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DREADDs mini review

DREADDs

Overview

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. (For practical advice on using these ligands in the lab, please see our other reviews: Stability of Water-Soluble DREADD Ligands in Solution: A Technical Review and Clozapine N-Oxide (freebase) - a technical review on stability, solubility and use in the lab)

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 [1]. 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).

Categorization of DREADDs according to signal transduction mechanisms that they tether

Figure 1. Categorization of DREADDs according to signal transduction mechanisms that they tether (Gi, Gq, Gs, or β-arrestin)

Gq-DREADDs

One of the first DREADDs was based on the human M3 muscarinic receptor (hM3) (Figure 2) [1]. 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 [1], and neuronal cells in vitro and in vivo [2].

Activation of Gq coupled DREADDs leads to an increase in intracellular calcium levels [3]. 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 [4]. 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 [5].

Gi-DREADDs

The most commonly used inhibitory DREADD is hM4Di, derived from the muscarinic M4 receptor that couples with Gi protein [1]. 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 [1] 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) [6]. KORDs were created by modifying the human KORs with the D138N point mutation in the third transmembrane domain [6]. SALB has modest affinity for KOR, and therefore its concentration in studies must be controlled to avoid non-KORD effects [6].

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 [9]. Activation of Gi coupled DREADD in neurons was also shown to suppress synaptic release probability [6,10] .

Gs-/β-arrestin-DREADDs

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 [2]. 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 [12]. 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 [12].

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 [11].

Gs-DREADDs were used to study circadian rhythms [5], and cell signaling pathways that may be involved in addiction [13].

DREADDs are activated by physiologically inert molecules

Figure 2. Designer compounds (CNO, perlapine, Compound 21, SALB) used to activate various DREADDS and their downstream pathways

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 [13].

DREADD ligands

CNO is the prototypical DREADD activator. CNO activates the excitatory Gq- coupled DREADDs: hM3Dq, hM1Dq and hM5Dq (pEC50 values are 7.26 and 8.61 at hM3Dq and hM1Dq respectively) and also the inhibitory hM4Di and hM2Di Gi-coupled DREADDs (pEC50 = 6.89 at hM4Di). CNO also activates the Gs-coupled DREADD (GsD) and the β-arrestin preferring DREADD: rM3Darr (Rq(R165L).

CNO has been administered intraperitoneally (i.p.), subcutaneously, directly infused intracranially, via drinking water, osmotic mini-pump and recently via eye drops. It is also available in a water soluble form, CNO dihydrochloride, which is highly advantageous for use in biological assays

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 [16]. Clozapine (also available as water-soluble clozapine dihydrochloride) is an atypical antipsychotic which has been indicated to show high DREADD (hM3Dq and hM4Di) affinity and potency. Subthreshold injections of clozapine itself can be utilised to induce preferential DREADD-mediated behaviors [16]. Therefore when using CNO, care must be taken in experimental design and proper controls should be incorporated.

DREADD agonist 2 (Compound 21) (DREADD agonist 21), represents an alternative chemogenetic actuator for muscarinic -based DREADDs and an alternative to CNO for in vivo studies in which metabolic conversion of CNO to clozapine is an issue. DREADD agonist 21 (Compound 21) is a potent and selective agonist at muscarinic based DREADDs such as the excitatory hM3Dq, hM1Dq and inhibitory hM4Di DREADDs (pEC50 values are 8.48, 8.91 and 7.77 at hM3Dq, hM1Dq and hM4Di respectively). DREADD agonist 21 (Compound 21) has excellent bioavailability, pharmacokinetic properties and brain penetrability. It has been reported that DREADD agonist 21 does not undergo back metabolism to clozapine.[17]. A water soluble dihydrochloride salt of DREADD agonist 21 (Compound 21) is now also commercially available from Hello Bio.

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 is a potent agonist at muscarinic based DREADDs such as the excitatory hM3Dq, hM1Dq and inhibitory hM4Di DREADDs (pEC50 values are 8.08, 8.38 and 7.27 at hM3Dq, hM1Dq and hM4Di respectively). [14,17]. It has been reported that perlapine does not undergo back metabolism to clozapine.A water soluble dihydrochloride salt  is now commercially available from Hello Bio.

Salvinorin B (SALB), an inactive metabolite of the KOR-selective agonist salvinorin A (SALA), is the selective activator of the KOR DREADD [6]. 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 [6].

Hello Bio has also introduced J60 dihydrochloride and J52 dihydrochloride - novel DREADD ligands, active in vivo, with high potency and affinity for hM3Dq and hM4Di DREADDs. Read more about these novel DREADD ligands

View the full range of DREADDs ligands available from Hello Bio.

Additional resources

We have also published these technical guides to offer practical advice on using DREADDs ligands in the lab:

References

  1. Armbruster BN et al (2007) 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(12) 5163-8. Pubmed ID:17360345
  2. Conklin BR et al (2008) Engineering GPCR signaling pathways with RASSLs. Nat Methods 5(8) 673-8. Pubmed ID:18668035
  3. Agulhon C et al (2013) Modulation of the autonomic nervous system and behaviour by acute glial cell Gq protein-coupled receptor activation in vivo. J Physiol 591(22) 5599-609. Pubmed ID:24042499
  4. Garner AR et al (2012) Generation of a synthetic memory trace. Science 335(6075) 1513-6. Pubmed ID:22442487
  5. Brancaccio M et al (2013) A Gq-Ca2+ axis controls circuit-level encoding of circadian time in the suprachiasmatic nucleus. Neuron 78(4) 714-28. Pubmed ID:23623697
  6. Vardy E et al (2015) A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior. Neuron 86(4) 936-46. Pubmed ID:25937170
  7. Carter ME et al (2013) Genetic identification of a neural circuit that suppresses appetite. Nature 503(7474) 111-4. Pubmed ID:24121436
  8. Parnaudeau S et al (2013) Inhibition of mediodorsal thalamus disrupts thalamofrontal connectivity and cognition. Neuron 77(6) 1151-62. Pubmed ID:23522049
  9. Kozorovitskiy Y et al (2012) Recurrent network activity drives striatal synaptogenesis. Nature 485(7400) 646-50. Pubmed ID:22660328
  10. Stachniak TJ et al (2014) Chemogenetic synaptic silencing of neural circuits localizes a hypothalamus to midbrain pathway for feeding behavior. Neuron 82(4) 797-808. Pubmed ID:24768300
  11. Farrell MS et al (2013) A Gαs DREADD mouse for selective modulation of cAMP production in striatopallidal neurons. Neuropsychopharmacology 38(5) 854-62. Pubmed ID:23303063
  12. Nakajima K et al (2012) Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor. Mol Pharmacol 82(4) 575-82. Pubmed ID:22821234
  13. Ferguson SM et al (2013) Direct-pathway striatal neurons regulate the retention of decision-making strategies. J Neurosci 33(28) 11668-76. Pubmed ID:23843534
  14. Chen X et al (2015) The first structure-activity relationship studies for designer receptors exclusively activated by designer drugs. ACS Chem Neurosci 6(3) 476-84. Pubmed ID:25587888
  15. Guettier JM et al (2009) A chemical-genetic approach to study G protein regulation of beta cell function in vivo. Proc Natl Acad Sci U S A 106(45) 19197-202. Pubmed ID:19858481
  16. Gomez et al (2017) Chemogenetics revealed: DREADD occupancy and activatoin via converted clozapine. Science 357(6350):503-507. Pubmed ID:28774929
  17. Thompson et al (2018) ACS Pharmacol.Transl.Sci. DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in Vivo. https://pubs.acs.org/doi/10.1021/acsptsci.8b00012

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REVIEW UPDATED AUGUST 2018