15 February 2010

Surface Plasmon Resonance (SPR)

Fragment-based drug discovery took off with NMR in the 1990s and went mainstream with X-ray crystallography in the 2000s. Now surface plasmon resonance (SPR) is becoming increasingly popular as a primary means of identifying hits. The technique has been mentioned more than a dozen times on Practical Fragments, but we’ve never devoted an entire post to it until now.

This post follows up on two recent publications. The first is an excellent summary of SPR by our friends at FBDD-Lit. Peter Kenny gives an overview of the technique and reports on a workshop given by SPR mavens Dave Myszka and Rebecca Rich. He also covers some of the seminal papers in the field.

The second report is in the brand new journal ACS Medicinal Chemistry Letters. In it, Iva Navratilova and Andrew Hopkins of the University of Dundee provide practical advice on using SPR for fragment-screening.

The authors describe their work on using SPR to identify fragments that bind to carbonic anhydrase II, a popular target for proof-of-concept studies. They screened a library of 656 fragments with molecular weights between 94 to 341 Da, with an average of 187 Da, or about 13 non-hydrogen atoms. The entire screen, which was done at three concentrations (16.6, 50, and 150 micromolar) took 4 weeks from assay development to hit confirmation on a Biacore T100, and consumed a total of 27 micrograms of protein.

Importantly, Navratilova and Hopkins were keenly aware of the potential for false positives or nonspecific binders (of which there were 230 at the highest concentration!) One way they controlled for such artifacts was to include an unrelated reference protein; data could be corrected by subtracting the response to the reference protein from the response to the target protein. Another analytical method to reduce the number of false positives was to only consider compounds that exceeded a minimum threshold for ligand efficiency (a metric invented by Hopkins and co-workers), a decision justified here given the often high affinities observed for carbonic anhydrase inhibitors. After these filters, an examination of the stoichiometry of binding revealed a dozen specific binders and four non-specific binders, a hit rate of 1.8%.

My one reservation with this paper is that carbonic anhydrase is a particularly easy test case, unlikely to fairly represent many of targets that people screen. Indeed, the confirmed hits (all of which contain sulfonamides), have affinities from 0.13 to 14 micromolar – far better than a typical fragment screen, and comparable to many HTS screens. Still, the tools and analyses described should apply to more challenging targets.

Finally, it is worth noting that if you want access to SPR technology but don’t have the resources or expertise to do it yourself, at least a couple companies (Beactica and Graffinity) specialize in applying SPR to FBDD.

2 comments:

KineticDiscovery said...

Kinetic Discovery, founded by Iva Navratilova and Andrew Hopkins, is a new company that offers SPR fragment screening services.
http://www.kineticdiscovery.com

Sriram Kumaraswamy said...

SPR is referred to almost exclusively in the few blogs that discuss fragment screening using biophysical techniques. Other methods such as ForteBio's Biolayer interferometry (BLI) are also in use for screening fragments.
BLI is also a label-free direct binding method that employes the target ligand on a biosensor surface and probes fragments in solution. BLI in fact offers higher throughput compared to SPR, employs a Dip and Read method where the biosensors dip into analyte samples in microwell plates (no microfluidics) and probes 16 samples at once.
You can catch some expert presentations on BLI for fragment screening at SBS conference in April this year.
John Wang, until recently at Novartis, Emeryville will talk about small molecule and fragment screening. Roche will present their work with BLI for fragment screening in a poster:
POSTER B209
Rapid Fragment Screening with a Robotically Automated ForteBio Octet RED384
Frank Podlaski, Charles Wartchow, Lin Gao, Shirley Li, Sean Walker, Sevan Ibabekci, Kuo-Sen Huang, David Mark
Discovery Technologies, Roche Inc.