Optogenetics applied to HTS

At Axxam, we have applied all the advantages of optogenetic technologies to the early phases of drug discovery, in order to build High-Throughput Screening (HTS) assays with significant improvements in content, precision and biological relevance.

Optogenetics' Principles

Optogenetics is one of the fastest growing and most promising technologies of recent years: the use of genetically encoded elements which activity 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.

We have developed cellular assays using either light actuators or light sensors to study several drug targets:

  • light-gated Channelrhodopsin-2 was used to modulate NCX1 sodium-calcium exchanger as well as CaV1.2, CaV1.3 or CaV3.3 calcium channels and to identify state-dependent blockers
  • light activated bPAC adenylyl cyclase was used to stimulate HCN2 channel
  • genetically encoded indicators for voltage or calcium were used for single- or multi-color detection of KIR2.3, TMEM16A, NaV1.5, CaV1.2, CaV3.3, ASIC, TRPM2 and muscarinic receptor activity

All of these assays were fully miniaturized in a 384-well format and completely HTS adapted using the FLIPR Tetra® (Molecular Devices) instrumentation, providing innovative drug screening tools of improved biological significance and effective operational costs and timelines.

FLIOP – Fluorescence Imaging and Optical Pacing

Our researchers and engineers have also developed FLIOP – “Fluorescence Imaging and Optical Pacing” (patent pending), a system that provides the FLIPR Tetra® instrument with a programmable, millisecond-switched optogenetic pacing, enabling for the first time simultaneous 384-well optical pacing, fluorescence imaging and compound addition.

FLIOP allowed to synchronize and repeatedly activate diverse cellular processes: voltage-gated calcium channels, voltage-gated sodium channels and cardiac action potential of iPS-derived cells were synchronously stimulated by light-gated Channelrhodopsin-2 at different repetitions and frequencies, enabling the study of blockers effect and use-dependency under normal or pathological firing conditions.

With FLIOP, High-Throughput Screening campaigns and Hit-to-Lead activities can be run with the enriched biological relevance of electrophysiology-like investigations while maintaining efficient operational costs and timelines. From the beginning of the drug discovery program, FLIOP can deliver high quality hits, avoiding the selection of un-relevant and false active compounds often resulting from the use of un-reversible and un-physiological chemical stimuli.

 

Optogenetics in Electrophysiology

In addition, to complete the characterization of cell lines expressing optically controlled ion channels, we have also setup an “Opto manual patch-clamp platform”. The system integrates a broad-spectrum LED illumination device that allows to deliver light pulses of programmable intensity, duration and wavelength while performing voltage- or current-clamp experiments.

With this setup, we successfully check the purity of in-house engineered clones stably expressing light-gated channel (for instance: Channelrhodopsin-2) alone or together with voltage-, ligand-gated ion channels (for instance: NaV1.5).

This activity is mandatory not only to confirm the results obtained with the FLIPR Tetra® functional flux assay but also to establish a strong correlation between these techniques used along the drug discovery cascade.

How can we support your research programs?

We offer construction of optogenetic cell lines (both with light actuators and light sensors), which can be transferred to the customer or run as part of contract research studies at Axxam, including HTS campaigns with customer’s and/or Axxam compound collections.

Do you need more information on optogenetics?

To learn how optogenetics has revolutionized the drug screening world, read the following articles:

H. Zhang et al., Trends Biotechnol (2017)

V. Agus et al., Curr Opin Biotechnol (2017)

V. Agus et al., SLAS Discov (2018)