By Stephanie Baringer
We know that science isn’t always a perfect, well, science. Despite our best efforts and rationale, things don’t always work the way we expect them too. Maybe a cytotoxic drug isn’t toxic to your cultured cells, or maybe your mouse model doesn’t behave in the anticipated way. These encounters highlight why validation experiments are essential. One specific validation study performed 10 years after the development of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) had researchers around the world questioning the validity of their own data. This story of DREADDs’ fall from grace has everything you need in compelling academic drama: rigorous science and even a Twitter fight.
DREADDs are artificially engineered receptors, often G protein-coupled receptors (GPCRs), that are selectively activated by synthetic drugs. While the concept for designer receptors was first explored in 19981, it was the Roth lab that developed the next generation of these receptors and termed them DREADDs in 20072. Using protein engineering, scientists mutated receptors to alter the ligand, or biomolecule, binding site, resulting in the receptors being unresponsive to their endogenous ligand and instead activated by a synthetic small molecule (Figure 1). Roth and colleagues saw DREADDS as a revolutionary technique in neuroscience3. With DREADDs, scientists have the power to manipulate specific neuron populations in vivo, and by controlling the activity of receptors, scientists can switch neuron populations on and off with the ease of an injection. For example, behavioral neuroscientists use DREADDs in excitatory neurons that are active during fear conditioning4, allowing them to artificially simulate fear memory to understand the process of memory encoding. Within 10 years of their design, DREADDs were used in labs around the world and were referenced in over 800 papers5. However, the field was rocked in 2017 when the Michaelides lab revealed that DREADDs had many more caveats than previously known5.
I remember the day well. In August 2017, I was working as a post-bac fellow in Baltimore, Maryland at the National Institute on Aging and my roommate was at the National Institute on Drug Abuse. She burst in the front door and exclaimed “My lab just published a paper disproving the use of DREADDs in vivo and the DREADDs creator is attacking the lab on Twitter!” The Michaelides lab, where she was working, had just completed a number of DREADD validation experiments that had not been performed at the time of DREADD’s creation2. Most DREADDs at the time, and particularly the pioneering Roth lab, used clozapine N-oxide (CNO) to turn DREADDs on and off. The Michaelides group demonstrated that once in the body, CNO is rapidly converted to clozapine5. This conversion is problematic because, clozapine, an antipsychotic drug, has a lot of nonspecific binding and binds to many receptors when used at the common CNO injection concentration. If CNO is like finding your specific room in a hotel, clozapine is like hitting all of the elevator buttons and seeing where you end up. Using radiolabeled saturation binding and competition assays, the Michaelides lab showed clozapine had strong binding affinity for the DREADDs whereas CNO, the Roth-designed ligand, did not5. The researchers further showed that CNO does not cross the blood-brain barrier while clozapine does and that clozapine engages with the DREADDs. This is critical because in vivo, the process relies on the injected compound to reach the DREADDs by crossing the blood-brain barrier to bind the receptors for the desired effect. Lastly, the Michaelides group demonstrated that clozapine alone activates DREADDs and produces the DREADD-specific behavioral responses that were thought to only be induced by CNO administration5, providing the final evidence that CNO administration and DREADDs were not the precise technology the field believed them to be.
If you have used DREADDs or based your studies off of DREADD-heavy experiments, no need to be filled with dread just yet. There are many things to consider when determining if the DREADDs will be or were used properly. First is the system of use. CNO is converted to clozapine via cytochrome P450 in the liver, thus using CNO DREADDs in cultured cells or mounted brain tissuewould not run into a receptor specificity problem nor would it have the nonspecific activation associated with clorapine6. If in vivo testing is required, the Michaelides groupsuggests using low doses of clozapine6. This removes the variability of CNO metabolism but could still have off-target affects, and it is important to consider how clozapine dosing could affect the behavior in testing6. Lastly, DREADDs can be designed for different synthetic ligands to bypass clozapine conversion. Many compounds other than CNO have been developed with DREADDs, such as perlapine, compound 21, and deschloroclozapine; however, none of these additional compounds have been validated for in vivo use yet6.
The Michaelides labemphasize that their discoveries do not discount conclusions drawn from all previous DREADD experiments, but the level to which every previous experiment’s environment was controlled should be critically examined5. In a well-controlled experimental design, DREADDs and CNO can still be powerful chemogenetic tools for scientists to manipulate cells and their activity. The story of DREADDs teaches us to never skip validation studies because at the least, it will give you confidence about your data, and at the most, it might just lead to a paper in the journal Science.
DREADDs are a popular chemogenetic tool for neuroscientists, but a validation study published 10 years after their development left the field questioning if they could trust the data.
1. Coward, P. et al. Controlling signaling with a specifically designed Gi-coupled receptor. Proc Natl Acad Sci U S A 95, 352–357 (1998).
2. Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci U S A 104, 5163–5168 (2007).
3. Roth, B. L. DREADDs for Neuroscientists. Neuron 89, 683–694 (2016).
4. Smith, K. S., Bucci, D. J., Luikart, B. W. & Mahler, S. V. DREADDs: Use and Application in Behavioral Neuroscience. Behav Neurosci 130, 137–155 (2016).
5. Gomez, J. L. et al. Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Science 357, 503–507 (2017).
6. Mahler, S. V. & Aston-Jones, G. CNO Evil? Considerations for the Use of DREADDs in Behavioral Neuroscience. Neuropsychopharmacology 43, 934–936 (2018).