Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, in particular tools for all-optical electrophysiology, now provide a means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline - from target identification and validation, to target-based and phenotypic screens, to clinical trials.
drug discovery; neuroscience; optogenetics; optopatch; phenotypic screening.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Conflict of interest statement
Conflict of Interest Statement: AEC is a founder of Q-State Biosciences. HZ is an employee of Q-State Biosciences.
Figure 1. Key Figure. Optogenetics throughout the discovery pipeline
Optogenetic assays can contribute at each stage of drug discovery. A) Comparative measures on rodent or human neurons +/- a disease-causing mutation can establish a screenable phenotype. Alternatively, optogenetic screens of genetic knockouts can identify novel targets which can then be the subject of optogenetic or conventional drug screens. B) Targets identified in culture-based assays should be validated via knockout and functional measurements in acute slice, and ideally
in vivo. Optogenetically triggered behaviors can provide a low-variance phenotype for testing the effects of mutations. C) Target-based optogenetic screens can be performed in a wide variety of cell-based assays. Alternatively, phenotypic optogenetic screens can identify compounds that modulate a disease-relevant functional phenotype regardless of mechanism. D) Screening hits should be validated in the same assays used for target validation. In addition, optogenetic assays in human iPSC-derived cardiomyocytes can help with cardiotoxicity assessment. E) Functional optogenetic measurements in human iPSC-derived neurons can seek to distinguish likely responders from non-responders prior to a clinical trial; or to match genetically heterogeneous patients with existing medications.
Figure 2. Optogenetic high-throughput target-based screens
A) Screens for state-dependent modulators of Na
V channels can be carried out in Optopatch spiking HEK cells . B) Screens for state-dependent modulators of Ca V channels use the gradual recovery of a step function channelrhodopsin, ChR2(D156A),  to induce a slowly varying membrane voltage . C) Screens for modulators of cyclic nucleotide gated (CNG) channels use a photo-activated adenylyl cyclase, bPAC,  to induce a step in cAMP concentration. D) Screens for modulators of receptor tyrosine kinase (RTK) signaling use a light-activated RTK and a transcriptional readout. 
Figure 3. Optical high-throughput screen of sodium channel modulators
A) HEK293 cell engineered for optogenetic studies on Na
V1.7. The cell stably expressed the test channel, Na V1.7, and K ir2.1 to lower the resting potential to near the K + reversal potential. The blue light-activated ion channel CheRiff induced action potentials upon blue illumination. Fluorescence of the voltage indicator QuasAr2 reported the dynamics. B) Optically induced and optically recorded spikes followed the stimulus pattern in the absence of drug, but in the presence of a state-dependent blocker, lidocaine, the spikes failed at high stimulus frequency (arrows). Scale bar 1 s. C) All-optical screen of 320 FDA-approved compounds for use-dependent block. Each response was characterized by the overall decay in the spike amplitude (use-dependence index) and the standard deviation in the spike amplitude. State-dependent blockers showed clear functional clustering. Image adapted from .
Figure 4. Optogenetics-enabled phenotypic assays
A) High-throughput measures of excitability in cultured neurons can characterize rodent and human iPSC-derived neurons. Here Optopatch recordings are shown from human iPSC-derived motor neurons comparing a wild-type genotype with cells containing an ALS-causing mutation, SOD1(A4V). The mutation causes differences in spontaneous and optogenetically induced firing patterns. Figure adapted from . B) All-optical measures of synaptic transmission can identify mutations or compounds that modulate excitatory or inhibitory signaling in cultured neurons. Here a single rat hippocampal neuron is stimulated with patterned illumination, and excitatory post-synaptic potentials are recorded in a neighboring cell. Figure adapted from . C) All-optical measures of excitability in acute brain slice can identify characteristic firing patterns in genetically defined neuronal sub-types in defined brain regions. Here an Optopatch recording from a somatostatin-positive interneuron is overlaid on an image of a brain slice expressing Optopatch under control of somatostatin-Cre. Figure adapted from . D) A mouse expressing channelrhodopsin 2 in its sensory neurons exhibits nocifensive behavior upon blue light illumination of its left hindpaw (left), while a control mouse does not (right). Figure adapted from .
Figure 5. Optogenetic excitability assays can be used throughout the discovery pipeline
A) Target identification. Each well contains neurons with a distinct genetic perturbation (e.g. gene knockout). Optogenetic measurements can identify genetic modulators of neuronal excitability. B) Target-based screen. Each well contains a mixture of cells expressing and not expressing a specific drug target, and a fluorescent marker to distinguish the populations (green). Optogenetic excitability measurements are performed with single-cell resolution. Compounds that modulate the excitability only of the cells with the target are judged to be functionally selective for the target in the native cellular milieu. C) Cell type-selective screen. Each well contains a mixture of two or more cell types with fluorescent markers identifying the cell type (green vs. red). Optogenetic excitability measurements are performed with single-cell resolution. Compounds that modulate the excitability of only one cell type are judged to have cell type-selectivity, regardless of mechanism of action. D) Patient stratification. Each well contains iPSC-derived neurons from a different patient. Optogenetic excitability measurements in the presence of a test compound can determine how the patient's cells will respond to the compound.
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Research Support, Non-U.S. Gov't
Drug Discovery / instrumentation*
Drug Discovery / methods*
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Optogenetics / instrumentation*