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Home , Mescaline

How structure can facilitate GPCR discovery OUTLINE

• 5-HT2C: Making safe and effective anorectic and antipsychotic • : using structural information to design novel probes • Or: a tale of two receptors Team 2B 5-hydroxytryptamine controls many central and peripheral functions (Berger, Gray and Roth, Annual Reviews in Medicine, 2009) 5-HT2 Receptors G-Protein Coupled Receptors

5-HT1 5-HT2A

5-HT2 5-HT2B

5-HT 5-HT3

5-HT4 Cation Channel

5-HT5

5-HT6

5-HT7 5-HT2 Receptors G-Protein Coupled Receptors LSD- like

5-HT1 5-HT2A

5-HT2 5-HT2B

5-HT 5-HT3 2C

5-HT4 Cation Channel

5-HT5

5-HT6

5-HT7 Hallucinogens mediate their effects on via agonism at 5-HT2A receptors (LSD, , mescaline)

Nichols, Pharmacology and Therapeutics, 2003 Science 2009

Ki for 1 receptor ~14 M

Using the Similarity Ensemble Approach (SEA) genome-wide predictions of drug off-target actions made (Keiser et al, Nature Biotech 2007; Keiser et al, Nature, in press) SEA predicts 5-HT receptors as target (Keiser et al, Nature, 2009)

Activity Class E-value

5-HT1B 3.1E-21

DMT 5 HT2A 1.2E-13 5 HT5A Agonist 1.1E-7

5 HT7 5E-6

5-HT1B: 129 nM 5-HT2A: 127 nM 5-HT7A: 206 nM DMT induces HTR in WT Mouse but not 5HT2A knockout (Keiser et al, Nature, 2009)

B. Roth et al, unpub 5-HT2 Receptors G-Protein Coupled Receptors

5-HT1 5-HT2A

5-HT2 5-HT2B Valvular heart disease 5-HT 5-HT3 2C

5-HT4 Cation Channel

5-HT5

5-HT6

5-HT7 5-HT2 Receptors G-Protein Coupled Receptors

Appetite 5-HT1 5-HT2A

5-HT2 5-HT2B

5-HT 5-HT3 2C

5-HT4 Cation Channel

5-HT5

5-HT6

5-HT7 The „valvulopathy receptor‟

1992: Study shows that and , when taken together, are effective appetite suppressants with fewer side effects than either drug alone. The combination of phentermine and fenfluramine becomes known as “fen/phen”

1997: In July, Connolly et al reported 24 cases of valvular heart disease in women treated with fenfluramine and phentermine. Soon after, similar reports accumulate in patients taking either fenfluramine or fenfluramine with phentermine

2010: Single largest pharmaceutical product liability suit ($26 Billion and counting) http://www.gwc.maricopa.edu/class/ bio202/cyberheart/hartint0.htm

A2 A1

- -

B A

- -

3 1

- -

HT6 HT1D HT7 HT5 HT2C HT2B HT2A HT1E HT1B HT1A

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ALPHA1A AMPA D5 5 5 EP EP ADENOSIN ADENOSIN ALPHA1B BETA2 BETA1 ALPHA2C ALPHA2B ALPHA2A PCP NMDA H2 H1 OXYTOCIN V3 V2 V1 BZP GABA GABA DAT NET SERT M5 M4 M3 M2 M1 KAPPA DELTA MU D4 D3 D2 D1 5 5 5 5 5 5 5 5 Ki (nM) (+/-) Fenfluramine HCI 5-HT2B (+) Fenfluramine HCI

(-) Fenfluramine HCI

Methysergide Maleate

Ergotamine Tartrate

Phentermine

Fluoxetine

Norfluoxetine

(+/-)

(+) Norfenfluramine

(-) Norfenfluramine

Rothman et al. Circulation. 2000;102:2836; O’Connor and Roth, Nature Drug Discovery 2005 Agonist Activity Of Fenfluramine At Cloned Human 5-HT2B Receptors

5-HT (EC50 1 nM)

Fenfluramine (EC50 = 600 nM)

Norfenfluramine (EC50 = 80 nM)

120

90 PI Accumulation (% max 5-HT 60 response) 30

0 -2 -1 0 1 2 3 4 Log (drug [nM])

RothmanRothman et etal. al, Circulation Circulation,. 2000;102:2836. 2000; Setola et al, Mol Pharmacol 2003; Roth NEJM 2007 5-HT2B transactivates TGF- pathway

Roth, NEJM, 2007 Why does S-norfenfluramine

favor the 5-HT2B receptor?

• 5-HT2B model constructed using validated 5-HT2A-rhodopsin-based model • Non-conserved residues near the putative binding pocket identified • Each mutated to corresponding 5-

HT2A or 5-HT2C sequence • Binding affinity of S-norfenfluramine measured The $26 billion methyl

Setola et al, Mol Pharm 2005 “The most fruitful basis for the discovery of a new drug is to start with an old drug” Sir James Black

Psilocybin—orally bioavailable, nontoxic —5-HT2A/2B/2C prodrug agonist: anorexia and anti-OCD actions + 2 additional, novel chemotypes

Compound 5-HT2A 5-HT2B 5-HT2C

Lorcaserin 406 +/- 8 158+/- 40 6 +/- 2 (93%) (Phase III)

O-4284 Inactive Inactive 16±3.7 (80±2%)

O-3944 520±275 (38±8%) Inactive 6.9±0.7 (88±5%)

WAY 161503 56 +/- 1 20 +/- 5 2 +/- 0.8 (92%) (Phase II)

“SAR of Psilocybin Analogs: Discovery of a Selective 5-HT2C Agonist” H. Sard, G. Kumaran, C. Morency, B. Roth, B. Toth, P. He, and L. Shuster Bioorg. Med. Chem. Lett. 2005, 15, 4555. Orally-active 5-HT2C-selective agonists have anti-OCD and anorectic actions in vivo Sard et al, Bioorg Med Chem Lett, 2005 U.S. and World-wide Patents Licensed to Galenea Pharmaceuticals 2008 NH NH N NH Cl N N NH Cl Cl N O HTS -> H (2) (3) WAY 161503 (4) WAY 629 (5)

N F a F H NH NH2 NHBoc Et H N NH2 OH OH H 9 10 O CF3 8 BMS pyrazinoisoindoline (6) (-)-trans-PAT (7) F F . b NHBoc c NH2 HCl O O

11 12

(+) 12 Antipsychotic drug with (+) 12 anorectic and (+) 12 actions (Cho et al, J Med Chem 2009; Kozikowski et al, revision) Team Sally D A B

Salvinorin A is one of the components isolated

Sheffler and Roth. Trends Pharmacol Sci. 2003;24:107. Effects Of Salvinorin A • Effects are nearly instantaneous (ie, before smoked material is exhaled) • Memory of having ingested a “drug” is frequently lost (ie, anterograde amnesia) • Users transported to “alternative/parallel universe” • Effects wear off quickly (eg, rapid ) • Unlike effects of traditional LSD-like hallucinogens LSD

Roth et al. Proc Natl Acad Sci USA. 2002;99:11934. In Vivo Effects Of Salvinorin A Abolished In - Receptor Knock-Out Mice 10 Vehicle Salvinorin A Naloxone 8

Tail 6 Flick Latency 4

2

0 Wild-Type KOR Knock-Out

Chavkin et al,.JPET 2004. A B

Roth et al. Proc Natl Acad Su USA. 2002;99:11934; Yan et al, Biochem 2005; Vortherms et al, JBC 2008; Yan et al, Biochem 2009 The C-2 Position of Salvinorin A Interacts with Y313 in the Binding Pocket, and C315 is Nearby

C315

Y313 C-2

Yan, et al. Biochemistry, 2009 RB-64 is Extraordinarily Potent

Salvinorin A RB-48 RB-64

2 1

3 35 Ki ([ H]U69593 binding) EC50([ S]GTPγS binding)

Salvinorin A 1.85 1.49 nM 17 6 nM

RB-48 2.10 0.84 nM 0.19 0.01 nM

RB-64 0.59 0.21 nM 0.077 0.016 nM The Mechanism for Affinity Labeling – Nucleophilic Substitution Reaction

isothiocyanate

Yan, et al. Biochemistry, 2009 Flag Separation by SDS-PAGE

His6

175 kDa

83 kDa

62kDa 52 kDa 47.5 kDa 47kDa

32.5 kDa

25 kDa

Coomassie Staining Anti-Flag Anti-KOR AB 4800 MALDI TOF/TOF

Resolve

Detection

Source

Applied Biosystems Mass Spec of Chymotryptic Peptides by MALDI TOF/TOF REVEALS SITE OF ADDUCT FORMATION Prepulse Inhibition (PPI) study in mice

Background 62 dB

Prepulse +4 dB +8 dB +12 dB +16 dB /20ms www.med-associates.com/startle/ en.wikipedia.org Pulse 120 dB/100 ms RB-64 is 20-fold more potent than salvinorin A for disrupting PPI

2 mg/kg Salvinorin A 0.1 mg/kg RB-64 GPCRs exist in multiple conformations which can be differentially stabilized by G-protein, ligands and scaffolding proteins (Urban et al, JPET 2007)

Gαi βArr Gβγ Gβγ

cAMP Internalization GIRK activation

FUNCTIONAL-SELECTIVITY The Modified Ternary Complex Model Predicts One Active State

R + L RL

R* + L R*L

R*G + L R*GL

Constitutively active GPCR Unitary active state CAN G PROTEINS DIFFERENTIALY ALTER LIGAND BINDING, AND EFFICACY, BY INDUCING RECEPTOR- SPECIFIC CONFORMATIONAL CHANGES? Extended Model

R+G16 + L R + L RL

R*-G16 + L R* + L R*L

R*G16-L R*G + L R*GL

++ Ca Mobilization Chavkin et al, JPET 2004; Yan et al, Biochem 2005 cAMP Over-expressed G proteins Force Distinct Patterns of G - Protein Coupling

Yan, et al. Biochemistry, 2008 G protein-Coupling Selectively Modulates

Salvinorin A Binding Affinity

in in Affinity Binding

Fold -

Yan, et al. Biochemistry, 2008 Conformational Changes can be Detected by Substituted Cysteine Accessibility Mapping

Conformation Active conformation Inactive conformation

Protein Surface

Water Accessible Surface

SCAM Pattern

Inhibition of Binding Pseudo-1st order rate constants for reactivity derived Mapping of 1st order rate constants reveals residues differentially altered by G protein environment

Side View Top View

Yan, et al. Biochemistry, 2008 Conclusions

• GPCR model-based drug design can be remarkably successful • Rational medicinal chemistry and SEA (cheminformatics/computational) essential • Compounds developed have demonstrated efficacy in vivo •NIMH Screening Program (NO180002) •NIMH-National Collaborative Drug Discovery Group (U19MH82114; UNC-Duke-Wyeth) •NIDA RO1DA017204 •Michael Hooker Chair

•Salvinorin story: •Feng Yan and Tim Vortherms •Rick Westkaemper lab (MCV) •Jordan Zjawiony lab (Univ Mississippi)

•5-HT2B story •Niels Jensen, John Allen, XP Huang, Vincent Setola •Richard Rothman lab (NIDA) •Alan Kozikowski lab (UIC) •Howard Sard (Organix)