DREADDs WEEK 6

Question 1

DEFINE DREADDs

Answer 1

Designer Receptors Exclusively Activated by Designer Drugs

Question 2

Controlling neural activity…PROBLEM? = 5

Answer 2

1. • Neural activity is the foundation of the nervous system
2. • Manipulating neural activity allows us to breakdown the function of the nervous system, the role of specific cells and circuits, and behaviour
3. • Historically, lesion studies or direct electrical stimulation were the main
     ways of manipulating neural
     activity

4 * Global vs local control

5 * Problem: How can we manipulate neural activity in specific circuits while leaving other circuits intact?

Question 3

CHEMOGENETICS …PROCESS… METHOD…

Answer 3

1. • A process where macromolecules can be engineered to interact with a previously unrecognised small molecules
2. • A method where proteins are engineered to interact with small molecular chemical actuators that they did not previously recognise

Question 4

Chemogenetic receptor criteria: 3

Answer 4

1) Not be receptive to endogenous ligands

2) Have minimal/no endogenous activity in the absence of ligand binding

3) Have high affinity for the ligand

Question 5

CHEMOGENETIC = Wide range of engineered proteins: 4

Answer 5

1. Kinases
2. Non-kinase enzymes
3. ’ GPCRs ‘
4. ‘Ligand gated ion channels’

Question 6

DREADDs = 5

WHAT ARE THEY?

Answer 6

1. • A subclass of engineered GPCRs
2. • Activation of DREADDs alters neuronal activity
3. • Clozapine-N-Oxide (CNO), a pharmacologically inert* metabolite
     of the antipsychotic drug clozapine is commonly used to activate DREADDs
4. • DREADDs were first derived by mutating the human M3 muscarinic
     receptor (hM3)
5. • hM3Dq refers to human M3 muscarinic DREADD receptor couple to Gq (one of the excitatory G proteins)

Question 7

hM3Dq = 3

Answer 7

1. • Activation of hM3Dq enhances neuronal firing by activating Gq signalling in neuronal and nonneuronal cells
2. • CNO activation of hM3Dq
     depolarises neurons which can be used to drive behaviours (e.g. feeding)
3. • Activation of hM3Dq also
     induces intracellular calcium release > can be used to alter astrocyte function/activity

Question 8

WHAT IS hM4Di? = 4

Answer 8

1. • hM4Di refers to human
     muscarinic DREADD receptor
2. • Used to inihibit neural activity by activating Gi-mediated signalling
3. • Activates potassium channels and hyperpolarises cells
4. • Can “silence” synapses by inhibiting neurotransmitter release

Question 9

Silencing synapses with hM4Di… 2

Answer 9

• Applying CNO to presynaptic neurons transfected with hM4Di increases the rate of synaptic failure
• (the rate at which presynaptic action
  potential firing fails to produce a current in the postsynaptic cell)

Question 10

Axon selective hM4Di = 2

Answer 10

1. • hM4Di causes hyperpolarisation (K+ efflux)
     and synaptic release inhibition
2. • If hM4Di is selectively located on axons/axonal terminals, its primary effect will be to inhibit synaptic release

Question 11

K opioid derived DREADD (KORD) = 5

Answer 11

1. Newer subtype of DREADD
2. • Activated by salvorin B which is pharmacologically inert and has no known other molecular target
3. • Activation of KORD induces hyperpolarisation and inhibits neurotransmitter release
4. • Strong inhibitor of
     neurotransmitter release
5. • Can be combined with a CNOactivated hM3Dq DREADD for bidirectional control of neural activity

Question 12

what is CNO? WHAT DOES DO? DOSES? = 4

Answer 12

1. • Activates DREADDs with low nanomolar concentrations of CNO
     – ~0.5-10mg/kg
2. • Long lasting- 1 injection lasts
     ~60 minutes in vivo
     – EC50~8.1nM
3. • Low doses of CNO used when you want short lasting
     DREADD activation
4. • CNO can back metabolise to clozapine and could have clozapine side effects, but the extent of back metabolism is low at the concentrations of CNO administered

Question 13

CNO – STUDIES = 4

Answer 13

1. • Studies have shown that CNO is not as exclusive as initially reported
2. • Reports of off-target effects throughout the CNS
3. • Some targets include serotonin receptors, muscarinic receptors, histamine receptors,
     dopamine receptors
4. • Higher doses doesn’t always result in changes in behaviour

Question 14

CNO alternatives? = 6
Compound 21:

Answer 14

1. • Compound 21:
2. • A more potent agonist of the hM3Dq DREADD than CNO
3. – EC50 ~ 2.95
4. • Can use lower concentrations for a similar response
     <0.5mg/kg
5. • Doesn’t seem to metabolise into CNO or CLZ

6 * Minimal off-target behavioural effects

Question 15

CNO alternatives?

• Deschloroclozapine:

Answer 15

1. • Can be used a very low doses
     – ~0.001-0.1mg/kg
2. • Minimal off target effects

Question 16

Salvorin B = 3

Answer 16

1. • Salvorin A is a psychotropic agent
2. • Salvorin B is an inert ligand
3. • Short lasting effect (~5 minutes)

Question 17

DREADDs to interrogate behaviour = 4

Answer 17

1. • Express DREADDs in specific cell types and in brain regions/locations
2. • The time of DREADD activation can be Controlled (i.e. when the drug is administered). Can observe behaviours before/during/after DREADD activation
3. • Salvorin B activated DREADDs are more
     useful when you want to observe the effects of transiently changing neural
     activity
4. • CNO activated DREADDs are more useful when you want to observe the effect of
     tonically changing neural activity (e.g. serotonergic neurons)

Question 18

DREADDs in drug development = 3

Answer 18

1. • DREADDs can identify which GPCR pathways are useful therapeutic targets
2. • “Biased” drug design
3. • Targeting specific cell types, within a certain circuit narrows down the effect and
     consequences of targeting a specific G protein pathway

Question 19

Synthetic ion channel: GluCl
= 6

Answer 19

1. • Aimed at modifying the membrane potential
2. • Modified glutamate gated chloride channel
3. • Ivermectin (anti-parasitic drug) activates GluCl
4. • Ivermectin activation occurs at low concentrations (5mg/kg)
5. • High concentrations of ivermectin activates GABAA
     receptors>tremors and paralysis
6. • Long lasting behavioural effects (days) after a single administration

Question 20

Synthetic ion channel: Glycinergic channel = 4

Answer 20

1. • Glycine gated chloride channels regulate inhibitory neurotransmission in the spinal cord and brainstem
2. • Glycine gated chloride channels also expressed non-synaptically in the brain
3. • Very low concentrations (10nM) increases the amount of chloride ion flow/post-synaptic inhibition
4. • So far, only appears to be effective in regulating spinal cord neurons