The scientist who co-founded drug-discovery firm Odyssey Thera and invented its core protein-fragment complementation assay for screening intracellular protein-protein interactions has published research demonstrating the enhanced sensitivity of a Gaussia luciferase-based version of the assay.
Despite the advantages of the new assay – namely, it is fully reversible and sensitive even at low cellular protein levels – it likely will not displace the fluorescent protein and Renilla luciferase-based versions of PCA currently used in Odyssey’s cell-based drug-discovery programs, an executive from the company said this week.
However, it is likely that Odyssey, whose intellectual property portfolio covers the new technology, will adopt it as it begins to develop new PCAs for in vivo imaging applications, he said.
The PCA technology was invented by Stephen Michnick, co-founder of Odyssey Thera and current head of its scientific advisory board. In PCA, two intracellular proteins of interest are fused to complementary fragments of a reporter protein. If the proteins interact, the reporter comes together to produce a detectable signal.
The original PCA was based on a fluorescent reporter protein such as GFP, and this is still the most common version of the assay. Odyssey has also successfully tested a version of the assay based on the well-known Renilla luciferase, which confers some advantages over fluorescence in terms of sensitivity, but does not provide sub-cellular localization information.
In a paper published in the December issue of Nature Methods [2006 Dec; 3(12): 977-9], Michnick, who is also a professor of biochemistry at the University of Montreal, described a new version of PCA based on a humanized version of luciferase derived from the copepod Gaussia princeps, also known as hGLuc.
Like Renilla luciferase, or hRLuc, hGLuc catalyzes the oxidation of luciferin to produce a detectable blue light. However, hGLuc generates 100-fold higher bioluminescence than hRLuc, which suggests it would make an even more sensitive PCA reporter.
Michnick provided evidence in the paper that hGLuc’s higher bioluminescence signal indeed resulted in a more sensitive PCA, and that it was able to detect interactions between proteins expressed at far-below-endogenous cellular levels.
Furthermore, Michnick demonstrated that the hGLuc assay was reversible. This is important because a perceived disadvantage of fluorescent-based PCA is that once the fluorescent protein complex is reformed because of a protein-protein interaction, it will not re-split into fragments even if the proteins in question would normally dissociate.
“Fluorescent proteins … must be expressed at high levels to assure that the signal is above background cellular fluorescence, and fluorescent protein PCAs are irreversible, which can be useful but also can lead to misinterpretation of turnover or localization of interacting proteins,” Michnick wrote in the paper. “We thus sought to develop a PCA that is sensitive enough that component proteins could be expressed at or below endogenous levels and for which the PCA reporter does not trap complexes. The G. princeps luciferase-based PCA presented here meets these two criteria.”
Despite the evidence presented by Michnick, it appears unlikely that Odyssey will incorporate the new technology in its proven cell-based PCA assays, which serve as the basis for its internal drug-discovery programs and contract drug-discovery and assay development programs.
According to Odyssey President and CSO John Westwick, luciferase-based PCAs in general are highly sensitive but tend to be inferior for the types of high-content drug-discovery screens Odyssey is conducting.
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“Fluorescence PCAs may be less reversible than luciferase … but that is more than compensated for by the large increase in spatial [and] high-content information you gain from fluorescence-based PCAs. |
“Luciferase has the advantages [of] low background, mainly, but the key factor is that you don’t get sub-cellular localization [information],” Westwick wrote in an e-mail to CBA News. “The fluorescence PCAs may be less reversible than luciferase – though not, as Michnick states, ‘irreversible’ – but that is more than compensated for by the large increase in spatial [and] high-content information you gain from fluorescence-based PCAs.”
In addition, Westwick wrote, “a significant portion of biological events measured by PCA are not highly dynamic in terms of the level of the protein complex, but are highly dynamic in terms of complex localization,” with the interaction between Akt and NFκB being classic examples.
“So in the balance, except for in vivo applications, we’d still lean towards fluorescence PCA as the most powerful approach overall,” Westwick wrote.
Despite this, Westwick conceded that the higher activity of hGLuc demonstrated in the Nature Methods paper could be advantageous in some cases, but that “in terms of expressing low levels of proteins and obtaining a detectable signal, we were already achieving that with Renilla.”
A PCA based on hGLuc may eventually have the greatest impact in small-animal imaging applications for drug discovery. Odyssey currently does not incorporate small-animal PCAs in its research program, but does have some interest in this area, Westwick wrote.
“It’s always better to express the lowest possible levels [of proteins], so it is likely that we will employ Gaussia when we engineer additional PCAs for in vivo imaging,” Westwick wrote in his e-mail.
Because of the reversible nature of hGLuc-based PCAs, this may eventually allow scientists to use small-animal imaging instrumentation such as Caliper’s Xenogen offers to detect protein complex formation and disassembly in living animals in real time.
Westwick noted that all forms of PCA, including those that utilize any type of luciferase, are protected by Odyssey’s patent estate.