Bi/CNS 150 Lecture 29.5 Friday, December 7, 2012 “Inside-out” Actions for Psychiatric Drugs

1. Bi/CNS 150 Lecture 29.5 Friday, December 7, 2012 “Inside-out” Actions for Psychiatric Drugs Henry Lester

2. How do psychiatric drugs work? • Statement of the problem: • Antispychotics, Antidepressants, Bipolar drugs • Lessons from nAChRS; • 3. Pharmacokinetics • 4. Detailed hypotheses: • Antipsychotic drugs • SSRI Antidepressant drugs • “Fast” NMDA blocker antidepressants • 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

3. Eroom’s law applies especially to neural drugs Scannell, Nature Revs Drug Disc. 2012

4. Contemporary ideas about psychiatric drugs have 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

5. Some psychiatric drugs, their targets, logP values, and half lives antipschizophrenic antidepressant ketamine (“special K”) NMDA glutamate receptor logP 2.2, 3-5 hr chlorpromazine (Thorazine) dopamine D2 receptor, GPCR logP 5.2, 16-30 hr recreational / abused / addictive nicotine acetylcholine receptor logP 1.2, 0.5 -2 hr clozapine (Clozaril) 5-HT2A serotonin receptor, GPCR logP 3.2, 8-12 hr fluoxetine (Prozac) serotonin transporter logP 3.4, 24-72 hr logP = log (solubility in octanol / water)

6. Like most drugs, nicotine is a weak base. Its neutral form passes through 6 plasma membranes in ~ 20 s Alveolar epithelium Brain capillary Endothelial cells Astrocyte End-feet Lungs Blood CSF H+ logP = 1.1 = log (solubility in octanol / water)

7. Na+ “Inside-out” Drug Action by Nicotine at α4β2 nAChRs nAChR membrane Classical Pathway: Channel activation & desensitization Ca2+ Plasma Golgi Clathrin Early endosome Secretory vesicle Nicotine in CSF COPI ATF6 Lysosome Golgi complex ATF6 COPI Pharmacological Chaperoning→ upregulation COPII Endoplasmic reticulum COPII vesicle Sec 13/31 nAChR PERK IRE1 M3-M4 loop ATF4 Sec24 Sec23 eIF2α Unfolded protein response Sar1 → Do neurons survive Despite stressors? H+ XBP1 UPRE + BiP ER PERK Nucleus IRE1

8. Three possible results of nicotine-nAChR binding in the endoplasmic reticulum 2. Nicotine binding at subunit interface favors assembled nAChRs (a “matchmaker”) • Agonist binding eventually favors stable, • high-affinity states (a “chaperone”) agonist Bound states with increasing affinity unbound 106 channels “closed” AC ? “activated” Highest affinity nicotine 20 sec Free Energy “desensitized” Reaction Coordinate 3. Nicotine may displace lynx, directing nAChRs toward cholesterol-poor domains (an “escort”) nicotine lynx

9. The three arms of the ER stress / unfolded protein response pathway R. L. Wiseman, C. M. Haynes, D. Ron Cell 2010

10. Inside-out Pharmacology of Nicotine Effects at α4β2 nAChRs During chronic exposure to nicotine, α4β2 nAChRs are selectively upregulated. Now we’re assessing gene expression in identified neurons chronically exposed to nicotine. Pharmacological chaperoning is necessary but not sufficient for upregulation. Upregulation proceeds similarly in clonal cells, rodent brains, and smokers’ brains. Other sequelae of chaperoning: changed stoichiometry, reduced ER stress and reduced UPR. Inside-out pharmacology is a powerful concept for nearly all CNS drugs: They are all membrane-permeant weak bases.

11. The discovery criteria for psychiatric drugs lead to excellent intracellular chaperoning • 1. High bioavailability implies high membrane permeation • All 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 target • Kd < 1 μM, often ~ 10 nM • a. “Chaperoning”: • (i) Transporter ligands are • organic substrates • ions, • or antagonists, • They favor two major binding states, “inward” vs “outward”. • (ii) GPCR ligands (see next slide): • agonists • antagonists • allosteric 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

12. Pharmacological chaperoning of GPCRs

13. Two mechanisms for gene activation downstream from antipsychotic drugs Most papers suggest . . . We suggest . . . B. Intracellular pharmacological chaperoning of GPCR, and downstream effects • Inhibition of plasma membrane GPCR , • and downstream effects Golgi Drug+ in CSF + + bg a ATF6 Golgi H+ β- arrestin Enzyme or channel Neutral permeant drug Drug+ In CSF Intracellular messenger + ATF6, CREB-H Endoplasmic reticulum kinase cascade + + PERK + + ATF4 p-eIF2α IRE1 Transcription factors Transcription factors Nucleus Nucleus UPRE + + H+ ER BiP IRE1 PERK XBP1

14. “Nearly” cell-autonomous actions of SSRI antidepressant treatment Kellermann group

15. Adult Neurogenesis Inside-out actions would occur here Other diagrams Samuels & Hen, Eur J. Neurosci, 2011

16. 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 Mouse hippocampus Marks et al, 1985 Days of nicotine infusion Dendritically localized events

17. How does acute ketamine produce antidepressant effects within 2 hr? Inside-out (1) involve BDNF synthesis & release, (2) occur in the dendrites, (3) require protein synthesis, (4) do not require gene activation. The effects Monteggia & Duman groups suggest . . . We suggest . . . BDNF secretion Outside-in NMDA Receptor Ca2+ + BDNF secretion Decreased Ca2+ flux Escorting Dendritic Golgi pPERK↓ BDNF↑ kinases↓ + + + + + COPII Dendritic ER p-eIF2α↓ BDNF mRNA NMDA Receptor BDNF↑ BDNF mRNA H+ + ER BiP IRE1 PERK

18. nAChR • “Acid trapping” of nicotine might • keep nAChRs • desensitized until • they are exocytosed; • serve as a • reservoir for nicotine membrane Cell pH nic+ Clathrin nicCSF 5.2 100 Early endosome Secretory vesicle COPI 6.0 30 Nicotine in CSF Lysosome 6.3 20 Golgi complex COPI 6.5 10 COPII Endoplasmic reticulum 6.7 3 7.2 1 nAChR & 7.2 1 See detailed calculations for antipsychotics: Tischbirek et al, Neuron 2012

19. What knowledge do we need next? • As usual, we need cell biology & biochemistry • 1. Reconstituted, cell-free systems for ER exit and retrieval • Better real-time markers for compartmentalized receptors and transporters • a. Imaging mass spectrometry • b. Plasma membrane binding only? Possible with impermeant derivatives • c. ER binding only? More challenging, especially for antagonists. • Better measurements of pathway-specific gene activation (RNA-Seq) • Analyze newly synthesized proteins

20. Three concepts used in describing complex diseases such a schizophrenia Polygenic the disease occurs only if several genotypes are present together Genetically Multifactorial several distinct genes (or sets of genotypes) can independently cause the disease Partially penetrant nongenetic or epigenetic factors are required, or the disease is inherently stochastic Polygenic Genetically Multifactorial Partially Penetrant

21. Contemporary ideas about psychiatric diseases have 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.

22. Pyramidal Cells GABAergic “chandelier cell” in human cerebral cortex has many large axon terminals . . . . . . and plentiful somatic ER Ch axon Ch terminals Ch terminals ~ 100 μm Jones, J. Comp. Neurol., 1984 DeFelipe, Brain (1999) 122, 1807 (Cajal Institute, Madrid)

23. Bi/CNS 150 End of Lecture 29.5