For example, in hippocampal neurons, the receptor reserve for depolarization is high whereas the receptor reserve for NMDAR potentiation and for PI hydrolysis is much lowerThis becomes a particular consideration when one is trying to identify antagonists….
Historic: Nerenberg,S.T.,andPrasad,R.(1975). Radioimmunoassays for Ig classes G, A, M,D,and E in spinal fluids:normal values of different age groups. J. Lab.Clin.Med. 86, 887–898. Compromised BBB in neurodegenerative disease;
Druggability Considerations for GPCRs and Ion Channels Shaun R. Stauffer6th Drug Discovery for Neurodegeneration Conference February 12-14, 2012
Outline1. CNS drug development statistics and the hope for BACE inhibitors2. Druggability considerations for GPCRs Orthosteric versus Allosteric Approaches M1/M4 PAMs- receptor reserve and probe dependence mGluR5 PAMs- ‘mode switching’ and allosteric agonism3. Summary and Outlook
CNS drug development challengesTremendous need: neurological and psychiatric conditions account for 13% of the global burden ofdiseaseCNS drugs spend 8.1 yrs in human testing, more than 2 yrs longer than average for all agentsRegulatory approval of CNS drugs takes longer- 1.8 yrs vs1.2 yrs for all drugs8.2% of CNS drug candidates that begin human testing will reach marketplace vs. 15 % for drugsoverall46% of CNS candidates succeed in late-stage (phase III) trials, compared with 66% for all drugsEvaluation of clinical improvement more difficult- schizophrenic episodes or cognitiveimprovement in Alzheimer‟s patients more variable and require outcomes trials for therapies aimedat disease modificationNew coalitions emerging to bring government agencies, drug companies and patient advocacygroups together, to develop a standardized clinical trials database to allow researchers to designmore 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.
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
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
Key AchievementTang 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) 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
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 nO P1 O O P1 OH O O P1 O O P1hydroxyethylene (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 Oaminostatine (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)
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 HEACoburn 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)
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’
Emerging Chemical Methods Enables Truncation of HEA O O S O O N S N 1) Rh(acac)(C2H4)2 / rac-BINAPF O O O F O O NH HN (HO)2B O N NH HN OH R 2) LiBH4 Ar 35 / 48 successfully isolated ClAr = + meta F N isomer HO2C F H HN F N S O O 4 IC50 = 28 nM
SAR: Alkyl Branch and P1 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.
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 O O S N N N X PhLow 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 nMPSA >100 P-gp ratio(h) = 2, Papp =reduction in HV robust A 22Log P 2.5 – 4.0 HBD/HBA = 1/6 cLogP = 2.6, PSA = 120 Å2MW >500HBD/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. US20080103351Sankaranarayanan, S. et al. J.Pharm. Exp. Barrow, J. et al. J.Med.Chem. 2008, US20080200445Ther. 2009, 131-140. 6259. US20070287692
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
Druggability Challenges for Allosteric Ligands of GPCRsAllosteric modulators can act at multiple distinct, often overlapping, butalso non-overlapping sites on the same receptor. SAR fails to translate.Shallow (steep or flat) SAR and difficult to add polar and/or solublizinggroups to generally small, lipophilic chemotypesAllosteric modulators can differentially regulate coupling of mGlus todifferent signaling pathwaysMembers of a single structural class can have a range of activitiesfrom PAM to NAM and can include neutral ligands, ago-PAMs, allostericagonist to partial antagonist‘Molecular Switch’ – unexpected alteration of pharmacology within anestablished series due to subtle, single heavy atom modifications. 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 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.
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 M4 Allosteric Modulator LY2033298 conformations which impact interactions with orthosteric ligands andProbes: 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
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• 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
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.
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.
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
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.
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)
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
mGluR5 orthosteric and allosteric agonists induce epileptiform activity in hippocampal area CA3 VU0360172 VU0424465 (Pure PAM) (Ago-PAM)
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
Summary and Druggability Principles for Allosteric GPCR ModulationReceptor reserve: consider multiple recombinant systems with differentexpression levels, primary neurons or other native systemsSelectivity screening/Probe Dependence: Profile key compounds infunctional 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 fordramatic changes in activity with subtle structural changes. MetaboliteID and in vivo testing of metabolites is critical for key compounds andfinal candidates.Ago-PAM activity: Drive chemistry effort using cell lines with relativelylow receptor expression. Cross check in native systems.PET Ligand development: Develop PET ligand in same series ascandidate. However within detailed molecular pharmacology studiesneeded to validate utility of PET ligand.
Vanderbilt Center for Neuroscience Drug Discovery Prof. P. Jeff Conn, DirectorIn 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 GogliottiElizabeth J. Herman Joy Marlo James Salovich Michael Bubser Ashley Brady Yiu-Yin Cheung Merideth Noetzel Meredith Noetzel Dan Foster Karen GregoryOutside Collaborators: Robert Kessler(Vanderbilt), Marc Caron (Duke), Tanya Daigle(Duke)Supported by NIMH, NIDA, NINDS, NARSAD.
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
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-controlledtrials) (Cummings et al., 2001; Raskind et al., 1997; McKeith et al., 2000).
BIOLOGICS FORCHALLENGING TARGETS:UNIQUE CHALLENGESAND LESSONS LEARNEDGURIQ BASI, Ph.D.VP, ELAN PHARMACEUTICALS6TH DRUG DISCOVERY FOR NEURODEGENERATION CONFERENCE
Evolution of drug development for neurodegeneration:Symptomatic to disease modifying Neurodegenerative diseaseL-DOPA Restrictions imposed by BBB = small molecules main-stay for RxAChEI‟s No-go for neurotrophin biologics Access of biologics to CNSNeurotrophins HistoricImmunotherap AD immunotherapyy Targeted delivery Alternative routes (nasal insulin)Gene therapy Opportunity on case by case basisRNAi 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