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CSIRO s Wayne Leifert on Cell-Free Assays for GPCR Drug Discovery

Wayne Leifert
Senior Research Scientist

At A Glance

Name: Wayne Leifert

Position: Senior Research Scientist, Molecular Biosensing Project, Commonwealth Scientific and Industrial Research Organization (CSIRO), Division of Molecular and Health Technologies, Adelaide, South Australia, Australia, since 1988.

Background: Diploma in Medical Laboratory Science, University of South Australia; BSc, Flinders University; and PhD, University of Adelaide, 2001.

Wayne Leifert has been involved in broad areas of research investigating a biomarker of human hypertension in pre-hypertensive adolescents, as well as mechanisms of diabetic neuropathy. He has also investigated ion channels in cardiac myocytes and their modulation via fatty acids; and most recently, has been involved with setting up a cell-free GPCR biosensor project at Australia's CSIRO.

"Cell-free?" This is an unusual approach, since the recent increase in the use of cell-based assays in drug discovery has been especially apparent in GPCR screening. Many researchers believe that a cell-based assay is the only way to screen for GPCR modulators because of the complex signaling cascade within the cell that occurs.

According to Leifert, part of his cell-free project looks at screening a range of molecules that interact with G-proteins, and another aspect is designing GPCR biosensors incorporating nanotechnology approaches. He recently was lead author on a paper on this topic published in the Oct. 18 online issue of the Journal of Biomolecular Screening (on whose editorial board Leifert sits). Last week Leifert responded via email to some questions CBA News had about the paper and his project at CSIRO.

I am interested in this from the perspective of someone who has been covering the recent boom in cell-based assays, in particular high-content screening, for drug discovery. Obviously this has been a growing trend, particularly for GPCR-based drug discovery. So my first and really most important question regarding your paper is whether you, your colleagues, or CSIRO as an organization feel that cell-free assays are, or will be more conducive to GPCR drug discovery.

There appears to be segregation between groups claiming that one assay methodology is possibly more superior than another with reference to cell-based and cell-free or biochemical assay-type screening. I guess this healthy ongoing debate will continue in the future. We do not feel that either approach is superior to the other as a whole. Indeed, it is very much dependent on what information you are trying to gather from your system that will lead you to the correct choice of assay. Our research group feels that there is a strong need to develop technologies that would enable screening against multiple GPCR targets simultaneously. In a similar manner to that used with DNA arrays, where a multitude of information can be gathered, we envisage that the development of an array format for proteins such as GPCRs would enable higher-level screening capabilities. Admittedly, we still have a long way to go in terms of developing appropriate protein-compatible surfaces that would allow miniaturization to array formats, and therefore new methods for increasing the sensitivity of detection of signaling or binding events will also be required. A spin-off from this could also be the utilization of these technologies in the development of novel biosensors utilizing front ends containing biological receptor entities with a biomimetic or other transduction process. From our CSIRO perspective (with CSIRO being Australia's national research organization), there is the opportunity to bring together many different capabilities — for example molecular biology, materials science, surface chemistry, et cetera, to collaborate together in a manner that would be quite distinct from the approach taken by the pharmaceutical industry. Indeed we are now seeing the formation of research projects which embrace the theme of nanobiotechnology, and this has application in many areas, including the development of future drug discovery and biosensor assay platforms.

What are the major shortcomings of using a cell-based system for GPCR screening in a pharmaceutical discovery context?

It is likely that cell-free or biochemical-type assays will always remain an important adjunct to cell-based systems for GPCR screening in the pharmaceutical discovery context, particularly since there are ever-increasing methods available to miniaturize cell-based readouts such as high-density well plates. Nevertheless, it is apparent that the utilization of smart nano-biotechnologies is allowing us to work at the nanometer scale, which is the size of a protein molecule. Cell-based assays, due to the very nature of the size of the cell being examined, will not allow for this level of miniaturization. Perhaps another issue worth considering here is that cell-based assays require optimal culture conditions, proper handling, and issues around dispensing and storage of cells. Ideally, if we can develop appropriate arrays of proteins such as GPCRs, some of these issues may be overcome. Also, having multiple GPCR targets on a single array chip may allow screening which would be particularly important for low-volume test compounds. The multiplicity of targets in cells could lead to non-specific interactions which need to be subsequently confirmed as specific GPCR interactions or otherwise. Therefore, the targeted, cell-free, protein approach may give more information on specificity. However, as mentioned earlier, the cell-based secondary approach is a useful follow up for further profiling of primary specific hit compounds.

Many supporters of high-content screening have said that it's OK to sacrifice things like "assay density" and high throughput because of the rich information they get from their screens. Do you think this holds water in the context of GPCR screening?

As always, it is very much dependent on what you are trying to achieve. For example, the miniaturized cell-free approach would allow direct observation of other biologically relevant interactions such as protein-protein or peptide-protein interactions that may be useful not necessarily for drug discovery, but for discovery of novel signaling mechanisms. An additional point here may be with regard to allosteric modulators and/or compounds that might act on very early downstream events such as G-protein interactions or the interaction of G-proteins with regulatory proteins. Cell-free assays would overcome possible permeability problems and make accessible reaction surfaces that may not readily be available with a cell-based screening protocol.

Do you think the cell-based methods have the potential to be miniaturized and put on chip format just as well as cell-free assays?

Certainly. One could easily envisage single live-cell arrays that may exist on or within an appropriate substratum so that the isolated cells are conveniently arranged. Nanotechnologists are developing ways to keep cells alive on surfaces in predetermined array locations in order to attempt to make cell chips. To demonstrate this effectively I think that some of the main longer-term hurdles here might be cell attachment and storage or maintenance, somewhat similar issues that apply to cell-free array formats.

What in your mind is the most promising cell-free approach for GPCR screening?

I think future nanotechnologies are going to play an important role in GPCR screening. Questions are not just related to pharmacology and biochemical interactions, but are as diverse as 'How do you stick a protein on a surface and keep it in its active form?' And this is even more so the case with membrane-associated proteins such as the GPCRs. And how do you measure a ligand-activated GPCR protein? To answer these questions requires a lot of input from various scientific disciplines. Fluorescence methods are still routinely used and look like they will be used in the foreseeable future. However, if we can develop alternative, more sensitive, generic methods, that would be ideal. There is the possibility that nanoparticles or quantum dots may play an important part in the future with respect to signal transduction following receptor activation. There are many methods available to screen at the low-throughput scale that can provide very important information, but they may not be useful in a high-throughput program. So it is important when considering designing new approaches in GPCR screening that the process can be adapted to high throughput easily.

In reality, will drug-discovery organizations eventually move almost completely toward either cell-based or cell-free methods, or is there room for both within the budget of most organizations?

I cannot easily answer this question. However I would suggest that as would be expected with an established and proven technology for which the infrastructure and plant investment would be considerable, that the decision to entirely re-tool from the cell-based to the cell-free assay technology would not be taken lightly. One would probably expect that cell-free technologies would gradually find a niche and, depending on their comparative performance in drug discovery, de-orphanizing, and other parameters, may eventually be better embraced by pharma and biotech organizations.

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