Axxam researchers have developed or optimized innovative technologies and tools to successfully overcome the many challenges faced during the development of HTS assays for drug discovery programs:
- Optogenetics technology: the use of genetically encoded elements which activity can be controlled by light to modulate the functionality of a gene of interest in a more precise, reversible and physiological way compared to the use of chemicals
- Photina® and i-Photina® (proprietary technology): Ca2+ activated photoproteins optimized for the generation of HTS-assays for targets correlated to the intracellular increase of Ca2+
- chAMPion technology (proprietary technology): a universal GPCR reporter cell line capable of measuring the functional activation of any transfected GPCR, and a real time assay for cAMP and cGMP level variation
NOTE: proprietary technologies are available from Axxam for licensing.
In addition, other novel, non-proprietary technologies and assay platforms have been developed or optimized by Axxam’s researchers to further improve the performance of cell based assays:
- Fluorescent Calcium BioSensor: a fast, reliable, non-toxic and cost-efficient fluorescent system to detect intracellular calcium variations
- Novel and alternative chemical and biological fluorescent readouts for ion flux
- Odorant receptors platform
- Genome editing approach for the generation of stable cool and smart cell line development
- Epigenetics: platform for compound testing
- Assays for Enzymatic Complexes constituted by multi-protein and/or multi-subunits
Optogenetics is one of the fastest growing and most promising technologies in recent years: the use of genetically encoded elements with activity that can be modulated by light has in fact completely revolutionized the manipulation and investigation of biological processes. Optogenetic tools can provide significant improvement compared to the classical “chemical” approaches achieving a precise control of many cellular pathways and allowing the modulation of single biological processes in a not-invasive and reversible manner, with a milliseconds timescale, and a cellular specificity in a way you cannot reach with the classical drug application, or even with electrical stimulation.
Optogenetic technologies combine the two fundamental concepts of spatial resolution and temporal resolution, to obtain a technique able to control a cellular response in a fast and repetitive way, acting only on a specific gene, in a specific cell line or cell compartment.
Axxam’s researchers have applied all the advantages of optogenetic technologies to the early phases of drug discovery, in order to build HTS assays with significant improvements in content, precision and biological relevance.
Axxam’s researchers have developed cellular assays using light activated proteins to modulate ion channel activity: light-gated Channelrhodopsin-2 was used to modulate CaV1.3 calcium channel and to identify state-dependent blockers; light activated “bPAC” adenylyl cyclase was used to stimulate HCN2 channel. Axxam’s researchers also developed cellular assays using genetically encoded indicators for Voltage or Calcium, generating a multi-color, multi-parametric recipient cell line suitable for the HTS of different targets and so significantly improving the content of the screening experiments, while considerably cutting costs and timelines.
All these assays where fully miniaturized in a 384-well format and completely HTS adapted using the FLIPR instrumentation, providing powerful and innovative tools for the identification of drug molecules.
Axxam offers construction of optogenetic cell lines (both with light actuators and light sensors), which can be transferred to the client or run as part of contract research studies at Axxam.
Literature can provide you with more information on how optogenetics have already revolutioned drug screening (read an example).
Axxam’s researchers have developed the two proprietary Ca2+activated photoproteins optimized for the generation of precise Ca2+ mobilization assays for High-Throughput Screening (HTS). The expression and functionality of Photina® and i-Photina® have been validated in most of the cells frequently used in HTS, such as CHO, HEK293 and Jurkat, and using several flash luminescence plate readers such as – FLIPR3, FLIPRTETRA, CyBi® Lumax, Lumilux.
Photina® and i-Photina® are very well suited to measuring GPCR activation and are ideal for any other cellular targets capable of increasing the intracellular Ca2+ concentration such as Ca2+permeable ion channels or Na+/Ca2+ exchangers.
The chAMPion assay platform is specifically designed to offer a simple, homogeneous, universal, and fast system for the detection of any GPCR signalling. chAMPion technology is based on the stable co-expression of a Ca2+ activated photoprotein and of a cyclic nucleotide-gated (CNG) channel acting as a cAMP biosensor. chAMPion Technology is offered in two cellular backgrounds:
- CHO-K1 (Hamster)
In the chAMPion system, the CNG channel has been modified to display sensitivity to cAMP levels in the physiological range. The calcium influx through the opened channel is immediately detected by the recording of the photoprotein-emitted flash type luminescence signal.
The chAMPion cell line can measure the activation of a transfected Gαq-coupled receptor by direct detection of Ca2+ ions released from internal cellular stores through activation of the phospholipase C (PLC) pathway.
Additionally, the chAMPion cell line system can monitor the activation of transfected Gαi or Gαs-coupled receptors, which elicit changes in 3,’5′-adenosine cyclic monophosphate (cAMP) levels, which in turn are responsible for CNG opening and consequent Ca2+influx.
chAMPion Technology Advantages
- Rapid measurement of receptor activity after compound addition (within seconds)
- Intact live cell “Real-Time” assay for sensitive detection of cAMP
- Kinetic readings
- Signal detection by luminescence (reduced false positives hit-rate)
- Detection of GαiPCRs antagonist as on-signal
- Well suited for HTS
- Useful for “orphan” GPCRs screening
chAMPionTechnology represents an assay platform for several target classes:
- cGMP and cAMP-Phosphodiesterases
- Nitric Oxide Synthases
- Any Ca2+ permeable Ion Channel or Ion Exchanger