New screening technique uses fluorescence to shed light on the activities on little-understood proteins
Ned Stafford/Hamburg, Germany
Researchers have developed a new high-throughput screening (HTS) technique that could shed light on the biochemical activities of numerous proteins about which little is currently known.
Benjamin Cravatt, part of the research team based at the Scripps Research Institute in La Jolla, California, says the new method combines Activity-Based Protein Profiling (ABPP), a technique he helped develop several years ago, with fluorescent activity-based probes.
The new technique, dubbed fluopol-ABPP, will make it easier to find molecules that proteins interact with and inhibitors that might block protein activity, Cravatt told Chemistry World.
’Our initial applications of competitive ABPP were in the area of secondary assays to assess the selectivity of lead inhibitors emerging from more conventional HTS assays,’ he says. ’However, we quickly came to realise that, when combined with fluorescence polarisation, ABPP could be used as a primary HTS assay, which would open up a huge fraction of the uncharacterised proteome to inhibitor screening.’
The fluopol-ABPP technique uses fluorescent probes to tag proteins. A probe attaching to a protein results in a larger molecule with higher fluorescent polarisation values. When an inhibitor binds efficiently with a protein, it can overshadow a probe, slowing or eliminating a rise in fluorescence polarisation, indicating inhibition.
Unlike ABPP, which relies on laborious gel and mass spectrometry techniques, fluopol-ABPP does not require separation or washing steps, so is readily incorporated into automated high-throughput screening systems.
The Scripps team has already used fluopol-ABPP to successfully study two enzymes from different classes: retinoblastoma-binding protein-9 (RBBP9) and glutathione S-transferase omega 1 (GSTO1). Both are thought to play a role in some cancers, but researchers had made little progress in learning the focus of their biological activity.
In what could be considered a modest oversimplification, Cravatt describes the technique as ’pretty much a simple joining of the two technologies. One of the great features of the assay is that it is minimalistic in composition - all you need is a purified enzyme of interest and the cognate activity-based probe.’
Cravatt believes it might be possible to further refine the fluopol-ABPP technique to enable screening of ’unpurified’ enzymes, making it useful for membrane enzymes. ’It would be wonderful to be able to perform the assay on partially purified enzymes, since the principal bottleneck to its broad application is access to purified preparations of proteins,’ he says.
Robert Damoiseaux, scientific director of the Molecular Shared Screening Resources at the University of California, Los Angeles, called fluopol-ABPP as presented in the paper ’a huge step forward,’ adding that he is cautiously enthusiastic about the team’s findings.
Damoiseaux highlighted several sticking points, however. These included the fact that the method has an intrinsic time-constraint and that the fluorescence polarisation is very dependent on ambient temperature (which could mean less stable readings) and the loss-of-signal assays used tend to be less stable than gain-of-signal screens.
’Being able to screen a disease-modifying enzyme for which so far no substrate is available is a very strong asset in the arsenal of the high-throughput screener,’ Damoiseaux told Chemistry World. ’Technically speaking, the demonstrated methodology is powerful, and appears to be simple and robust enough for high-throughput screening. However, the devil is in the detail and a few questions remain open and only time - and a few screens run using this technology - will tell.’
et al., Nature Biotechnology, 2009. DOI: 10.1038/nbt.1531