Illuminating Activity - In Vivo and In Vitro

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Is there a correlation be between in vitro and in vivo performance?

In vitro cell culture has been a powerful tool for shedding light on cellular processes, but behavior in vivo—in the context of the cell’s native environment within the living organism—can be quite different. This difference is one of the many factors that can plague high-throughput drug screening efforts, as candidate compounds that perform well in vitro can show little to no activity in vivo.

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But what if the same quantitative in vitro cell assay could also be used in vivo?

Not only would readout be fast, but a direct correlation could be drawn between in vitro and in vivo performance. In addition, pharmacokinetic and pharmacodynamic studies would be easier to conduct and evaluate.

Above: Wild type HEK293 cells mixed with cells stably expressing SSTR2 and β-arrestin fused to split ELuc fragments. Courtesy of Mitsuru Hattori, The University of Tokyo.

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Did you know?
A recent model of the drug discovery and development process calculated overall attrition rates at 95 %, from discovery to approval. Total cost per drug: US $1.8 Billion.1
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Hamamatsu cameras in drug discovery
Researchers such as Kraft, et al,2 are using Hamamatsu cameras for cell based drug discovery. See how—search for Hamamatsu at PubMed Central.
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Takakura, et al,3 in the lab of Takeaki Ozawa, demonstrate the power of such a system.

This was accomplished by using a yellow light-emitting split firefly luciferase construct to assess GPCR activity in plate assays, live single-cell microscopy assays, and within the livers of intact, living mice.

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Live Cell Imaging Conditions

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20x dipping objective
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Upright bioluminescence microscope (BX61, Olympus Corp.)
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Emission long-pass filter
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ImagEM-1K EM-CCD Camera (Hamamatsu Photonics K.K.), cooled to -80°C
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Read the Paper (login may be required)
Takakura, H., Hattori, M., Takeuchi, M. & Ozawa, T. Visualization and quantitative analysis of G protein-coupled receptor-β-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation. Acs Chem. Biol. 7, 901–910 (2012).
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The biggest issue Takakura, et al,3 had to overcome was signal—

How do you non-invasively detect a signal generated from organs deep within the body? And using a system that can also provide quantitative information—such as IC50—in plate and single-cell assays?

While zebrafish researchers have the advantage of a transparent larval stage, enabling excitation and detection of fluorescent probes, fluorescence is not as straightforward for imaging in most intact organisms.6

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Instead of fluorescence, Takakaura, et al,3 turned to the less cytotoxic luminescence-producing split luciferase systems.6

With these constructs, the N and C termini of the luciferase protein are translationally fused to the reporter proteins—in this case the GPCR β2-adrenergic receptor (ADRB2) and its intracellular binding partner β-arrestin (ARRB2). When ADRB2 is activated, ARRB2 binds, the two luciferase halves are brought together, and bioluminescence is produced.

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Using a split firefly luciferase (North American Photinus pyralis) with λmax = 558 nm, Takakura, et al,3 showed detection of ADRB2 activiation in plate assays, single-cell microscopy assays using live cells, and in vivo assays, imaging bioluminescence originating in the livers of anesthetized mice and visualized with an external camera.

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Above: The split luciferase assay. The GPCR β2-adrenergic receptor (ADRB2) fused to the N-terminal luciferase domain and it’s cytoplasmic partner β-arrestin fused to the C-terminal luciferase domain. Upon binding and activation of ADRB2, β-arrestin binds, allowing complementation of intact luciferase, leading to bioluminescence.

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Technologies for Biology
CCD, EM-CCD, sCMOS—what do these different technologies mean for biological experiments? Find out.
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A better understanding bioluminescence.

While bioluminescence has been used to track tumors near the surface of nude mice, Takakura, et al,3 show it’s effectiveness at reporting on events occurring inside the mouse. In addition to speeding the path between screening hit to in vivo testing, this approach can also enable basic researchers to better understand the relationship between cultured cell models and the native system.

“Despite their tractability as drug targets, the majority of GPCR-based drug discovery programs have failed to yield highly selective compounds. The traditional approach to drug discovery has been to target the endogenous ligand (orthosteric)-binding site, to either mimic or block the actions of the endogenous neurotransmitter or hormone in a competitive manner. However, this approach has suffered from a paucity of suitably subtype-selective ligands.”

Karen J. Gregory, Elizabeth N. Dong, Jens Meiler, and P. Jeffrey Conn.
Allosteric Modulation of Metabotropic Glutamate Receptors: Structural Insights and Therapeutic Potential. Neuropharmacology. 2011 January; 60(1): 66-81. PMCID: PMC2981682.

”The existence of functional GPCR heteromers represents both a major challenge and an opportunity for neuroscience and drug discovery. The number of GPCRs for which evidence suggests a functional role of heteromerization is rapidly increasing. Given the large number of GPCR genes, their ability to form combinations raises the daunting possibility that there are tens or even hundreds of thousands of unique receptor heteromers in the brain and nervous system. Elucidating the function of each of these potential distinct receptor complexes represents a considerable challenge.”

Laura Albizu, Jose L. Moreno, Javier Gonzalez-Maeso, and Stuart C. Sealfon. Heteromerization of G Protein-Coupled Receptors: Relevance to Neurological Disorders and Neurotherapeutics.
CNS Neurol Disord Drug Targets. 2010 November 1; 9(5): 636-650. PMCID: PMC3066024.

“Therefore, in order to advance our fundamental mechanistic understanding of signal transduction in and on the plasma membrane, a means to accomplish exact quantification by accurately counting the number densities of receptors and obtaining a full description of the dynamic equilibrium between receptor monomers and dimers must be developed.”

Rinshi S. Kasai, Kenichi G. N. Suzuki, Eric R. Prossnitz, Ikuko Koyama-Honda, Chieko Nakada, Takahiro K. Fujiwara, and Akihiro Kusumi.
Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging. J Cell Biol. 2011 February 7; 192(3): 463-480. PMCID: PMC3101103.


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  2. 02. Kraft, R. et al. A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions. Dis. Model. Mech. 6, 217-235 (2013).
  3. 03. Takakura, H., Hattori, M., Takeuchi, M. & Ozawa, T. Visualization and quantitative analysis of G protein-coupled receptor-β-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation. Acs Chem. Biol. 7, 901-910 (2012).
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  7. 07. Kevles, B. Naked to the Bone: Medical Imaging in the Twentieth Century. (Rutgers University Press, 1997).
  8. 08. Sweet, W. H. The Uses of Nuclear Disintegration in the Diagnosis and Treatment of Brain Tumor. N. Engl. J. Med. 245, 875-878 (1951).
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  10. 10. Lauterbur, P. C. Magnetic resonance zeugmatography. Pure Appl Chem 40, 149-57 (1974).
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