FBDD is a maturing field and one sign this maturation is the publication of a volume of Methods in Enzymology devoted to the subject. The article in this collection that most interested me was the review by Anthony Giannetti on the use of Surface Plasmon Resonance (SPR) in Fragment Lead Generation. The review is described as a ‘comprehensive walk-through’ and in-depth treatment of topics such as target immobilization and buffer/compound preparation justifies this description. I’m still working my way through some of the data analysis sections...
The target is tethered to a surface in SPR and this is usually referred to as ‘immobilization’, which is an unfortunate term, albeit the one that is most commonly used in the literature. Vendors of competing assay technologies (who would naturally prefer you to use their technology instead) often present this as a weakness of SPR. One concern is that tethering will compromise the ability of the target to bind ligands and the review does cite a couple of articles which compare affinities measured with SPR to those measured using methods such as isothermal titration calorimetry.
The system in an SPR assay is heterogenous, which is another way of saying that the concentration of protein is not uniform, particularly in the direction perpendicular to the surface to which it is tethered and this creates some interesting possibilities. Tight binding occurs when the value of the ligand Kd is lower than the concentration of the protein to which it binds. We typically configure assays for measuring affinity and potency so that ligand concentration is significantly greater than protein concentration. This means that ligand binding does not affect the concentration of unbound ligand and the math is a whole lot easier if you can make this assumption. If, however, the protein concentration in your assay is 1nM and you want to measure the potency for a compound with an IC50 of 0.01nM you’re going have a problem because you’ll need the compound at a concentration of 0.5nM in order to occupy half the binding sites. In enzyme inhibition assays, the concentration of the enzyme limits sets an upper limit on the potency that you can measure and this may be an issue for attempts to estimate the maximum potency of ligands.
In a heterogeneous system, things are not quite as simple because concentration is less clearly defined and you need to think in terms of quantities (in molar terms of course) of protein and ligand. Localising a small amount of protein on the chip surface rather than having a larger amount of protein distributed evenly throughout the sample volume means less depletion of the reservoir of unbound ligand when 50% of binding sites become occupied. Also in the SPR assay, the solution of ligand flows over the chip, making depletion of unbound ligand even less of a problem.
Tight binding is not usually a problem when screening fragments and the main reason for bringing up the subject was to get you thinking a bit about assays. There are a number of technologies for detecting the binding of fragments and quantifying the affinity with which they bind. This raises a couple of questions. Firstly, to what extent do we need new screening technologies for FBDD? Secondly, which weaknesses in the current methodology should be addressed with the highest priority?
Literature cited
Giannetti, From experimental design to validated hits: A comprehensive walk-through of fragment lead identification using surface plasmon resonance. Methods Enzymol. 2012, 493, 169-218. DOI
Showing posts with label surface plasmon resonance. Show all posts
Showing posts with label surface plasmon resonance. Show all posts
Friday, 3 February 2012
Sunday, 7 March 2010
Surface Plasmon Resonance
General Reviews
Rich & Myszka, Grading the commercial optical biosensor literature – Class of 2008: ‘The Mighty Binders’ J. Mol. Recognit. 2010, 23, 1-64 Link | Review
Application to Fragment Screening
Giannetti, From experimental design to validated hits: A comprehensive walk-through of fragment lead identification using surface plasmon resonance. Methods Enzymol. 2012, 493, 169-218. DOI
Perspicace et al, Fragment-Based Screening Using Surface Plasmon Resonance Technology, J. Biomol. Screen. 2009, 14, 337-349 DOI | Review
Binding Pathologies
Giannetti et al, Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors, J. Med. Chem. 2008, 51, 574-580 DOI | Review
Rich & Myszka, Grading the commercial optical biosensor literature – Class of 2008: ‘The Mighty Binders’ J. Mol. Recognit. 2010, 23, 1-64 Link | Review
Application to Fragment Screening
Giannetti, From experimental design to validated hits: A comprehensive walk-through of fragment lead identification using surface plasmon resonance. Methods Enzymol. 2012, 493, 169-218. DOI
Perspicace et al, Fragment-Based Screening Using Surface Plasmon Resonance Technology, J. Biomol. Screen. 2009, 14, 337-349 DOI | Review
Binding Pathologies
Giannetti et al, Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors, J. Med. Chem. 2008, 51, 574-580 DOI | Review
Thursday, 11 February 2010
Ligand protein interactions by SPR
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.
Literature cited
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
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.
Literature cited
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
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