Fragments 2013

Fragments 2013, the 4^th RSC-BMCS fragment-based drug discovery meeting, took place this week at STFC Rutherford Laboratory in Oxfordshire, UK. These biennial meetings started in 2007; you can read impressions of Fragments 2009 here and here. With 14 speakers, 49 posters, multiple exhibitors, close to 200 attendees, and a pre-conference training course, this post can touch on just a few topics. Some of the talks and poster summaries are available here.

One of the first things I noticed was the number of new faces, always a good indicator for the health of a field. I was also struck by the number of attendees from big pharma, including a couple companies that had draconian travel policies in 2012. Hopefully this portends a thaw from the last few years.

False positives or false negatives?

A recurring theme was the (ir)reproducibility of fragment-finding methods. Practical Fragments recently discussed this here, and it seems many other folks are also finding that orthogonal methods can produce non-overlapping sets of hits. For example, Ursula Egner from Bayer Healthcare described two different targets screened using multiple methods. For thrombin, her team found the following from a library of 1891 fragments:

27 hits with IC₅₀ ≤ 650 μM using high-concentration activity screening

75 hits with IC₅₀ ≤ 2 mM using SPR

58 hits that stabilized the protein more than 2σ above baseline against thermal melting

Of these, only 2 were found by all three techniques, and of 114 fragments soaked into crystals, only 15 gave structures.

Another (unnamed) protease gave similar results with a library of 2031 fragments:

17 hits from high-throughput screening

48 hits from SPR

38 hits from thermal shift

None of the hits were found in all three assays!

In this case soaking was not possible, and of the 93 co-crystallization trials only 8 produced structures, of which none came from the thermal shift assays (also true for thrombin).

Al Gibbs from Jannsen R&D found similar results in a retrospective analysis of hits against ketohexokinase (see also here). Of 786 fragments tested, there were:

54 hits in an activity assay (mass-spectrometry based)

75 hits from SPR

44 hits that produced crystal structures

Of all these, only 2 were in common. There was also no correlation between affinity or solubility and the ability to obtain a crystal structure.

However, these observations were not universal. Rod Hubbard noted that of the 32 targets screened over the past 10 years at Vernalis, there tended to be good overlap between hits from SPR and NMR, and that these tended to produce X-ray crystal structures, though crystallography had plenty of false negatives too. He did single out thermal melt assays as being particularly unreliable, as have others.

How to reconcile these varied experiences? Rod stressed that assays required very careful optimization, and that subtle changes could dramatically improve the number and quality of hits. Indeed, the thrombin example above used different cutoffs for the activity and SPR screens, and the ketohexokinase crystals were soaked at pH 4.5 while other assays were run at pH 7.5. Tony Giannetti noted that his group at Genentech tries to closely match their SPR screening conditions with those that the crystallographers use.

All this does raise the question of what to do when your orthogonal assays don’t agree: do you go with less validated hits, risking false positives, or throw away potentially valuable fragments? There probably is no one right answer. If you have plenty of hits that confirm in all your assays you should probably stick with those, but if you’re working on a tougher target you may need to dig into the noise, but be especially wary of misleading, ultimately meaningless babble. Of course, the potential for false positives means you want to take even more care in your fragment library design.

Library design

On the topic of “three-dimensional” fragments, a concern raised as far back as 2009 is that they may have a lower hit-rate than “flatter”, more aromatic fragments. Dirk Ullman noted that over the course of 29 screens he and his colleagues at Evotec have obtained 15,687 fragment hits, with any given fragment rarely hitting more than 4 different targets. Reassuringly, Oliver Barker presented a poster in which he found that while these hits were slightly biased towards having fewer tertiary and quaternary carbons as well as a lower Fsp3, this was a very modest trend, probably not statistically significant.

Teddy’s recent poll asking how much diversity readers want in their fragment libraries found that though the majority of respondents (60%) wanted maximum diversity, target-focused libraries can be effective too. Paul Bamborough described how, in addition to a generic fragment screening library, he and his colleagues at GlaxoSmithKline also built a collection of 936 fragments geared for kinases (described here) and, more recently, 1326 fragments targeted to bromodomains. These have yielded much better hit rates than have their diverse fragment sets.

The targeted libraries also provide a nice example of fragment-assisted drug discovery: they were designed based on molecules derived from high-throughput screening, and the data generated by screening the fragments against several bromodomains have in turn informed the design of 25,000 new lead-like molecules for HTS and several billion DNA-encoded molecules.

There was plenty else of note, including some nice fragment-to-lead stories (such as this) and others that should be appearing in the literature soon, but I’ll end here. What were your impressions?