A receptor activated solely by a synthetic ligand (RASSL) or designer receptor exclusively activated by designer drugs (DREADD), is a class of artificially engineered protein receptors used in the field of chemogenetics which are selectively activated by certain ligands. [1] They are used in biomedical research, in particular in neuroscience to manipulate the activity of neurons. [2]
Originally differentiated by the approach used to engineer them, RASSLs and DREADDs are often used interchangeably now to represent an engineered receptor-ligand system. [3] These systems typically utilize G protein-coupled receptors (GPCR) engineered to respond exclusively to synthetic ligands, like clozapine N-oxide (CNO), [4] and not to endogenous ligands. Several types of these receptors exists, derived from muscarinic or κ-opioid receptors. [1]
One of the first DREADDs was based on the human M3 muscarinic receptor (hM3). [5] Only two point mutations of hM3 were required to achieve a mutant receptor with nanomolar potency for CNO, insensitivity to acetylcholine and low constitutive activity and this DREADD receptor was named hM3Dq. M1 and M5 muscarinic receptors have been mutated to create DREADDs hM1Dq and hM5Dq respectively. [5]
The most commonly used inhibitory DREADD is hM4Di, derived from the M4 muscarinic receptor that couples with the Gi protein. [5] Another Gi coupled human muscarinic receptor, M2, was also mutated to obtain the DREADD receptor hM2D. [5] Another inhibitory Gi-DREADD is the kappa-opioid-receptor (KOR) DREADD (KORD) which is selectively activated by salvinorin B (SalB). [6]
Gs-coupled DREADDs have also been developed. These receptors are also known as GsD and are chimeric receptors containing intracellular regions of the turkey erythrocyte β-adrenergic receptor substituted into the rat M3 DREADD. [7]
A growing number of ligands that can be used to activate RASSLs / DREADDs are commercially available. [8]
CNO is the prototypical DREADD activator. CNO activates the excitatory Gq- coupled DREADDs: hM3Dq, hM1Dq and hM5Dq and also the inhibitory hM4Di and hM2Di Gi-coupled DREADDs. CNO also activates the Gs-coupled DREADD (GsD) and the β-arrestin preferring DREADD: rM3Darr (Rq(R165L). [9]
Recent findings suggest that systemically administered CNO does not readily cross the blood-brain-barrier in vivo and converts to clozapine which itself activates DREADDs. Clozapine is an atypical antipsychotic which has been indicated to show high DREADD affinity and potency. Subthreshold injections of clozapine itself can be utilised to induce preferential DREADD-mediated behaviors. Therefore, when using CNO, care must be taken in experimental design and proper controls should be incorporated. [10]
DREADD agonist 21, also known as Compound 21, represents an alternative agonist for muscarinic-based DREADDs and an alternative to CNO. It has been reported that Compound 21 has excellent bioavailability, pharmacokinetic properties and brain penetrability and does not undergo reverse metabolism to clozapine. [11] Another known agonist is perlapine, a hypnotic drug approved for treating insomnia in Japan. It acts as an activator of Gq-, Gi-, and Gs DREADDs that has structural similarity to CNO. [12] A more recent agonist of hM3Dq and hM4Di is deschloroclozapine (DCZ). [13]
On the other hand, SalB B is a potent and selective activator of KORD. [14]
JHU37160 and JHU37152 have been marketed commercially as novel DREADD ligands, active in vivo, with high potency and affinity for hM3Dq and hM4Di DREADDs.[ citation needed ]
Dihydrochloride salts of DREADD ligands that are water-soluble (but with differing stabilities in solution) have also been commercially developed (see [15] [16] for aqueous stability).
RASSLs and DREADDs are families of designer G-protein-coupled receptors (GPCRs) built specifically to allow for precise spatiotemporal control of GPCR signaling in vivo . These engineered GPCRs are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the interrogation of several GPCR signaling pathways, including those activated by Gs, Gi, Gq, Golf and β-arrestin. [18] A major cause for success of RASSL resources has been open exchange of DNA constructs, and RASSL related resources.
The hM4Di-DREADD's inhibitory effects are a result of the CNO's stimulation and resulting activation of the G-protein inwardly rectifying potassium (GIRK) channels. This causes hyperpolarization of the targeted neuronal cell and thus attenuates subsequent activity. [19]
This chemogenetic technique can be used for remote manipulation of cells, in particular excitable cells like neurons, both in vitro and in vivo with the administration of specific ligands. [2] Similar techniques in this field include thermogenetics and optogenetics, the control of neurons with temperature or light, respectively. [2]
Viral expression of DREADD proteins, both in-vivo enhancers and inhibitors of neuronal function, have been used to bidirectionally control behaviors in mice (e.g odor discrimination). [20] Due to their ability to modulate neuronal activity, DREADDs are used as a tool to evaluate both the neuronal pathways and behaviors associated with drug-cues and drug addiction. [21]
Strader and colleagues designed the first GPCR which could be activated only by a synthetic compound [22] and has gradually been gaining momentum. The first international RASSL meeting was scheduled for April 6, 2006. A simple example of the use of a RASSL system in behavioral genetics was illustrated by Mueller et al. (2005) where they showed that expressing a RASSL receptor in sweet taste cells of the mouse tongue led to a strong preference for oral consumption of the synthetic ligand, whereas expressing the RASSL in bitter taste cells caused dramatic taste aversion for the same compound. [23]
The attenuating effects of the hM4Di-DREADD were originally explored in 2007, before being confirmed in 2014. [19]
G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors (GPLR), form a large group of evolutionarily related proteins that are cell surface receptors that detect molecules outside the cell and activate cellular responses. They are coupled with G proteins. They pass through the cell membrane seven times in the form of six loops of amino acid residues, which is why they are sometimes referred to as seven-transmembrane receptors. Ligands can bind either to the extracellular N-terminus and loops or to the binding site within transmembrane helices. They are all activated by agonists, although a spontaneous auto-activation of an empty receptor has also been observed.
Acetylcholine (ACh) is an organic compound that functions in the brain and body of many types of animals as a neurotransmitter. Its name is derived from its chemical structure: it is an ester of acetic acid and choline. Parts in the body that use or are affected by acetylcholine are referred to as cholinergic.
A neurotransmitter receptor is a membrane receptor protein that is activated by a neurotransmitter. Chemicals on the outside of the cell, such as a neurotransmitter, can bump into the cell's membrane, in which there are receptors. If a neurotransmitter bumps into its corresponding receptor, they will bind and can trigger other events to occur inside the cell. Therefore, a membrane receptor is part of the molecular machinery that allows cells to communicate with one another. A neurotransmitter receptor is a class of receptors that specifically binds with neurotransmitters as opposed to other molecules.
Behavioral neuroscience, also known as biological psychology, biopsychology, or psychobiology, is part of the broad, interdisciplinary field of neuroscience, with its primary focus being on the biological and neural mechanisms underlying behavior. Cognitive neuroscience is similar to behavioral neuroscience, in that both fields study the neurobiological functions related to psychology, as in experiences and behaviors. Behavioral neuroscientists examine the biological bases of behavior through research that involves neuroanatomical substrates, environmental and genetic factors, effects of lesions and electrical stimulation, developmental processes, recording electrical activity, neurotransmitters, hormonal influences, chemical components, and the effects of drugs. Important topics of consideration for neuroscientific research in behavior include learning and memory, sensory processes, motivation and emotion, as well as genetic and molecular substrates concerning the biological bases of behavior.
Nicotinic acetylcholine receptors, or nAChRs, are receptor polypeptides that respond to the neurotransmitter acetylcholine. Nicotinic receptors also respond to drugs such as the agonist nicotine. They are found in the central and peripheral nervous system, muscle, and many other tissues of many organisms. At the neuromuscular junction they are the primary receptor in muscle for motor nerve-muscle communication that controls muscle contraction. In the peripheral nervous system: (1) they transmit outgoing signals from the presynaptic to the postsynaptic cells within the sympathetic and parasympathetic nervous system, and (2) they are the receptors found on skeletal muscle that receive acetylcholine released to signal for muscular contraction. In the immune system, nAChRs regulate inflammatory processes and signal through distinct intracellular pathways. In insects, the cholinergic system is limited to the central nervous system.
Muscarinic acetylcholine receptors, or mAChRs, are acetylcholine receptors that form G protein-coupled receptor complexes in the cell membranes of certain neurons and other cells. They play several roles, including acting as the main end-receptor stimulated by acetylcholine released from postganglionic fibers. They are mainly found in the parasympathetic nervous system, but also have a role in the sympathetic nervous system in the control of sweat glands.
Functional selectivity is the ligand-dependent selectivity for certain signal transduction pathways relative to a reference ligand at the same receptor. Functional selectivity can be present when a receptor has several possible signal transduction pathways. To which degree each pathway is activated thus depends on which ligand binds to the receptor. Functional selectivity, or biased signaling, is most extensively characterized at G protein coupled receptors (GPCRs). A number of biased agonists, such as those at muscarinic M2 receptors tested as analgesics or antiproliferative drugs, or those at opioid receptors that mediate pain, show potential at various receptor families to increase beneficial properties while reducing side effects. For example, pre-clinical studies with G protein biased agonists at the μ-opioid receptor show equivalent efficacy for treating pain with reduced risk for addictive potential and respiratory depression. Studies within the chemokine receptor system also suggest that GPCR biased agonism is physiologically relevant. For example, a beta-arrestin biased agonist of the chemokine receptor CXCR3 induced greater chemotaxis of T cells relative to a G protein biased agonist.
G protein-gated ion channels are a family of transmembrane ion channels in neurons and atrial myocytes that are directly gated by G proteins.
The Gs alpha subunit is a subunit of the heterotrimeric G protein Gs that stimulates the cAMP-dependent pathway by activating adenylyl cyclase. Gsα is a GTPase that functions as a cellular signaling protein. Gsα is the founding member of one of the four families of heterotrimeric G proteins, defined by the alpha subunits they contain: the Gαs family, Gαi/Gαo family, Gαq family, and Gα12/Gα13 family. The Gs-family has only two members: the other member is Golf, named for its predominant expression in the olfactory system. In humans, Gsα is encoded by the GNAS complex locus, while Golfα is encoded by the GNAL gene.
The human muscarinic acetylcholine receptor M5, encoded by the CHRM5 gene, is a member of the G protein-coupled receptor superfamily of integral membrane proteins. It is coupled to Gq protein. Binding of the endogenous ligand acetylcholine to the M5 receptor triggers a number of cellular responses such as adenylate cyclase inhibition, phosphoinositide degradation, and potassium channel modulation. Muscarinic receptors mediate many of the effects of acetylcholine in the central and peripheral nervous system. The clinical implications of this receptor have not been fully explored; however, stimulation of this receptor is known to effectively decrease cyclic AMP levels and downregulate the activity of protein kinase A (PKA).
Dopamine receptor D2, also known as D2R, is a protein that, in humans, is encoded by the DRD2 gene. After work from Paul Greengard's lab had suggested that dopamine receptors were the site of action of antipsychotic drugs, several groups, including those of Solomon H. Snyder and Philip Seeman used a radiolabeled antipsychotic drug to identify what is now known as the dopamine D2 receptor. The dopamine D2 receptor is the main receptor for most antipsychotic drugs. The structure of DRD2 in complex with the atypical antipsychotic risperidone has been determined.
The muscarinic acetylcholine receptor M1, also known as the cholinergic receptor, muscarinic 1, is a muscarinic receptor that in humans is encoded by the CHRM1 gene. It is localized to 11q13.
The muscarinic acetylcholine receptor M2, also known as the cholinergic receptor, muscarinic 2, is a muscarinic acetylcholine receptor that in humans is encoded by the CHRM2 gene. Multiple alternatively spliced transcript variants have been described for this gene. It is Gi-coupled, reducing intracellular levels of cAMP.
Psychosine receptor is a G protein-coupled receptor (GPCR) protein that in humans is encoded by the GPR65 gene. GPR65 is also referred to as TDAG8.
A GPCR oligomer is a protein complex that consists of a small number of G protein-coupled receptors (GPCRs). It is held together by covalent bonds or by intermolecular forces. The subunits within this complex are called protomers, while unconnected receptors are called monomers. Receptor homomers consist of identical protomers, while heteromers consist of different protomers.
Gene therapy is being studied for some forms of epilepsy. It relies on viral or non-viral vectors to deliver DNA or RNA to target brain areas where seizures arise, in order to prevent the development of epilepsy or to reduce the frequency and/or severity of seizures. Gene therapy has delivered promising results in early stage clinical trials for other neurological disorders such as Parkinson's disease, raising the hope that it will become a treatment for intractable epilepsy.
Chemogenetics is the process by which macromolecules can be engineered to interact with previously unrecognized small molecules. Chemogenetics as a term was originally coined to describe the observed effects of mutations on chalcone isomerase activity on substrate specificities in the flowers of Dianthus caryophyllus. This method is very similar to optogenetics; however, it uses chemically engineered molecules and ligands instead of light and light-sensitive channels known as opsins.
Perlapine, sold under the brand names Hypnodine and Pipnodine, is a hypnotic and sedative of the tricyclic group which is marketed in Japan. It acts primarily as a potent antihistamine, and also has anticholinergic, antiserotonergic, antiadrenergic, and some antidopaminergic activity. The drug has relatively weak affinity for the dopamine D2 receptor (IC50Tooltip Half-maximal inhibitory concentration = 1,803 nM) and, in accordance, is said to be ineffective as an antipsychotic. However, it retains higher affinity for the dopamine D1 receptor (IC50 = 198 nM). Its IC50 values are 19 nM for the α1-adrenergic receptor, 4,945 nM for the α2-adrenergic receptor, and 70 nM for the serotonin 5-HT2A receptor. Perlapine is closely related to clotiapine, clozapine, fluperlapine, loxapine, and tilozepine.
Clozapine N-oxide (CNO) is a synthetic drug used mainly in biomedical research as a ligand to activate Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), despite the initial belief that it was biologically inert. However, it has been shown to not enter the brain after administration and to reverse metabolize in peripheral tissues to form clozapine. Clozapine can bind to a number of different serotonergic, dopaminergic and adrenergic receptors within the brain. These off-target effects mean behavioral data using the CNO-DREADD system have to be interpreted with caution.
Bryan L. Roth is the Michael Hooker Distinguished Professor of Protein Therapeutics and Translational Proteomics, UNC School of Medicine. He is recognized for his discoveries and inventions in the general areas of molecular pharmacology, GPCR structure, and function and synthetic neurobiology. He is a member of the American Academy of Arts and Sciences (AAAS) and the National Academy of Medicine (NAM)