It is many scientists’ new favorite tool: optogenetics, a relatively recent discipline that makes it possible to control cells with light signals. Bayer researchers want to use it to improve substance screening and to discover active substances that might otherwise never have been found.
The search for new active ingredients is a complex matter. More than 4 million chemical compounds are stored in Bayer’s substance library. The existing screening methods have limitations.
Researchers are using the new method of optogenetics to determine whether a substance has the desired effect. They use these molecular switches to activate test cell, thus increasing the accuracy of the screening process.
Researchers do not require chemical additives which can cause side effects in the test results. Screening is more precise and potential active ingredients can be identified more quickly.
Controlling molecules at the touch of a button – that sounds like science fiction. In the still young research field of optogenetics, however, precisely this is becoming reality. Optogenetics is a union of two disciplines that, at first glance, have little to do with one another: optics and genetics. Modern methods in genetics enable scientists to make cells light-sensitive and then control them with optical impulses. A mere ten years have passed since neurobiologist Karl Deisseroth from Stanford University in the United States astonished experts with one of the first optogenetic experiments. He managed to control brain cells in a mouse using light rays and to make the animal go round in circles. The potential uses nowadays extend far beyond neurobiology. Scientists in many different disciplines are working on this promising method worldwide, including Dr. Linn Schneider from Bayer’s Pharmaceuticals Division and Dr. Arunas Damijonaitis from the Crop Science Division.
Anyone entering Schneider’s laboratory in Wuppertal cannot fail to notice a complex robot system taking up a good third of the large room. “That’s our octopus,” says Schneider. Unlike its deep-sea namesake, the machine has only four arms instead of eight. The octopus is a fully automated high-throughput screening (HTS) system. Bayer researchers use it to test the pharmacological action of millions of substances. After identifying a target protein that might be associated with a particular disease, the scientists place it in a test cell together with a fluorescent indicator. In the octopus, these cells then encounter potential active substances, in other words molecules from Bayer’s own substance libraries. If a substance binds to the target protein as desired, the indicator lights up indicating pharmacological activity. A camera records the corresponding spot on the microtiter plate in which the substance is held in one of 1,536 wells, and one important first step in the development of a new medicine is done.
In the best-case scenario, we want to activate cells with one wavelength and measure them with another.
Finding a suitable drug candidate for a medicine is a time-consuming task, in view of the sheer number of potential active substances; to start with, there are 4.1 million substances stored in Bayer’s libraries alone. With the aid of optogenetics, however, Schneider and Damijonaitis want to improve the HTS process as part of the cross-divisional “Life Science Collaboration” initiative, as the conventional approach using chemical indicators has a number of disadvantages.
The biggest drawback is the unreliable hit rate. The cells that are the focus of biochemists’ attention exist naturally in different states. “Some are active, others are passive,” explains Damijonaitis. “But binding often takes place only in the active state.”
Optogenetic methods could be useful in research into various biochemical processes. Scientists are focusing in particular on ion channels, i.e. proteins that, depending on their state, allow the passage of electrically charged particles. This is where scientists see the most common ground across divisions. A multitude of biological processes take place via ion channels. Their state depends on the membrane potential of the cell, in other words the electrical voltage between the inside of the cell membrane and its surroundings. In cell cultures, the distribution of ion channel state is random. Since active compounds are often better able to bind to active ion channels, passive ion channels are not deemed important in providing any usable information in screening.
Scientists would therefore like to have a switch for cells. On a small scale, this already exists in electrophysiology, where researchers influence the membrane potential of cells with electrodes, resulting in their activation. However, this is a complex, rather slow process and one that is often not feasible for high-throughput screening.
Cell switches made possible through optogenetics, on the other hand, are much better. Light impulses activate ion channels in cells in thousandths of a second, switching them on more or less instantaneously. To do this, researchers use a light-sensitive molecule, known as the “optogenetic tool”. In recent years, the optogenetic toolbox has been expanded to include a number of light-sensitive molecules. The best-researched of these is channelrhodopsin, used by the fresh-water alga Chlamydomonas reinhardtii to seek out sites with favorable light conditions. Channelrhodopsin is a light switch that researchers use to activate ion channels to increase their screening hit rate.
Introducing the switch into the cell is the genetic aspect of optogenetics. Researchers achieve this by inserting the part of the algal genome responsible for production of channelrhodopsin into the cell. The DNA acts as a kind of guide by which the cell pieces together proteins – in this case specifically the molecular light switch (see also “Light switches for molecules”).
active substances are stored in Bayer’s substance libraries.
U.S. Researchers Control Mouse with Light Signal
Optogenetics originated in neuroscience. It is the answer to a question that has been puzzling researchers for decades: how to address individual nerve cells without affecting the cells around them. The 2005 experiment was one of the milestones in the development of optogenetics.
The U.S. neurologist and psychiatrist Karl Deisseroth and his team at the University of Stanford succeeded in placing nerve cells in the brain of a living organism under remote control. The film clips are as impressive today as when they were made, twelve years ago.
The mouse wanders randomly around its cage, looking here, sniffing there, standing still. A light conductor - a transparent cable - protrudes from its head. When a ray of blue light contacts the conductor, the small rodent is set into motion. It traces an almost perfect clockwise circle, as if it were remote-controlled. It only stops moving when the light goes off. The U.S. researchers had implanted a kind of switch in the mouse’s brain and can now make it run in circles at the flick of a switch.
The light switch is a protein called channelrhodopsin. This substance originates in the fresh-water alga Chlamydomonas reinhardtii, which uses it to seek out sites with favorable light conditions. The scientists analyzed the DNA of the algae and copied the part that is responsible for the production of channelrhodopsin. The DNA is like the recipe that the cell uses to make proteins. As the final step, the researchers inserted the copied DNA segment into the mouse’s brain cells that regulate motion. There are many ways of modifying the DNA of cells. In this case, they packed the genetic material into a virus which was designed to infect the desired cells. This meant that only the nerve cells for motion to the left received the instructions for the molecular light switch.
For Schneider’s optogenetic research, the octopus screening robot was equipped with LED lights. This allows it to irradiate the substances with light of any color. The light spectrum also includes ultraviolet, in other words light that is invisible to the human eye. “In the best-case scenario, we would want to activate with one wavelength and measure with another,” says Schneider.
This works if optogenetics is used to introduce not just switches but also sensor molecules into the cell. They perform the role of indicators and show whether binding between the active substance and the target protein occurs. Unlike chemical indicators in conventional HTS, they are not applied to the substance/cell mixture as a chemical but are introduced into the cell itself as DNA. “That, too, is progress, as it avoids misleading incidental effects,” says Damijonaitis. “In addition, the new sensors measure ion flows faster and more selectively,” adds Schneider. Until now, scientists had only been able to demonstrate electrical charges, irrespective of their element. With suitable sensors, they can now distinguish ions from one another.
In their work, Schneider and Damijonaitis are guided by recent scientific developments. Initial results with the new screening method are so promising that Bayer is providing additional funding for their work for two years. With two postdocs appointed specifically for the “Life Science Collaboration”, up to eight scientists are now working across divisions on optogenetic tools.
The potential applications are diverse. For Damijonaitis and his research for Crop Science, the top priority is ion channels. “Many of the insecticidal crop protection agents on the market address ion channels. Since ion channels in insects are completely different in some respects from those of vertebrates, we can target these ion channels specifically, without posing a risk to humans.”
Key progress: with optogenetic methods, we avoid misleading incidental effects.
Even though Schneider and Damijonaitis work in different Bayer research fields, they share a common goal: “We want to discover something that we couldn’t find with conventional systems,” says Damijonaitis. In other words, both want to shed light in the darkness.
Interview: Prof. Alexander Gottschalk
Biochemist Professor Alexander Gottschalk from the University of Frankfurt is one of the leading scientists in the field of optogenetics worldwide. research talked to him about problems and opportunities in this young research discipline.
For what purposes are optogenetic methods being used?
Optogenetics was originally used in neuroscience. Light-sensitive proteins are now also being used in cell biology. Any number of things can be controlled with them from the outside, whether gene expression, modification of the structure of cells or cell movement.
How does the protein know which cell to modify?
Genes, the “blueprints” for proteins, bear a kind of access code for this. A gene consists essentially of two segments: the promoter, a type of address, followed by the DNA sequence, which codes for the protein.
How do you introduce the light-sensitive proteins into the nerve cell?
To do this, we have to genetically modify the cell. In more complex organisms, such as a mouse, the genome that is to be incorporated and that is to make the cell light-sensitive is packed into a virus. The harmless gene vector introduces its genome, often in the form of RNA, into the cell, where it is translated into DNA and becomes permanently integrated into the host-cell genome.
From then on, the modified genetic information serves as the source for the optogenetic tool, i.e. the protein.
Could this be used in the future in medicine?
Potentially, yes. The proviso, however, is that the light for activation is able to reach even the affected cells. The process might work, for example, in degenerative diseases of the eye’s retina. If photoreceptor cells die off with advancing years and no longer sense light, the cells that have previously only transmitted the light signal could themselves be made light-sensitive. Similar approaches are available for hearing.
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