Manetsch (Northeastern, Bcl-x_L and MCL-1), and Andrew Petros (AbbVie, BCL-2 and MCL-1). Of course, it was AbbVie (neé Abbott) that pioneered BCL inhibitors as well as FBLD in general, and I was happy to hear that there is a renaissance occurring there, with fragment approaches being applied to all targets, even those undergoing HTS.

Finally, there were some interesting practical lessons on library design. Peter Kutchukian described how the Merck fragment library was rebuilt to incorporate more attractive molecules that chemists would be excited to pursue. There is an ongoing debate as to whether a fragment library should be maximally diverse or contain related compounds to provide some SAR directly out of the screen, and in the case of the Merck library the decision was to target roughly five analogs in the primary library, with a secondary set of available fragments for follow-up studies.

The utility of having related fragments in a library was illustrated in a talk by Mark Hixon (Takeda) about their COMT program. A HTS screen had failed, and even a screen of 11,000 fragments came up with only 3 hits (with an additional close analog found by catalog screening). Remarkably, all of these are extremely closely related, but other analogs in the library didn’t show up; had they not had multiple representatives of this chemotype in their library they would have come up empty-handed.

In the interest of space I’ll close here. Teddy will post his thoughts later this week, and please share your own. CHI has announced that next year’s meeting will be held in San Diego the week of April 19. And there are still several great events on the calendar for this year!

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.”

Fragment specificity

A frequent topic in fragment roundtable discussions concerns specificity: do fragments hit lots of targets, or just a few? Isabelle Krimm and colleagues at the Université de Lyon in France studied this question experimentally and report their results in a recent issue of J. Med. Chem. The paper provides data for the ongoing debate of whether and how much specificity a fragment should exhibit before being pursued for further lead development.

The researchers assembled a diverse set of 150 fragments and used NMR techniques to determine whether they bind to five different proteins. Three of the proteins, Bcl-xL, Bcl-w, and Mcl-1 are related members of the Bcl-2 family of antiapoptotic proteins, and at least the first of these has been successfully targeted using fragment-based methods. The fourth protein, PRDX5, has proven to be much less yielding to inhibitor discovery, while the fifth, human serum albumin (HSA), binds a wide variety of small molecules.

After applying 1D-NMR techniques (WaterLOGSY and STD) to all of their fragments against each of the five proteins, the researchers used more rigorous but less sensitive 2D-NMR (HSQC) to determine the binding sites of the hits. (This later study revealed, in agreement with previous results from the same lab, that the fragments all bind in the “hot spots” or active sites of the proteins.)

More than two-thirds of the fragments bound to at least one protein, a rather high hit rate. However, the hit rates for each protein varied considerably, with only 7 hits for PRDX5 and 72 for HSA (with a close second of 71 for Bcl-xL). Within the Bcl-2 family there was little specificity observed: Mcl-1, with 29 hits, shared all but one hit with either Bcl-xL or Bcl-2 or both; such non-specificity among related proteins has been discussed previously. In the case of HSA and Bcl-xL, although both proteins had similar numbers of hits, just over half of these were in common, demonstrating that fragment specificity is not difficult even with small-molecule sponges such as HSA. That said, many fragments were remarkably nonspecific, with 22 hitting four of the 5 proteins. Amazingly, all 7 of the hits against PRDX5 also hit all four other proteins.

The physicochemical properties of the fragments that hit one or more proteins were compared with those of the library as a whole, and although most of the parameters were similar, the ClogP values (a measure of hydrophobicity) were considerably higher for hits, and highest of all for the non-specific hits.

These findings are more evidence that, as predicted almost a decade ago, fragments can bind to more proteins than can larger, more complex molecules. The follow-up question, how much does this matter, is still up for debate. There are plenty of examples of developing specific inhibitors from non-specific starting points during the course of fragment optimization. But how non-specific is too non-specific? Would you feel comfortable pursuing any of the fragments that hit all of the proteins?