1. Druggability Considerations for GPCRs
and Ion Channels
Shaun R. Stauffer
6th Drug Discovery for Neurodegeneration Conference
February 12-14, 2012

2. Outline
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

3. CNS drug development challenges
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.

4. Amyloid Precursor Protein (APP) Proteolysis:
A Fork in the Road
• -Secretase pathway – Predominant, sAPP neurotrophic (non-amyloidogenic)
• -Secretase pathway – Minor, normal in development/repair and pathologic
(A ) roles (oligomerization, “amyloidogenic”)
-secretase -secretase -secretase
VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK
LVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK
A 1-40 (major)
sAPP A 1-42 (minor)
sAPP
Inhibition of -secretase (BACE) should impede the production
of the peptide A , hence slowing the progress of Alzheimer‟s
disease

5. 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 -strand inhibitor/substrate to achieve potency
P2
P1'
NH2
CO2H CO2H
O
H O H OH Me H O H
N N N N CO2H ~1 nM BACE-1
H2N N N
O H O Me O Me H O
Me Me
Me
P3 P1
L. Hong, J. Tang et al, Science 2000, 290, 150

6. Key Achievement
Tang and coworkers, Science 2000, 290, 150-153.

7. 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)
P2
P1'
NH2
CO2H CO2H
O
H O H OH Me H O H
N N N N CO2H ~1 nM BACE-1
H2N N N
O H O Me O Me H O
Me Me
Me
P3 P1
L. Hong, J. Tang et al, Science 2000, 290, 150
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

8. Aspartyl Protease Transition State Analogues
HHO OHP1' H O
N N
N
O P1 H O P2'
transition state
H OH P1' H OH P1' H NH2 P1' H OH O
N N N N
n
O P1 O O P1 OH O O P1 O O P1
hydroxyethylene (HE) di-hydroxyethylene (DHE) aminoethylene (AE) statine-based
H NH2 O OH H O H OH P1' O H P1'
H
N N N N P N
n N
O H
O P1 O P1 P1' O P1 P1 ' O P1 O
aminostatine (AS) hydroxyethyamine (HEA) phospinate-based reduced amide (RA)
Rich, D. J. Med.Chem., 2002,45, 541.; Greenlee, W. J. J. Med. Res. Rev. 10, 173, (1990)

9. Merck-Neogenesis Collaboration
• Screening a 5 million member compound library yields a single lead
P2 Opt.
O O
S
N
N O P1 /TSA Opt.
O O
OH H
O O NH HN N
O P1 '
NH NH HEA incorporation
Rich, D. et al P3
NH2 JMC 2002, 45, 541 P1
1
2 BACE-1 IC50 = 10 nM
BACE-1 IC50 = 25,000 nM
MW 506 MW 578
3 HBD P3 + P1 amides
4 HBA PGP P2 sulfonamide
substrate HEA
Coburn et al, J. Med. Chem., 2004, 47, 6117 V. John et al, J. Med Chem., 2004, 47, 158 (Elan)
Stachel et al, J. Med. Chem., 2004, 47, 6447 S. Kaldor et al, Bioorg. Med. Chem. Lett., 1995, 5, 721 (HIV)

10. X-Ray of HydroxyEthylAmine (HEA)
P2
O O
S
N
O O
OH H
Ph NH HN N
Ph
P3 P1'
P1
2
S1, S3
hydrophobic
regions:
S1, S3 and S2’
S2’

11. Emerging Chemical Methods Enables
Truncation of HEA
O O
S O O
N S
N
1) Rh(acac)(C2H4)2 / rac-BINAP
F O O
O F O O
NH HN (HO)2B
O N NH HN
OH
R
2) LiBH4
Ar
35 / 48 successfully isolated
Cl
Ar =
+ meta
F N isomer
HO2C
F H
HN F N
S O
O
4
IC50 = 28 nM

12. SAR: Alkyl Branch and P1
Stauffer, S. R. et al. Bioorg. Med. Chem. Lett., 2007, 17, 1788.

13. 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.

14. Pocket Collapses via Ligand-Dependent Conformational Change
Ser10
Thr232
G. McGaughey

15. Pocket Collapses via Ligand-Dependent Conformational Change

16. Fast forward: Carbinamines, Spiropiperidines, and Acyl Amidines
O O
S
N
N
N X
Ph
Low CNS penetration 0.05 b/pIC50 = 330 nM IC50 = 0.4 nM
BACE-1 BACE-1 fragment-based discovery
sAPP _NF IC50 = 4200 nM ICMK-8931 entering PhII
sAPP _NF 50 = 40 nM
PSA >100 P-gp ratio(h) = 2, Papp =reduction in HV
robust A 22
Log P 2.5 – 4.0 HBD/HBA = 1/6
cLogP = 2.6, PSA = 120 Å2
MW >500
HBD/HBA = 2/4 40% reduction Rhesus CSF A 40
after IV infusion
high metabolism, low %F
Persistence and serendipity!
Zhu, Z. et al. J.Med.Chem. 2010, 951.
Stanton, M. et al. J. Med. Chem. 2007, 3431. US20080103351
Sankaranarayanan, S. et al. J.Pharm. Exp. Barrow, J. et al. J.Med.Chem. 2008, US20080200445
Ther. 2009, 131-140. 6259. US20070287692

17. G protein-coupled receptors
orthosteric NH2 allosteric
binding site binding site(s)
transmembrane
heptahelical domain
(7TM domain)
COOH
• 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: Allosteric Modulators Offer Advantages
• Selectivity
– Negative allosteric modulators • Mimic physiological conditions
• No desensitization, down regulation or
– Positive allosteric modulators internalization
• Less side effects
– Allosteric agonists
Challenges: Steep/Flat SAR, ‘mode switching’
– Neutral cooperativity

18. Druggability Challenges for Allosteric Ligands of GPCRs
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.
Wood, et al., Biochemistry 2011, 50, 2403-2410.

19. 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
VU0364572 allosteric agonist/PAM
High 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 37
Brain AUC/Plasma AUC = 1.4
Potentiate NMDA currents in hippocampal CA1
Lebois, E.P. et al. Bioorg. Med. Chem. Lett. 2011, 6451.

20. 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

21. Probe Dependence
• Allosteric ligands induce
distinct GPCR
M4 Allosteric Modulator LY2033298
conformations which
impact interactions with
orthosteric ligands and
Probes: intracellular signalling
partners
orthosteric agonist • Surrogate probes may
LY2033298 > M4 Potentiator be preferred however
[3H]-QNB antagonist
Selective PAM undesired pharmacology
LY2033298 > M4 „Neutral‟
may occur
• Utilize more native
systems during LO
transition
M1/M4 preferring agonist (Xanomeline)
LY2033298 > M4 Potentiator
non-selective (M2 modulator)
Melancon, B. J. J. et. al. J. Med. Chem. 2012 in press

22. 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.

23. Evidence for Therapeutic Potential for Schizophrenia via
Facilitation of mGluR5 Function
• Modulating dopamine release → positive symptoms
Renoldi et al., 2007; Liu et al., 2008
• Affecting dopamine-mediated behaviour
Liu et al., 2008; Spear et al., 2011
• Enhancing cognitive function → cognitive symptoms
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
• Hedonic processes → negative symptoms
Vardigan et al., 2010

24. mGlu5 PAMs – in the beginning….
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.

25. mGlu5 PAMs – A New Series, A new ‘Switch’
Nature of HBA and amide steric bulk can promote „switches‟
Western basic pyridine routinely instills NAM character- „Molecular lock‟
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.

26. mGlu5 NAMs – A New Series, A new ‘Switch’
VU0364289 Reversal of Amphetamine Induced Hyperlocomotor Activity
20%BCD vehicle i.p./Amphetamine 1.0 mg/kg; n=14
1600 10e 56.6 mg/kg i.p./Amphetamine 1.0 mg/kg; n=12
(Total Beam Breaks/5 min interval) 1400
1200
Ambulations
1000
800 # # # # #
# # # #
600
# #
400
200
0
0 20 40 60 80 100 120
Time (min)
Rodriguez et al.,Bioorg. Med. Chem. Lett., 2009, 19, 3209-3213
Zhou et al. ACS Med. Chem. Lett. 2010, 1, 433-438.
Xionget al.,Bioorg. Med. Chem. Lett., 2010, 20, 7381-7384

27. PAM Versus Ago-Potentiator Pharmacology

28. Ago-PAMs vs PAMs: PAMs could maintain spatial and
temporal aspects of mGluR5 signaling
LTD – Cognition
impairment?
Epileptiform
activity?
Theoretically, pure positive allosteric modulators should maintain
activity-dependence of mGluR5 activation and reduce adverse
effect liability relative to mGluR5 agonists.

29. 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
VU0360172 VU0361747
PAMs and ago-PAMs
have identical activity
in animal models of
antipsychotic-like
efficacy..
Noetzel, M. Mol. Pharmacol. 2011, in press (doi:10.1124/mol.111.075184)

30. Finding true Ago-PAMs: VU0424465 is a robust agonist in low
expressing cell lines and native systems
EC50 = 7 nM (69%) cLogP = 3.6
rmGlu5: Ago-PAM PPB (h, r) 97.8, 97.2%
Astrocytes: Ago-PAM AHL- beh. disturbances
VU0424465
VU0424465

31. mGluR5 orthosteric and allosteric agonists induce
epileptiform activity in hippocampal area CA3
VU0360172 VU0424465
(Pure PAM) (Ago-PAM)

32. ‘Molecular Locks’ provide pure PAMs with no epileptiform activity:
Oxetane Amide VU0430644 (ML254)
PAM CRC Agonist CRC
VU0424465
VU0424465
ML254
EC50 = 8.7 nM
Fold-Shift ~ 2 VU0430644
VU0430644 cLogP = 3.1
PPB (h, r): 97, 96%
Clhep (h, r) : 0.2, 1.6 mL/min/kg
LTD Epileptiform activity
VU0430644 VU0424465
VU0424465
VU0430644

33. 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 PTZ threshold cpd 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
 Monitor Ago-PAM activity to identify compounds free from potential pro-convulsive activity

34. Summary and Druggability Principles for Allosteric
GPCR Modulation
Receptor reserve: consider multiple recombinant systems with different
expression levels, primary neurons or other native systems
Selectivity screening/Probe Dependence: Profile key compounds in
functional GPCR assays with full agonists CRCs (select Millipore panel),
utilize multiple probes and/or native orthosteric ligand
Mode 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.

35. Vanderbilt Center for Neuroscience Drug Discovery
Prof. P. Jeff Conn, Director
In vivo/Ephys Molecular Pharm Med Chem DMPK
Carrie Jones Colleen Niswender Craig Lindsley Scott Daniels
Jennifer Ayala Dave Weaver Satyawan Jadhav
Shaun Stauffer
Jana Shirey Evan LeBois Annie Blobaum
Corey Hopkins
Zixiu Xiang Alice Rodriguez Usha Menon
Kyle Emmitte
Alexis Hammond Paige Vinson Matt Mulder
Michael Wood
Paulianda Jones Greg Digby Katrina Brewer
Sameer Sharma
Alex Kane Tom Utley Ryan Morrison
Richard Williams
Analisa Thompson Daryl Venable Frank Byers
Phil Kennedy
Jerri Rook Kari Johnson Tom Bridges
Darren Engers
Jay Rosanelli Doug Sheffler Tammy Santomango
Rocco Gogliotti
Elizabeth J. Herman Joy Marlo James Salovich
Michael Bubser Ashley Brady Yiu-Yin Cheung
Merideth Noetzel Meredith Noetzel
Dan Foster Karen Gregory
Outside Collaborators: Robert Kessler
(Vanderbilt), Marc Caron (Duke), Tanya Daigle
(Duke)
Supported by NIMH, NIDA, NINDS, NARSAD.

36. Backup Slides

37. GABA-A receptor PAMs provide precedent for different
in vivo effects of pure PAMs versus ago-PAMs
Pure PAMs: anxiolytic, Ago-PAMs: general
sedative - safe, large anesthetic; potentially
therapeutic window lethal adverse effects,
narrow therapeutic window
Other Potential Mechanisms for CNS Adverse Effect Liability
- Agonist activity at Ionotropic glutamate receptors (Kainate, AMPA, NMDA)?
- mGlu3 antagonist activity?
- Glutamate transporter inhibition?
- Excessive fold-shift of glutamate CRC on mGluR5?
Confidential-Janssen-Vanderbilt mGluR5 PAM Project

38. Xanomeline Induces Robust Improvement in
Behavioral Disturbances in AD Patients
Xanomeline (LY246708)
Bodick et al., Arch Neurology (1997) 54(4):465-73.
M1/M4 preferring agonist
 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).

39. BIOLOGICS FOR
CHALLENGING TARGETS:
UNIQUE CHALLENGES
AND LESSONS LEARNED
GURIQ BASI, Ph.D.
VP, ELAN PHARMACEUTICALS
6TH DRUG DISCOVERY FOR NEURODEGENERATION CONFERENCE

40. Evolution of drug development for neurodegeneration:
Symptomatic to disease modifying
 Neurodegenerative disease
L-DOPA  Restrictions imposed by BBB = small molecules main-stay for
Rx
AChEI‟s  No-go for neurotrophin biologics
 Access of biologics to CNS
Neurotrophins
 Historic
Immunotherap  AD immunotherapy
y  Targeted delivery
 Alternative routes (nasal insulin)
Gene therapy  Opportunity on case by case basis
RNAi  Antibody Technology Platforms
 CM&C, costs, and timelines to IND

41. 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