DREADD Ligands

DREADD Mechanism of Action

The first DREADDs to be developed were derived from human muscarinic acetylcholine receptors (mAChRs) and termed hM1-5. These receptors have mutations in their orthosteric binding site, abolishing their affinity for the endogenous ligand ACh, while rendering them responsive to small molecule DREADD ligands, such as Clozapine-N-oxide (CNO, Cat. No. 4936) or Deschloroclozapine (DCZ, Cat. No. 7193).

The effect of ligand binding depends on the G protein-coupling of the DREADD. Binding of a ligand to DREADDs coupled to G_q signaling pathway (hM₁D_q, hM₃D_q and hM₅D_q) activates phospholipase C, which catalyzes the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP₂) to inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG). Both IP₃ and DAG have second messenger functions: IP₃ binds to its intracellular receptors to elicit Ca²⁺ release from intracellular stores, while DAG activates multiple forms of protein kinase C (PKC). When activated by ligand binding, DREADDs coupled to the G_i signaling pathways cause inhibition of adenylyl cyclase (AC), leading to a decrease in intracellular cAMP levels. cAMP activates protein kinase A (PKA) and EPAC, therefore ligand binding at G_i DREADDs inhibits PKA and EPAC downstream signaling (Figure 1). G_i-coupled DREADDs are also known as inhibitory DREADDs (iDREADDs).

Following the development of muscarinic DREADDs, another iDREADD has been developed from the κ-opioid receptor, termed KORD. KORD is activated by the pharmacologically inert small molecule Salvinorin B (Cat. No. 5611), and like hM₄D_i, is coupled to the G_i signaling pathway. The simultaneous use of KORD and hM₃D_q with their associated DREADD ligands enables researchers to have bidirectional control over neuronal activity, and the simultaneous interrogation of κ-opioid and mAChR signaling.

Figure 1: Binding of DREADD ligands to G_q-DREADDs provokes neuronal firing, whereas binding to G_i-DREADDs results in inhibition of neuronal activity. Clozapine N-oxide and DREADD agonist 21 are non-selective muscarinic DREADD agonists and so can activate or inhibit neuronal activity, depending on the specific receptor being expressed. Salvinorin B is selective for the KORD receptor, which is coupled to G_i signaling; consequently binding results in inhibition of neuronal activity.

In addition to coupling to G proteins, GPCRs can also activate β-arrestin mediated signaling pathways. This non-canonical pathway can be controlled by CNO acting at a mutated M₃ muscarinic receptor known as Rq(R165L), which activates arrestin signaling pathways without changes to G protein-mediated pathways.

More recently a DREADD derived from the human free fatty acid receptor 2 (FFA2) has been developed. Bearing mutations that alter the length of free fatty acid that acts at the receptor, FFA2-DREADD responds to Sorbic acid (Cat. No. 7119), but not short chain free fatty acids. Using FFA-DREADD knockin mice researchers have been able to investigate the role of FFA2 in the gut.

DREADD Ligand Evolution and CNO Alternatives

CNO is a metabolite of Clozapine (Cat. No. 0444); research suggests that this metabolism is bidirectional, and that CNO may undergo reverse metabolism to clozapine in vivo in mice, rats, guinea pigs and non-human primates. Clozapine is an atypical antipsychotic that displays antagonism at dopamine receptors and 5-HT_2A/2C receptors, so its presence in in vivo investigations of neuronal pathways may confound experimental results. Additionally, CNO is a substrate for the P-glycoprotein (P-gp) multidrug transporter, which is expressed on the blood brain barrier where it helps maintain the integrity of the blood brain barrier, and prevents the entry of drugs and removes them from the CNS.

The potential generation of clozapine and efflux of CNO from the brain has necessitated the development of DREADD ligand alternatives to CNO. DREADD agonist 21 (Cat. No. 6422) is a potent hM₃D_q and hM₄D_i DREADD agonist that displays excellent blood brain barrier penetration. Similarly, Deschloroclozapine (DCZ, Cat. No. 7193) is a high affinity and highly potent activator hM₃D_q and hM₄D_i DREADDs, which displays low 'off-target' binding to other GCPRs, ion channels and transporters. DCZ is particularly suited to in vivo use as it displays 100-fold greater affinity for hM₃D_q and hM₄D_i compared to CNO, and exhibits rapid (approximately 10 minutes after intraperitoneal injection) binding and activation of hM₃D_q and hM₄D_i in mice and non-human primates.

Literature for DREADD Ligands

Tocris offers the following scientific literature for DREADD Ligands to showcase our products. We invite you to request* your copy today!

*Please note that Tocris will only send literature to established scientific business / institute addresses.

Chemogenetics Research Bulletin

Produced by Tocris, the chemogenetics research bulletin provides an introduction to chemogenetic methods to manipulate neuronal activity. It outlines the development of RASSLs, DREADDs and PSAMs, and the use of chemogenetic compounds. DREADD ligands and PSEMs available from Tocris are highlighted.

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Allosteric GPCR Pharmacology Poster

G protein-coupled receptors (GPCRs) are intrinsically allosteric proteins. This poster provides insights into allosteric mechanisms of GPCR biology, highlighting key facets of GPCR allostery and therapeutic applications of allosteric modulators.

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GPCR Efficacy and Biased Agonism Poster

GPCRs can interact with multiple distinct transducers or regulatory proteins and these can be preferentially engaged in an agonist-specific manner giving rise to biased agonism. This poster discusses cutting edge GPCR signaling pharmacology and highlights therapeutic applications of biased agonism.

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