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Bi/CNS 150

Lecture 29.5

Friday, December 7, 2012

“Inside-out” Actions for Psychiatric Drugs

Henry Lester

How do psychiatric drugs work?

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1. Statement of the problem: Antispychotics, Antidepressants, Bipolar drugs

2. Lessons from nAChRS; 3. Pharmacokinetics

4. Detailed hypotheses: Antipsychotic drugs SSRI Antidepressant drugs “Fast” NMDA blocker antidepressants

5. Tests of “inside-out” mechanisms for psychiatric drugs

Psychiatric drugs bind to classical targets within early exocytotic pathways: Therapeutic effects

Biological Psychiatry, Dec 2012 Henry A. Lester, Julie M. Miwa, and Rahul Srinivasan

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Eroom’s law applies especially to neural drugsScannell, Nature Revs Drug Disc. 2012

Contemporary ideas about psychiatric drugshave emphasized binding to

the classical targets at synapses. . .

“Inside-out” mechanisms emphasize binding to the same classical targets, but within the

endoplasmic reticulum and cis-Golgi

Some psychiatric drugs, their targets, logP values, and half lives

fluoxetine (Prozac)

serotonin transporter logP 3.4, 24-72 hr

clozapine (Clozaril)5-HT2A serotonin receptor, GPCR

logP 3.2, 8-12 hr

ketamine (“special K”)

NMDA glutamate receptor logP 2.2, 3-5 hr

chlorpromazine (Thorazine)

dopamine D2 receptor, GPCRlogP 5.2, 16-30 hr

nicotine acetylcholine receptor

logP 1.2, 0.5 -2 hr

recreational / abused / addictive

antipschizophrenic antidepressant

5logP = log (solubility in octanol / water)

BloodLungs

H+

Like most drugs, nicotine is a weak base.Its neutral form passes through 6 plasma membranes in ~ 20 s

logP = 1.1 = log (solubility in octanol / water) 6

NH+

N

N

N

N

N

CSF

Alveolar epithelium Brain capillary

AstrocyteEnd-feet

Endothelialcells

7Nucleus

UPRE

PlasmanACh

R

Nicotine in CSF

Classical Pathway:Channel activation & desensitization

→ Do neurons survive Despite stressors?

Unfolded protein response

membrane

COPII vesicleSec 13/31

Sar1

Sec24Sec23

ATF6

Golgi

PharmacologicalChaperoning→ upregulation

M3-M4 loop

H+ +

ERBiP

PERKIRE1

Clathrin

Secretory vesicle

COPII

Golgi complex

COPI

Early endosome

COPI

Lysosome

Ca2+

Na+

“Inside-out” Drug Action by Nicotine at α4β2 nAChRs

NH+

N

H+

N

N

Endoplasmic reticulum

nAChR

ATF6

IRE1

XBP1

eIF2α

PERK

ATF4

1. Agonist binding eventually favors stable, high-affinity states (a “chaperone”)

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channels

nicotine20 sec

Three possible results of nicotine-nAChR binding in the endoplasmic reticulum

“closed”

AC Highest affinity

Fre

e E

ne

rgy

Reaction Coordinate

“activated”

“desensitized”

Bound states with increasing affinityunbound

agonist

?

2. Nicotine binding at subunit interface favors assembled nAChRs (a “matchmaker”)

3. Nicotine may displace lynx, directing nAChRs toward cholesterol-poor domains (an “escort”)

nicotine

lynx

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R. L. Wiseman, C. M. Haynes, D. Ron Cell 2010

The three arms of the ER stress / unfolded protein response pathway

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During chronic exposure to nicotine, α4β2 nAChRs are selectively upregulated.

Pharmacological chaperoning is necessary but not sufficient for upregulation.

Other sequelae of chaperoning: changed stoichiometry, reduced ER stress and reduced UPR.

Upregulation proceeds similarly in clonal cells, rodent brains, and smokers’ brains.

Inside-out Pharmacology of Nicotine Effects at α4β2 nAChRs

Now we’re assessing gene expression in identified neurons chronically exposed to nicotine.

Inside-out pharmacology is a powerful concept for nearly all CNS drugs:They are all membrane-permeant weak bases.

The discovery criteria for psychiatric drugs lead to excellent intracellular chaperoning

1. High bioavailability implies high membrane permeationAll psychiatric drugs have logP > 2

2. Good stability in the body implies simple or little enzymatic breakdown.Half-life is ~ 1 day.

3. Good selectivity, few off-target effects imply high-affinity binding to the targetKd < 1 μM, often ~ 10 nM

a. “Chaperoning”: (i) Transporter ligands are

organic substratesions,or antagonists,

They favor two major binding states, “inward” vs “outward”.

(ii) GPCR ligands (see next slide): agonistsantagonistsallosteric modulators“inverse” agonists

b. “Matchmaking”:(i) Neurotransmitter transporters must homodimerize before leaving the ER(ii) GPCRs homo- and heterodimerize, in some cases required for ER export,

in some cases favored by ligands 11

Pharmacological chaperoning of GPCRs

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receptor mutant /WT drug class reference

adenosine A1 mutant agonists; antagonists (Malaga-Dieguez et al., 2010)

dopamine D4 bothtransported dopamine; quinpirole; antagonists

(Van Craenenbroeck et al., 2005

)gonadotropin-

releasing hormonemutant antagonists

(Conn and Ulloa-Aguirre, 2011

)

histamine H2 both agonist, inverse agonist (Alewijnse et al., 2000)

opsin mutant -- (Noorwez et al., 2008)

δ-opioid mutant antagonist (Leskela et al., 2012)

μ-opioid mutant agonists, antagonists (Chaipatikul et al., 2003)

melanin conc. hormone

mutant antagonist (Fan et al., 2005)

melanocortin-4 both antagonist, inverse agonist (Tao, 2010)vasopressin V1a both antagonist (Hawtin, 2006)

vasopressin V1b/V3 both antagonist (Robert et al., 2005)vasopressin V2 both antagonists (Wuller et al., 2004)

Enzyme or

channel

Transcription factors

Nucleus

Intracellular messenger

kinase cascade

Drug

+ In CSF

+

Endoplasmic reticulum

GolgiH+

ATF6,

CREB-H

IRE1

XBP1

p-eIF2α

PERK

ATF4

NucleusUPRE

Drug

+ in CSF

Transcription factors

ATF6

+ +

Neutral permeant drug

+

+ +

+

BiP

PERKIRE1

++

H+

ER

Golgi

β-arrestin

A. Inhibition of plasma membrane GPCR , and downstream effects

B. Intracellular pharmacological chaperoning of GPCR, and downstream effects

Most papers suggest . . . We suggest . . .

Two mechanisms for gene activation downstream from antipsychotic drugs

“Nearly” cell-autonomous actions of SSRI antidepressant treatment

Kellermann group 14

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Other diagrams

Samuels & Hen, Eur J. Neurosci, 2011

Adult Neurogenesis

Inside-out actions would occur here

Gene activation is too brief to account for the “therapeutic lag”

Axonal transport provides a natural delay in the “inside-out” mechanism.

Speed: ~ 1 mm / day.

Suggests that equivalent effects would require briefer delays in animals with shorter axons

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Marks et al, 1985

Dendritically localized events

Days of nicotine infusion

Mouse hippocampus

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How does acute ketamine produce antidepressant effects within 2 hr?

Monteggia & Duman groups suggest . . .

(1) involve BDNF synthesis & release, (2) occur in the dendrites,(3) require protein synthesis, (4) do not require gene activation.

The effects

NMDA Receptor

kinases↓

BDNFsecretion

BDNF mRNA

BDNF↑

Dendritic Golgi

Outside-in

Ca2++

Decreased Ca2+ flux

DendriticER

COPII

BDNF mRNA

p-eIF2α↓

pPERK↓

Escorting

BDNF secretion

BDNF↑

Inside-out

BiP

PERKIRE1

H+

+

ER

+ +

+

+ +

NMDA Receptor

NH2+

H3C

H+

O

Cl

NHH3C

O

Cl

We suggest . . .

“Acid trapping” of nicotine might1.keep nAChRs

desensitized untilthey are exocytosed;

2.serve as a reservoir for nicotine

Cell nACh

R membrane

Clathrin

Endoplasmic reticulum

nAChR

Secretory vesicle

COPII

Golgi complex

COPI

Early endosome

COPI

Lysosome

pH

5.2 100

6.0 30

6.7 3

6.3 20

6.5 10

7.2 1

nic+

nicCSF

Nicotine in CSF

NH+

N

H+

N

N

See detailed calculations for antipsychotics:Tischbirek et al, Neuron 2012

7.2 1 &

What knowledge do we need next?

As usual, we need cell biology & biochemistry

1. Reconstituted, cell-free systems for ER exit and retrieval

2.Better real-time markers for compartmentalized receptors and transportersa. Imaging mass spectrometryb. Plasma membrane binding only? Possible with impermeant derivativesc. ER binding only? More challenging, especially for antagonists.

3.Better measurements of pathway-specific gene activation (RNA-Seq)

4.Analyze newly synthesized proteins

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Polygenicthe disease occurs only if several genotypes are present together

Genetically Multifactorialseveral distinct genes (or sets of genotypes) can independently cause the disease

Partially penetrantnongenetic or epigenetic factors are required, or the disease is inherently stochastic

PolygenicGenetically Multifactorial

PartiallyPenetrant

Three concepts used in describing complex diseases such a schizophrenia

Contemporary ideas about psychiatric diseaseshave emphasized synaptic and signaling deficits . . .

“Inside-out” mechanisms emphasize that ~30% of a cell’s proteins enter the ER,

and additional nuclear and cytoplasmic proteins control their synthesis & trafficking.

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GABAergic “chandelier cell” in human cerebral cortex hasmany large axon terminals . . .

DeFelipe, Brain (1999) 122, 1807 (Cajal Institute, Madrid)

PyramidalCells

Ch terminals

~ 100 μm

Ch terminals

Ch axon

. . . and plentiful somatic ER

Jones, J. Comp. Neurol., 1984

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Bi/CNS 150

End of Lecture 29.5