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Photina® is an improved Ca2+ activated
photoprotein optimized for the generation of precise Ca2+ mobilisation assays for High-Throughput Screening (HTS). Developed by Axxam researchers from their extensive experience in photoprotein biology and screening assays, Photina® is validated for use with a wide range of plate readers and is commercially available from PerkinElmer through an exclusive license granted by Axxam for worldwide commercialization.
While the mechanism of action of Photina® is similar to other photoproteins, the total light release is greater and the reaction kinetic is slower - opening new opportunities for the application of flash
luminescence assays in HTS. The inherent flash luminescence of Photina® offers several advantages compared to classical Ca2+ fluorescent dyes:
- low background levels result in a large signal to noise ratio
- Ca2+ concentrations can be measured at specific cellular sites
- tested compounds only require short incubation periods
- reaction kinetics can be followed.
Axxam scientists tested the expression and functionality of Photina® in various cell lines most frequently used in HTS, such as CHO, HEK293 and Jurkat cells. Results demonstrate that the expression of the protein is stable over time, does not affect cell growth and viability and no toxicity is detected - even after long periods of cell culture.
Photina® has been targeted to different cell compartments such as plasma membranes and mitochondria. The choice between cytoplasmic or mitochondrial localization of Photina® greatly expands the range of targets addressable by the use of flash luminescence. Localization of a Ca2+ activated photoprotein within the mitochondria has the advantage of amplifying the luminescent response triggered by the activation of GPCRs coupled to the Gαq/PLC pathway.
While Photina® is well-suited to measure GPCR activation, it also is ideal for any other cellular targets capable of increasing the intracellular Ca2+ concentration. Photina® has been validated for addressing other important drug discovery target classes, such as Ca2+ permeable ion
channels or Na+/Ca2+ exchangers. Members of these target classes have been transfected in CHO cells that express either the mitochondrial or the cytoplasmic version of Photina® and results clearly demonstrate that the functional activation of these targets can be detected easily and reliably by Photina® using a range of flash luminescence plate readers - FLIPR3, FLIPRTETRA, CyBi® Lumax, Lumilux, CCD camera - and with different MTP formats (96, 384, 1536).
In all tested conditions, the very high light intensity generated by Photina® allows
the use of very few cells/well versus the larger requirements of most other photoproteins
and HTS protocols. The signal intensity obtained with 250 cells/well is strong
enough to guarantee reliable signal detection. Photina® is
therefore particularly attractive for HTS campaigns involving large compound collections since the required production quantity of the cellular assay material
can be reduced significantly. An average Z' factor value of 0.7 for most of the
tested assays also provides the required robustness for application to HTS.
Photina® is covered under the granted European patent EP1413584 entitled "Photoprotein with Improved Bioluminescence" and corresponding patent applications and issued patents elsewhere.
In May 2007, Axxam granted an exclusive global license to PerkinElmer whereby PerkinElmer became the exclusive provider of the Axxam Photina® photoprotein technology to the drug discovery market.
Axxam retains the rights to use the technology for its discovery services to third parties, as assay development, high-throughput screening (HTS) and compound profiling, as well as for any further application of development of this technology.
The terms of the agreement also provide for a formal research and development program between Axxam and PerkinElmer to develop additional Photina® GPCR and ion channel cell lines for use in high throughput screening and compound profiling.
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