Last week NovAliX held its biophysics meeting outside of Strasbourg for the first time. Naturally they chose Boston, one of the most European of US cities and a major hub of drug discovery. The event brought together 118 participants from 15 countries, roughly 80% from industry. Although the food and drink could not compare to France, the science and discussion were every bit as satisfying. With 30 talks and 22 posters I won’t attempt to be comprehensive, but as with last year just try to capture a few themes.
One particularly noteworthy session was devoted to single particle cryo-electron microscopy (cryo-EM), which was recently reviewed in Nat. Rev. Drug Discov. by conference chairman Jean-Paul Renaud and a multinational team of experts. The approach involves flash-freezing a thin film of sample and using transmission electron microscopy to capture two-dimensional “projection” images of your target. If the protein is randomly oriented you can computationally combine thousands of individual images into a three dimensional structure. Although the technique has been around for decades, until recently the resolution was too low to be useful for structure-based drug design. Recent advances in hardware and computation have led to what’s come to be known as the “resolution revolution,” explained Gabe Lander (Scripps).
One advance is the 300 keV Titan Krios – a massive (and massively expensive) instrument that is so widely coveted that Gabe showed pictures of happy scientists hugging newly delivered crates. Indeed, of the ~1000 structures solved to < 4 Å resolution, the vast majority of them were solved on one of more than 130 Krios instruments throughout the world. But Gabe showed that high resolution structures can be obtained with more common 200 keV instruments, including a 2.6 Å resolution structure of aldolase (150 kD), a 2.9 Å structure of hemoglobin (64 kD), and a 2.9 Å resolution structure of alcohol dehydrogenase (81 kD) with bound NAD+ cofactor. Although only a handful of sub-2 Å structures have been reported, he thought these would become routine in the next few years.
Bridget Carragher (New York Structural Biology Center) described challenges and how to overcome them. Currently it takes at best eight hours to go from data to structure, but she thought getting this to under one hour would be achievable. Moreover, cryo-EM can be used to characterize different conformational or oligomeric states present in a single sample, as Giovanna Scapin (Merck) demonstrated with insulin binding to its receptor. Indeed, even simple visualization – without fancy computational processing – can provide useful information about protein aggregation, as demonstrated by Wen-ti Liu (NovAliX).
Although primary fragment screening still looks a long way off for cryo-EM, it should start to provide useful structural information for fragments bound to targets less amenable to conventional biophysical techniques, such as membrane proteins – the topic of another session.
Miles Congreve (Heptares) discussed how their stabilized “StaR” GPCRs can provide high-resolution crystal structures suitable for FBDD (see for example here). This has allowed them to discover less lipophilic, more ligand-efficient drug candidates against a variety of targets.
According to Anass Jawhari, it isn’t even necessary to make mutant GPCRs: Calixar has developed proprietary detergents that can stabilize full length adenosine A2A receptor for a week – more than enough time to perform STD NMR screens of 100 fragments and identify 19 hits, some of which turned out to be functional antagonists. Matthew Eddy (University of Southern California) used two-dimensional NMR on this same protein to reveal dramatic differences in conformational dynamics when bound to agonists vs antagonists.
Indeed, conformational changes and dynamics were a running theme throughout the conference. Keynote speaker and Nobel-laureate Martin Karplus (Harvard) quoted fellow Nobelist Richard Feynman: “everything that living things do can be understood in terms of the jiggling and wiggling of atoms.” (As an aside, Martin’s MCSS method pioneered computational FBDD approaches, predating SAR by NMR.) Göran Dahl (AstraZeneca) described how large scale conformation changes well outside of the active site of PI3Kgamma were responsible for freakishly high selectivity of a class of inhibitors.
But how do you detect conformational changes? We’ve previously mentioned Biodesy’s SHG approach, and Parag Sahasrabudhe (Pfizer) described how this proved useful for classifying ligands for IL-17A. Gerrit Sitters (Lumicks) described a completely different “dynamic single-molecule” (DSM) approach, which involves trapping a single fluorescently labeled protein between DNA strands tethered to two microspheres. Changes in protein conformation caused by ligand binding change the distance between microspheres, and these can be detected to within 1 Å.
Kinetics is intimately linked to dynamics, but the factors responsible for slow binding and dissociation are still poorly understood. Chaohong Sun (AbbVie) examined an archive of 8000 data points and found that on-rates and off-rates each varied by more than five orders of magnitude. There was no correlation with ClogP of the ligands, though larger ligands were more likely to have slower kinetics. There were also significant target effects; on-rates were consistently slow for one target.
As we’ve previously discussed, off-rate screening (ORS) can be used to identify hits in crude reaction mixtures, and Menachem Gunzburg (Monash University) described how this technique is being used in hit-to-lead efforts. Lowering the temperature to 4 °C and adding 5% glycerol further slows dissociation, allowing weaker hits to be discovered.
At the extreme, irreversible inhibitors have an off-rate of 0, and Gregory Craven (Imperial College London) described quantitative irreversible tethering of electrophilic fragments to cysteine residues in proteins using a fluorimetric plate-based assay. As we’ve noted, one challenge with irreversible tethering is deconvoluting intrinsic reactivity from proximity-directed reactivity, which Gregory addresses using a reference thiol such as glutathione.
There is much more to say but in the interest of time I’ll stop here. If you missed the conference you have two chances next year: June 4-7 when it returns to Strasbourg, and November 20-22 when it will be held in Kyoto. And there are still excellent events coming up this year – hope to see you at one!