Druggability Considerations for GPCRs and Ion Channels

Shaun R. Stauffer

6th Drug Discovery for Neurodegeneration Conference

February 12-14, 2012

1. CNS drug development statistics and the hope for BACE inhibitors

2. Druggability considerations for GPCRs

Orthosteric versus Allosteric Approaches

M1/M4 PAMs- receptor reserve and probe dependence

mGluR5 PAMs- ‘mode switching’ and allosteric agonism

3. Summary and Outlook

Outline

Tremendous need: neurological and psychiatric conditions account for 13% of the global burden of

disease

CNS drugs spend 8.1 yrs in human testing, more than 2 yrs longer than average for all agents

Regulatory approval of CNS drugs takes longer- 1.8 yrs vs1.2 yrs for all drugs

8.2% of CNS drug candidates that begin human testing will reach marketplace vs. 15 % for drugs

overall

46% of CNS candidates succeed in late-stage (phase III) trials, compared with 66% for all drugs

Evaluation of clinical improvement more difficult- schizophrenic episodes or cognitive

improvement in Alzheimer’s patients more variable and require outcomes trials for therapies

aimed at disease modification

New coalitions emerging to bring government agencies, drug companies and patient advocacy

groups together, to develop a standardized clinical trials database to allow researchers to design

more efficient studies for new treatments and share the risk for development.

A Dearth of New Meds: Drugs to treat neuropsychiatric disorders have become too risky for Big Pharma.K. I. Kaitin, C. P. Milne Scientific American, Aug. 2011.

CNS drug development challenges

VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK

b-secretase g-secretase

Ab1-40 (major)

Ab1-42 (minor)sAPPb

• a-Secretase pathway – Predominant, sAPPa neurotrophic (non-amyloidogenic)

a-secretase

sAPPa

LVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK

• b -Secretase pathway – Minor, normal in development/repair and pathologic

(Ab) roles (oligomerization, “amyloidogenic”)

Amyloid Precursor Protein (APP) Proteolysis: A Fork in the Road

Inhibition of b-secretase (BACE) should impede the production of the peptide Ab, hence slowing the progress of Alzheimer’s disease

BACE Active Site Properties

• Membrane associated aspartyl protease within pepsin family• First cloned and purified in 1999• Large, open, hydrophilic cleft • Complementary binding to extended b-strand inhibitor/substrate to achieve potency

H2N

HN

NH

HN

HN

NH

HN CO2H

CO2H

OMeMe

O O

NH2

O

OH Me

O Me

O

O

CO2H

Me

Me

P1'

P1

P2

P3L. Hong, J. Tang et al, Science 2000, 290, 150

~1 nM BACE-1

Key Achievement

Tang and coworkers, Science 2000, 290, 150-153.

BACE Inhibitor Challenges

• Best known inhibitory motifs are Transition State Analogues (TSA’s)• Problem: TSA’s historically have poor brain penetration (Ritonavir®),

CYP inhibition and poor oral bioavailability (Renin inhibitors)

How can we achieve BACE inhibition in the CNS?Average properties of marketed CNS drugs:small, rigid, Pgp <2.5, LogP >2, MW ~320; HBD ~1, HBA ~2, PSA ~41

K.M. Mahar Doan, J.W. Polli, et al. J. Pharmacol. Exp. Ther., 2002, 1029-1037

H2N

HN

NH

HN

HN

NH

HN CO2H

CO2H

OMeMe

O O

NH2

O

OH Me

O Me

O

O

CO2H

Me

Me

P1'

P1

P2

P3L. Hong, J. Tang et al, Science 2000, 290, 150

~1 nM BACE-1

Aspartyl Protease Transition State Analogues

Rich, D. J. Med.Chem., 2002,45, 541.; Greenlee, W. J. J. Med. Res. Rev. 10, 173, (1990)

HN

O P1

OH P1'

O

HN

O P1

OH P1'

O

hydroxyethylene (HE)

OH

di-hydroxyethylene (DHE)

HN

O P1

OH O

statine-based

n

HN

HN

O P1

OH

hydroxyethyamine (HEA)

P1'

O HN P

O P1

OH

phospinate-based

P1'

O

HN

O P1

NH2

aminoethylene (AE)

P1'

O

HN

O P1

NH2 O

aminostatine (AS)

n O

P1' HN

NHO P1

P1'

O

reduced amide (RA)

HN

NH

HN

O P1

P1'

O P2'

OHO OH

transition state

Coburn et al, J. Med. Chem., 2004, 47, 6117

• Screening a 5 million member compound library yields a single lead

Merck-Neogenesis Collaboration

Stachel et al, J. Med. Chem., 2004, 47, 6447V. John et al, J. Med Chem., 2004, 47, 158 (Elan)S. Kaldor et al, Bioorg. Med. Chem. Lett., 1995, 5, 721 (HIV)

NH

O

HN

OOH H

N

NS

O O

BACE-1 IC50 = 10 nMMW 578

HEA incorporation

Rich, D. et alJMC 2002, 45, 541

P1'

P1

P3

P3 + P1 amidesP2 sulfonamideHEA

PGPsubstrate

1

2

ON

ONH

O

NH

O

NH2

BACE-1 IC50 = 25,000 nMMW 5063 HBD4 HBA

P2 Opt.

P1 /TSA Opt.

NHPh

O

HN

OOH H

N

Ph

NS

O O

P3P1

P1'

P2

2

hydrophobic regions: S1, S3 and S2’

S2’

S1, S3

X-Ray of HydroxyEthylAmine (HEA)

Emerging Chemical Methods Enables Truncation of HEA

NH

O

HN

OO

NS

O O

F

O

1) Rh(acac)(C2H4)2 / rac-BINAP

(HO)2B

R

N

2) LiBH4

NH

O

HN

O

NS

O O

F

OH

Ar

Ar =

35 / 48 successfully isolated

F

Cl

HO2CN

SO

HN

+ meta isomer

FF H

N

O 4 IC50 = 28 nM

SAR: Alkyl Branch and P1

Entry

O

HN

N

NS

OO

NH

R'

NH2

R'

BACE-1(IC50 nM)

sAPPNF(IC50 nM)

10 n-Bu 9 110

9 n-Bu 20 110

11

n-Bu 5 38

13

-CH2CH2CF3 2 71

R

R

S

F

F

12

CH3 32 54"

Ph

Ph

8 H 34 279Ph

Stauffer, S. R. et al. Bioorg. Med. Chem. Lett., 2007, 17, 1788.

Origin of P3 Potency Enhancement?

• New H-bonding manifold for aminopyridine?• 10s loop conformational change (S3 pocket, residues 9-14)?

Apo BACE-1,10s dynamics: J. Yon, et al. J. Mol. Biol. (2004) 343, 407.Renin S3sp: J. Rahuel, et al. Chem. Biol., 2000, 493.McGaughey, G. B. et al. Bioorg. Med. Chem. Lett., 2007, 17, 1117.Stauffer, S. R. et al. Bioorg. Med. Chem. Lett., 2007, 17, 1788.

BACE-1 IC50 = 351 nMsAPP_NF IC50 = 518 nM

O

HN

NS

OO

S

NH2O

NH

FO

HN

N

NS

OO

HN

BACE-1 IC50 = 5 nMsAPP_NF IC50 = 38 nM

S

NH2

Pocket Collapses via Ligand-Dependent Conformational Change

Ser10

Thr232

G. McGaughey

Pocket Collapses via Ligand-Dependent Conformational Change

Fast forward: Carbinamines, Spiropiperidines, and Acyl Amidines

N

N X

Ph

NS

O O

Low CNS penetration 0.05 b/pPSA >100Log P 2.5 – 4.0MW >500HBD/HBA = 2/4

BACE-1 IC50 = 0.4 nMsAPPb_NF IC50 = 40 nMP-gp ratio(h) = 2, Papp = 22HBD/HBA = 1/6cLogP = 2.6, PSA = 120 Å2

40% reduction Rhesus CSF Ab40after IV infusion high metabolism, low %F

N

N

NS

O O

ON

N

PhNH2

O Cl

Stanton, M. et al. J. Med. Chem. 2007, 3431.Sankaranarayanan, S. et al. J.Pharm. Exp. Ther. 2009, 131-140.

Barrow, J. et al. J.Med.Chem. 2008, 6259.

NN

N

O

NH

F

CH3

BACE-1 IC50 = 330 nMsAPPb_NF IC50 = 4200 nM

N

NH

O NH

R3R2

R1

fragment-based discoveryMK-8931 entering PhIIrobust A b reduction in HV

Zhu, Z. et al. J.Med.Chem. 2010, 951.US20080103351US20080200445US20070287692

Persistence and serendipity!

• Classical GPCR ligands modulate signaling through the orthosteric site by:– Blocking the native agonist (competitive antagonist)– Directly stimulating a receptor response (agonist)– Blocking constitutive activity (inverse agonist)

• Functional assays identify:– Negative allosteric modulators– Positive allosteric modulators– Allosteric agonists– Neutral cooperativity

Allosteric Modulators Offer Advantages• Selectivity• Mimic physiological conditions• No desensitization, down regulation or internalization• Less side effects

Challenges: Steep/Flat SAR, ‘mode switching’

COOH

NH2

transmembraneheptahelical domain

(7TM domain)

orthostericbinding site

allostericbinding site(s)

G protein-coupled receptors

Allosteric modulators can act at multiple distinct, often overlapping, but also non-overlapping sites on the same receptor. SAR fails to translate.

Shallow (steep or flat) SAR and difficult to add polar and/or solublizing groups to generally small, lipophilic chemotypes

Allosteric modulators can differentially regulate coupling of mGlus to different signaling pathways

Members of a single structural class can have a range of activities from PAM to NAM and can include neutral ligands, ago-PAMs, allosteric agonist to partial antagonist

‘Molecular Switch’ – unexpected alteration of pharmacology within an established series due to subtle, single heavy atom modifications.

Druggability Challenges for Allosteric Ligands of GPCRs

Wood, et al., Biochemistry 2011, 50, 2403-2410.

Receptor Reserve: Excess Receptors Beyond Those Necessary for a Maximal Response

• High Receptor Reserve: Potency < Affinity

• Low Receptor Reserve: Potency ≈ Affinity

• In vivo there is a large range of mAChR receptor reserve levels

• In a given cell, mAChR coupling to distinct pathways can have different receptor reserves

NNOEt

ONH

O VU0364572 allosteric agonist/PAMHigh reserve M1 EC50 = 110 nM (96% AcH max)Low reserve M1 EC50 = 1300 nM

Highly selective (M2-M5, Ricerca)Rat CLp = 14.7 mL/min/kg, %F 37Brain AUC/Plasma AUC = 1.4Potentiate NMDA currents in hippocampal CA1

Lebois, E.P. et al. Bioorg. Med. Chem. Lett. 2011, 6451.

Receptor Reserve – Weak Partial Agonist Considerations

• Weak partial agonists can have increased efficacy and potency in high receptor reserve

• Weak partial agonists can look like antagonists in low receptor reserve

• High receptor reserve systems set the highest bar for identifying antagonists

• This is critical for an antagonist program as it is the safest way to identify true antagonists

Probe Dependence

• Allosteric ligands induce distinct GPCR conformations which impact interactions with orthosteric ligands and intracellular signalling partners

• Surrogate probes may be preferred however undesired pharmacology may occur

• Utilize more native systems during LO transition

N+ O

O

NMeO

Cl

S

NH2O

HN

NT3C

O

O

OH

orthosteric agonistLY2033298 > M4 Potentiator Selective PAM [3H]-QNB antagonist

LY2033298 > M4 ‘Neutral’

M4 Allosteric Modulator LY2033298

N

NS

N

O

CH3

Probes:

M1/M4 preferring agonist (Xanomeline)LY2033298 > M4 Potentiatornon-selective (M2 modulator)

Melancon, B. J. J. et. al. J. Med. Chem. 2012 in press

Metabotropic Glutamate Receptor 5 and Schizophrenia

Schizophrenia- Afflicts 1% of the worldwide population- Three symptom clusters: positive, negative and cognitive

NMDA receptor hypofunction hypothesis- PCP and ketamine (NMDA receptor antagonists) induce schizophrenia-like

symptoms in humans and rats (Krystal JH et al., 1994; Gaspar PA et al., 2009)

Metabotropic Glutamate Receptor 5- Close signalling partner with NMDA receptors; regulating NMDA receptor

function, cognition enhancement- non-dopminergic approach required to develop more effective antipsychotics

that will target negative and cognitive symptoms.

Evidence for Therapeutic Potential for Schizophrenia via Facilitation of mGluR5 Function

→ positive symptoms• Modulating dopamine release Renoldi et al., 2007; Liu et al., 2008• Affecting dopamine-mediated behaviour Liu et al., 2008; Spear et al., 2011

→ cognitive symptoms• Enhancing cognitive function Balschun et al., 2006; Liu et al., 2008; Uslaner et al., 2009; Ayala et al., 2009

• Enhancing synaptic plasticity Le Vasseur et al., 2008; Kwon and Castillo, 2008; Rebola et al., 2008

→ negative symptoms• Hedonic processes Vardigan et al., 2010

mGlu5 PAMs – in the beginning….

NN

FF

DFBPAM, EC50 = 2.6 M

NN

OMeMeO

DMeOBNAM, EC50 = 3.0 M

NN

ClCl

DCBSAM, 7.6 M vs. DFB

Br

HNN

O

O

O

N

Non-MPEP site PAM

Cl

HNN

O

O

OOH

NN

HN

OCN

iterative libraries

fragmentlibraries

CPPHAPAM, EC50 = 250 nM (h)

CDPPBPAM, EC50 = 130 nM (h)

in vivo POC

Non-MPEP

MPEP

O’Brien et al., Mol. Pharm. 2003, 64, 731-740; O’Brien et al. J. Pharm. Exp. Ther. 2004, 309, 568-579; Lindsley et al. J. Med. Chem. 2004, 47, 5825-5829; Kinney et al. J. Pharm. Exp. Ther. 2005, 313, 199-212; Hemstapat, et al. Mol. Pharm. 2006, 70, 616-626.

NN

HN

O

VU-71selective mGlu1 PAM

NO2

phenylmigration

mGlu5 PAMs – A New Series, A new ‘Switch’

Rodriguez et al. Mol. Pharmacol. 2010, 78, 1105-1123.Williams et al. Bioorg. Med. Chem. Lett. 2011, 21, 1350-1353.

Sams et al. Bioorg. Med. Chem. Lett. 2011, 21, 3407-3410.

N

NH

O

PAM, EC50 = 19 nM

N

NH

O

SAMN

N

NH

O

NAM, IC50 = 227 nM

Ar

N

N

O

R

PAMs, ago-PAMs, NAMs, SAMs

N

O

OH

VU0092273HTS Hit

EC50 = 10 nM

iterativeparallel

synthesisN

NH

O

VU0360172EC50 = 16 nMF

Nature of HBA and amide steric bulk can promote ‘switches’Western basic pyridine routinely instills NAM character- ‘Molecular lock’

mGlu5 NAMs – A New Series, A new ‘Switch’

N

N

O

N

NAMIC50 = 990 nM

N

N

O

N

S

NAMIC50 = 540 nMKi = 440 nM

N

N

O

N

O

PAMEC50 = 5.4 M

%Glu max EC20 = 86%

##

VU0364289 Reversal of Amphetamine Induced Hyperlocomotor Activity

Time (min)

0 20 40 60 80 100 120

Am

bu

lati

on

s

(To

tal B

ea

m B

rea

ks

/5 m

in in

terv

al)

0

200

400

600

800

1000

1200

1400

1600 20%BCD vehicle i.p./Amphetamine 1.0 mg/kg; n=1410e 56.6 mg/kg i.p./Amphetamine 1.0 mg/kg; n=12

###

##

# ##

#

Rodriguez et al.,Bioorg. Med. Chem. Lett., 2009, 19, 3209-3213Zhou et al. ACS Med. Chem. Lett. 2010, 1, 433-438.

Xionget al.,Bioorg. Med. Chem. Lett., 2010, 20, 7381-7384

N

N

O

OCN

In vitro DMPK prof ile:

Plasma Protein Binding (human, rat): 94, 90%

Cell Permeability (PAMPA) = 22 x 10-6 cm/sec

Metabolic Stability (h, r): < 5% remaining

Kinetic solubility (pH 2, 4, 7.4): >28 g/mL (>80 M)

Vehicle 20% -CD: 1-6 mg/mL, solution pH 6-7

6-10 mg/mL, microsuspension

VU0364289 cLogP = 3.2

N

N

OO

NF

ClDMPK prof ile:

Protein Binding (human, rat): 53, 74%Permeability = 25 x 10-6 cm/sec

In vitro metabolic stability: poor

Fassif/SGF: > 1 mg/mLIn vivo behavior models:

reverses MK801-induced hyperlocomotion

reduces conditioned avoidance response

CPPZ

In vitro prof ile:EC50 = 0.55 M (pure-PAM)mGlu1-4, 6-8: inactive at 25 MKi = 2.37 M, full [3H]MPEP

PAM Versus Ago-Potentiator Pharmacology

Ago-PAMs vs PAMs: PAMs could maintain spatial and temporal aspects of mGluR5 signaling

LTD – Cognition impairment?

Theoretically, pure positive allosteric modulators should maintain activity-dependence of mGluR5 activation and reduce adverse

effect liability relative to mGluR5 agonists.

Epileptiform activity?

Allosteric agonist activity is dependent on mGluR5 expression levels and may have no impact in native systems

• No agonist activity in cultured astrocytes

• No agonist activity in neuronal populations assessed using electrophysiology

• Representative pure PAMs and ago-PAMs have identical activity in animal models of antipsychotic-like efficacy..

VU0360172 VU0361747

Noetzel, M. Mol. Pharmacol. 2011, in press (doi:10.1124/mol.111.075184)

Finding true Ago-PAMs: VU0424465 is a robust agonist in low expressing cell lines and native systems

VU0424465

VU0424465

N

F

O

NH OH

EC50 = 7 nM (69%)rmGlu5: Ago-PAMAstrocytes: Ago-PAM

cLogP = 3.6PPB (h, r) 97.8, 97.2% AHL- beh. disturbances

mGluR5 orthosteric and allosteric agonists induce epileptiform activity in hippocampal area CA3

VU0360172 (Pure PAM)

VU0424465(Ago-PAM)

N

F

O

NH

O

‘Molecular Locks’ provide pure PAMs with no epileptiform activity: Oxetane Amide VU0430644 (ML254)

PAM CRC Agonist CRC

LTD Epileptiform activity

VU0430644

VU0430644VU0430644

VU0430644

VU0424465

VU0424465

VU0424465

VU0424465

ML254EC50 = 8.7 nMFold-Shift ~ 2cLogP = 3.1PPB (h, r): 97, 96%Clhep (h, r) : 0.2, 1.6 mL/min/kg

Is There a Big Enough Safety Window?• Group I agonist DHPG is epileptogenic (Merlin and Lisa, 2002)

Relative Incidence of Behavioural Effects (%)Observation Compound A Compound B

Excitation 77 0

Forepaw trampling 60 0

Salivation 50 0

Chewing movements 40 0

Flat body posture 33 0

Tremor 23 0

Piloerection 10 0

Sniffing 7 3

Body twitches 3 0

Spasms 3 0

Clonic convulsions 3 0

Wet urogenital region 3 0

PTZ threshold cpd B

Monitor Ago-PAM activity to identify compounds free from potential pro-convulsive activity

Receptor reserve: consider multiple recombinant systems with different expression levels, primary neurons or other native systemsSelectivity screening/Probe Dependence: Profile key compounds in functional GPCR assays with full agonists CRCs (select Millipore panel), utilize multiple probes and/or native orthosteric ligandMode Switching: Avoid scaffolds that show a strong tendency for dramatic changes in activity with subtle structural changes. Metabolite ID and in vivo testing of metabolites is critical for key compounds and final candidates.Ago-PAM activity: Drive chemistry effort using cell lines with relatively low receptor expression. Cross check in native systems. PET Ligand development: Develop PET ligand in same series as candidate. However within detailed molecular pharmacology studies needed to validate utility of PET ligand.

Summary and Druggability Principles for Allosteric GPCR Modulation

Vanderbilt Center for Neuroscience Drug DiscoveryProf. P. Jeff Conn, Director

Supported by NIMH, NIDA, NINDS, NARSAD.

Outside Collaborators: Robert Kessler (Vanderbilt), Marc Caron (Duke), Tanya Daigle (Duke)

Molecular PharmColleen Niswender

Dave WeaverEvan LeBois

Alice Rodriguez Paige VinsonGreg DigbyTom Utley

Daryl VenableKari JohnsonDoug Sheffler

Joy MarloAshley Brady

Meredith NoetzelKaren Gregory

In vivo/EphysCarrie JonesJennifer AyalaJana Shirey Zixiu Xiang

Alexis HammondPaulianda Jones

Alex KaneAnalisa Thompson

Jerri RookJay Rosanelli

Elizabeth J. HermanMichael Bubser

Merideth Noetzel Dan Foster

Med ChemCraig Lindsley

Shaun StaufferCorey HopkinsKyle EmmitteMichael Wood

Sameer SharmaRichard Williams

Phil KennedyDarren EngersRocco GogliottiJames SalovichYiu-Yin Cheung

DMPKScott Daniels

Satyawan JadhavAnnie Blobaum

Usha MenonMatt Mulder

Katrina BrewerRyan Morrison

Frank ByersTom Bridges

Tammy Santomango

Backup Slides

GABA-A receptor PAMs provide precedent for different in vivo effects of pure PAMs versus ago-PAMs

Confidential-Janssen-Vanderbilt mGluR5 PAM Project

- Agonist activity at Ionotropic glutamate receptors (Kainate, AMPA, NMDA)?- mGlu3 antagonist activity?- Glutamate transporter inhibition?- Excessive fold-shift of glutamate CRC on mGluR5?

Other Potential Mechanisms for CNS Adverse Effect Liability

Pure PAMs: anxiolytic, sedative - safe, large therapeutic window

Ago-PAMs: general anesthetic; potentially lethal adverse effects, narrow therapeutic window

Xanomeline Induces Robust Improvement in Behavioral Disturbances in AD Patients

AChE inhibitors have antipsychotic efficacy in AD patients (double blind, placebo-controlled trials) (Cummings et al., 2001; Raskind et al., 1997; McKeith et al., 2000).

Bodick et al., Arch Neurology (1997) 54(4):465-73.

N

NS

N

O

CH3 Xanomeline (LY246708)M1/M4 preferring agonist

6TH DRUG DISCOVERY FOR NEURODEGENERATION CONFERENCE

BIOLOGICS FOR CHALLENGING TARGETS: UNIQUE CHALLENGES AND LESSONS LEARNED

GURIQ BASI, Ph.D.VP, ELAN PHARMACEUTICALS

Evolution of drug development for neurodegeneration: Symptomatic to disease modifying

L-DOPA

AChEI’s

Neurotrophins

Immunotherapy

Gene therapy

RNAi

Neurodegenerative disease Restrictions imposed by BBB = small

molecules main-stay for Rx No-go for neurotrophin biologics

Access of biologics to CNS Historic AD immunotherapy Targeted delivery Alternative routes (nasal insulin)

Opportunity on case by case basis Antibody Technology Platforms CM&C, costs, and timelines to IND

Neurotrophins Promises

Neuroprotection, Neuro-restoration

NGF, BDNF, Nerturin

Limitations Poor bio-availability in target organ following systemic peripheral delivery

Undesirable side effects from non-targeted central delivery, e.g. generalized sprouting promoting inappropriate connections, neuralgia

Solutions Localized (chronic) central delivery to affected region(s)

Surgical implants for localized infusion (GDNF)

Targeted delivery

Gene therapy (Tuczyinski 2004) via implantation of genetically modified fibroblasts; CERE-110 – viral delivery of NGF (recruiting P2, n=50 end May 2012) CERE-120 (AAV2-Neurturin) - P2 (Dec 2008): Failed on 1o endpoint (efficacy in motor function at 12

mo), may have benefit at 18 mo. OLE in progress