Category: High-Definition Biotechnology

1063-D - HTS-compatible optogenetic tools to assess cardiac toxicity in early phase of drug discovery

Tuesday, February 6, 2018
5:00 PM - 6:00 PM

A key step in the drug discovery process is the assessment of potential drug-induced cardiac toxicity. The recent development of induced-pluripotent stem cells-derived cardiomyocytes (iPS-CM) technology allows the possibility to study in-vitro cardiac targets in a human genetic background. On the other hand, since cardiac function strongly depends on beating frequency, state and use-dependency of drugs might have different impact on the overall toxicity. Therefore, it is essential to develop cost-effective in-vitro screening assays that can be used in an early stage to verify the use- and state-dependency of new molecules on human cardiomyocytes over the physiological frequency range (60 - 180 bpm). The use of light-sensitive proteins, such as channelrhodopsins, represents a new powerful alternative to commonly used systems to control beating frequency (i.e. electric field stimulation), being able to control cardiac excitation-contraction coupling with high spatial and temporal precision compatible with the needs of the HTS format.
Here, we present our new HTS-compatible platform based on the use of optogenetic to study cardiac-relevant targets expressed in both iPS-CM and/or heterologous systems.
In particular, we manage to obtain an optogenetic pacing of iPS-CM by co-culturing these latter with ChR2-expressing HEK-293 cells in 384-well MTP format. By custom application of light of two different wavelengths (one for ChR2 activation and one for probe excitation) we measured the effect of different stimulation frequency on calcium transients detected by standard fluorescent calcium dye. The “opto-syncytium” approach allows the possibility to analyze the use- and state- dependency of a drug in a cost-effective and patient-specific background in the early phases of drug screening.
Another novel application of our platform is the measurement of the electrogenic transporter NCX1 (sodium-calcium exchanger) reverse-mode activity suitable for HTS assays. Since NCX1 is a relevant modulator of cardiac calcium homeostasis, it is a promising target for drug discovery and cardiac safety, but the available assays are still not specific and involve complex solution exchange protocols. As a first proof of concept we have co-expressed ChR2 and NCX1 in HEK-293 cells and confirmed the functional presence of both proteins with standard protocols validated in-house, such as the measurement of blue-light induced depolarization and the measurement of calcium influx following the “sodium switch” protocol. Once we obtained a pure clone, by monitoring intracellular calcium, we were able to demonstrate the activation by light of NCX1, due to intracellular sodium load and partial membrane depolarization following ChR2 activation.
In conclusion, our integrated optical HTS-compatible platform represents a new promising tool to evaluate potential cardiotoxicity of molecules in the early phase of drug discovery, in both patient-specific and heterologous backgrounds.

Viviana Agus

Principal scientist
Axxam SpA
Milan, Lombardia, Italy

Dr Agus is responsible for the development of cellular-based assays for High Throughput Screening since 2001. She has broad experience on different cellular targets: GPCRs, nuclear hormone receptors, transporters, but mainly in many different ion channel classes (voltage-gated, ligand-gated, passive cation or anion channels). She developed and optimized functional assays either in a recombinant or native configuration, using constitutive or inducible expression system and state of the art Fluorescent and Luminescent readouts. In the last two years she has been fully dedicated to the setup of a platform of Optogenetic assays suitable for light-controlled High Throughput Screening, in particular using either Channelrhodopsins or genetically encoded voltage indicators for the study of ion channel target activity using FLIPR instrumentation. She obtained her graduation in Biological Sciences at the “University of Milan”. After she did her apprenticeship at “National Institute of Tumours of Milan”, then she joined Axxam in 2001.