I have now been in Melbourne for about a month and have found the city very much to my taste. I’m visiting some friends to help out with some fragment stuff and have already been wreck diving (on the HMAS Canberra) and watched the Australian Open and a rather one-sided ODI between Australia and the West Indies. On the science side of things, I was able to gatecrash Surface Plasmon Resonance (SPR) course, hosted by the Biomolecular Interaction Facility at CSIRO, Parkville, and taught by Rebecca Rich and David Myszka of the University of Utah. Not the whole course, I might add, because the participants spent the second day of the course in the lab and I’m sure there was a clause in my visa agreement that stipulated that I was not to enter a laboratory except as an observer accompanied by a responsible adult.
SPR has always represented a bit of a gap in my knowledge base so this was always going to be a great opportunity. As well as being experts in this field, Rebecca and David present their material with great clarity, enthusiasm, charm and humour. I particularly liked David’s take on the Maxwellian Demon (these molecules don’t have eyes).
When using SPR to screen ligands, the protein is typically immobilised on the surface of the sensor chip. The term ‘immobilised’ is actually a bit of a misnomer and ‘tethered’ would actually be a more appropriate term. The SPR technology can be used to look at diverse types of interaction over a wide range of affinities and kinetic parameters (e.g. on and off rates) can also be measured.
There is of course a slight catch. The experiments need to be performed carefully and this was a recurring theme in the lectures (and presumably in the practical sessions as well). Now it turns out that much of the SPR literature is perhaps based on experiments that have been performed less than perfectly and, as a public service, Rebecca and David have reviewed and graded the SPR literature of 2008. GRADED? Yes, GRADED, and there were some Fs! Of course David is just the person to do the grading since he sports whiskers of which a Victorian (historical context rather than geographical) head master would be justifiably proud and it is easy to imagine him summoning the hapless transgressors to his study.
A grading exercise like this is unlikely to win you many friends and the authors are realistic to accept that it is likely to reduce the likelihood of either being elected to the National Academy of Sciences although hopefully they will never have to employ the services of professional food tasters when they attend SPR conferences. Putting on my computational chemistry hat, I couldn’t help thinking that the QSAR and Virtual Screening fields might benefit from a similar treatment...
There are a number of articles describing the use of SPR to screen fragments against target proteins and the one I’ve chosen to take a look at is from some folk at Roche. One of the authors of this work is David Banner, whose talk at RSC BMCS 2009, I greatly enjoyed, not least because he made no reference to ligand efficiency except, if I recall correctly, to say that he would not be referring to it.
The Roche group screened a library of 2226 compounds against chymase at 200 micromolar and found 80 hits so clearly SPR technology can provide the throughput required to run a fragment screen. The compounds were screened against an inactive (zymogen) form of the protein as a check for non-specific binding. The authors also described cross-competition experiments which could be used to determine whether two fragments were binding at the same or different sites and it is worth remembering that you need to be able to measure binding very directly to get this sort of information. It would have been really interesting if the results of the cross-competition assays had been integrated with crystallography since 12 co-crystallised complexes showed fragments binding in the active site.
Both stoichiometry and kinetics of binding can be determined by SPR making it an appropriate technique with which to observe interactions between badly behaved ligands and proteins. In an excellent (A-graded by Rebecca and David) article, another Roche group exploit SPR to classify some of these binding pathologies. It is particularly good reading for anyone who has worked up results from high throughput screens but that is not a place I particularly want to go to right now since it’s getting rather late at night and I really don’t want to have nightmares about pathological fragments.
Rich & Myszka, Grading the commercial optical biosensor literature – Class of 2008: ‘The Mighty Binders’ J. Mol. Recognit. 2010, 23, 1-64 Link
Perspicace et al, Fragment-Based Screening Using Surface Plasmon Resonance Technology, J. Biomol. Screen. 2009, 14, 337-349 DOI
Giannetti et al, Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors, J. Med. Chem. 2008, 51, 574-580 DOI