DREADDs mini review
Advances in genetics have allowed molecular biologists to modify G-protein coupled receptors (GPCRs) to preserve much of the receptor's native functionality while altering specific properties such as agonist binding. Designer receptors exclusively activated by designer drugs (DREADDs) are genetically engineered GPCRs that are activated by physiologically inert synthetic small molecules (designer drugs) but not their endogenous ligand. DREADDS can be expressed in specific neuronal populations in vivo and can be used to study roles of the native receptor in cell signaling. Written by our expert PhD qualified technical team, and endorsed by our Scientific Advisory Board, this mini-review summarizes some of the features of DREADDs and their ligands.
In vivo use of DREADDs are particularly promising because cells that express the DREADDs can be modulated without the need for any implants or other invasive procedures while granting remote control of cellular signaling, neuronal activity and behavior. Expression of DREADDs can be controlled in order to achieve the spatial control required to study specific cell populations in organs such as brain and liver.
DREADDs were originally created using directed molecular evolution of human muscarinic receptors in yeast . This allowed the creation of a variety of mutant muscarinic receptors with nanomolar potency for clozapine-N-oxide (CNO), a physiologically inert metabolite of the atypical antipsychotic clozapine.
A variety of DREADDs now exist and can broadly be categorized based on the signal transduction mechanisms that they link to: Gi, Gq, Gs, or β-arrestin (Figure 1).
Figure 1. Categorization of DREADDs according to signal transduction mechanisms that they tether (Gi, Gq, Gs, or β-arrestin)
One of the first DREADDs was based on the human M3 muscarinic receptor (hM3) (Figure 2) . Only two point mutations of hM3 (Y3.33C and A5.46G) were required to achieve a mutant receptor with nanomolar potency for CNO, insensitivity to acetylcholine (ACh) and low constitutive activity. This DREADD receptor was named hM3Dq. M1 and M5 muscarinic receptors (that couple Gq/11 proteins) were also used to create DREADDs (hM1Dq and hM5Dq, respectively) because the Y3.33 and A5.46 residues are conserved in all five of the known muscarinic ACh receptors (M1-M5).
The hM3Dq DREADD receptor is coupled to the Gq/11 mediated signaling pathway and is widely used to induce firing of neurons following CNO binding in many biological assay systems including: stable and transiently transfected cells in vitro , and neuronal cells in vitro and in vivo .
Activation of Gq coupled DREADDs leads to an increase in intracellular calcium levels . This induces second messenger signaling which leads to activation of kinases such as protein kinase C (PKC) and Ca2+/calmodulin-dependent kinases (CAMKs), which are involved in subsequent signaling cascades that lead to gain in cell function.
Gq-DREADDs have been used to suggest that Gq signaling in hippocampal neurons may be required for formation of new memories . By expressing Gq-DREADDs in suprachiasmatic neurons, Gq signaling was also identified as being important for modulating circadian rhythms, and therefore suggests a new therapeutic target for treatment of circadian disorders .
The most commonly used inhibitory DREADD is hM4Di, derived from the muscarinic M4 receptor that couples with Gi protein . Another Gi coupled human muscarinic receptor, M2, was also mutated to obtain the DREADD receptor hM2D. In the cases of both M4 and M2, Y3.33C and A5.46G point mutations produced these DREADD receptors  with CNO sensitivity similar to the Gq coupled muscarinic receptors.
Another inhibitory Gi-DREADD is the Κ-opioid-receptor (KOR) DREADD (KORD) which is selectively activated by salvinorin B (SALB) . KORDs were created by modifying the human KORs with the D138N point mutation in the third transmembrane domain . SALB has modest affinity for KOR, and therefore its concentration in studies must be controlled to avoid non-KORD effects .
Gi coupled DREADDs have been shown to inhibit neuronal activity by induction of hyperpolarization through activation of G-protein inwardly rectifying potassium channels (GIRKs) [1,6].
Gi coupled DREADDs have been used to study signaling pathways involved in cognitive impairments and eating disorders [7,8]. Gi-DREADDs were also used to study the role of Gi-signaling pathway in neuronal plasticity in the striatum . Activation of Gi coupled DREADD in neurons was also shown to suppress synaptic release probability [6,10] .
Gs coupled DREADDs have also been developed. These receptors (known as GsD) are chimeric receptors containing intracellular regions of the turkey erythrocyte β-adrenergic receptor substituted into the rat M3 DREADD . The resulting Gs DREADD has modest constitutive activity and has been used to probe the roles of Gs and Golf coupled signaling pathways in vivo [2,11].
A DREADD that couples β-arrestin has also been created . This DREADD was shown to activate β-arrestin pathways in vitro, however, it requires high concentrations of CNO which may limit its use in vivo .
Gs coupled receptors mediate signaling through activation of adenylyl cyclase which increases levels of the second messenger cyclic AMP (cAMP). cAMP activates protein kinase A which leads to an increase in cell function .
Gs-DREADDs were used to study circadian rhythms , and cell signaling pathways that may be involved in addiction .
Expression of DREADDs in vivo
Cell type-specific expression of DREADDs is achieved by using viral vectors (most commonly an adeno-associated virus (AAV) vector) that encodes the DREADD protein. The viral vector containing the DREADD of interest can be injected into mice that are genetically engineered to express a recombinase enzyme under the control of a cell type-specific promoter of gene expression. After recombination in situ, the DREADD is only expressed in cells that express the cell type-specific promoter .
CNO is the prototypical activator of Gq-DREADDs with low nanomolar potency at hM3Dq (EC50 = ~10 nM) and modest selectivity for hM3Dq over native muscarinic receptors (EC50 = ~30 μM) . CNO is also used to activate Gs coupled DREADD and hM4Di receptors. CNO is a physiologically inert metabolite of the atypical antipsychotic clozapine [14,15] and is able to cross the blood brain barrier for high CNS availability, which makes it a suitable compound for in vivo studies. CNO is available in a water soluble form which is highly advantageous for use in biological assays.
Another activator of Gq-, Gi-, and Gs DREADDs that has structural similarity to CNO is perlapine, a drug already approved for treating insomnia in Japan. Perlapine has low nanomolar potency at hM3Dq (EC50 = 2.8 nM) and has > 10,000 fold selectivity for hM3Dq over muscarinic receptors .
While CNO is an inert metabolite of clozapine, it has the potential to reverse-metabolize to clozapine and other clozapine metabolites in some non-rodent species . In order to combat this problem, a compound structurally related to CNO, Compound 21 (DREADD agonist 21), was synthesized and biologically validated to have high selectivity for hM3Dq (EC50 = 1.7 nM) versus muscarinic receptors (maximum activation = ~20% at micromolar concentrations compared to acetylcholine) .
Salvinorin B (SALB), an inactive metabolite of the KOR-selective agonist salvinorin A (SALA), is the selective activator of the KOR DREADD . In addition to high potency of SALB at KORD (EC50 = ~10-100 nM) and high selectivity over human KOR (~100 fold selective for KORD over human KOR), SALB also has good CNS penetrability .
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