Receptor-channel Research at the Interface between Academia and Pharma Prof. Mihály Hajós

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1 Receptor-channel Research at the Interface 1, Pharm.D., Ph.D. Professor Adjunct Head of Translational europharmacology Laboratory Section of Comparative Medicine Yale School of Medicine, ew Haven, CT, USA Use of mind-altering substances dates to prehistoric times Coca leaves Peyote icotiana tabacum 2 Datura stramonium Amanita muscaria 2 th century: europharmacology 3 1

2 Outline of the presentation Drug-target selection Synaptic targets: Receptors/Ion-channels Drug discovery process (long, tedious and expensive) Discovery of selective ligands of nicotinic α7 receptors Current developments in drug discovery 4 5 Target selection History of neuropharmacology: Serendipity e.g., discovery of antipsychotic or anti-depressive drugs Ideally, it should be based on: Genetics or Understanding the pathophysiology of the disease However, even recently, it has not been the case; Target selection is mostly based on: Understanding the mode of action of currently used medicines (frequently leading to me-too-drugs ) Clinical off-target observations, recognition of potential therapeutic application of our currently used drugs Targeting synapses for CS drug treatment Presynaptic targets: Presynaptic autoreceptors Presynaptic heteroreceptors Various mechanisms of transmitter-release and up-take Postsynaptic targets: Postsynaptic receptors Various mechanisms for eliminating neurotransmitters: enzymatic degradation/take-up 6 2

3 Two types of receptors 1. Ionotropic receptors they form or are directly linked to ion-channels 2. Metabotropic linked to secondary messenger system Ligand-gated ion channels G-protein-couples receptors 7 Drug-binding sites at ionotropic receptors Orthosteric bindings site: Bindings site for endogenous neurotransmitter Allosteric bindings sites: Bindings sites for endogenous hormones, modulators α1 β2 γ2 α1 β2 8 Ion-channel, formed by 5 receptor subunits GABA A receptor Interactions between receptors and receptor ligands Resting stage, ion-channel closed Agonist, is binding to either orthosteric or allosteric site, receptor activated and ion-channel opens Modulator is binding to allosteric site, receptor stays inactive and ion-channel closed 9 3

4 Interactions between receptors and receptor ligands (2) Agonist is binding to orthosteric site receptor activated and ion-channel opened Agonist is binding to orthosteric site Positive allosteric modulator is binding to allosteric site, Activation of the receptor enhanced and ion-channel opened further 1 Agonist is binding to orthosteric site egative allosteric modulator is binding to allosteric site, Activation of the receptor diminished and ion-channel opened less 11 Javitt at el., 211 Detecting receptor binding in living human brain PET radiopharmaceutical for quantifying metabotropic glutamate 5 receptors Wong et al., Johns Hopkins University School of Medicine, Baltimore, MD Pfizer Inc., ew York, Y Vanderbilt University, ashville, T Merck Research Laboratories, West Point, PA 4

5 Proof of functional receptor engagement: europhysiological (EEG) signals as translational biomarkers 13 Chemically distinct inhibitors of mglu5 receptors elicit highly comparable neurophysiological (EEG) signals in rats, which are considered as translational functional biomarkers Harvey et al, 213 Inhibition of mglu5r for treatment of fragile X syndrome FXS: FMR1 gene mutation loss of fragile X mental retardation protein (FMRP): hyperactivity of mglu5-rs 14 Michalon et al., 213 Discovery of α7 nachr ligands at Pharmacia and Pfizer Connections between α7 nachrs and schizophrenia (established by academic teams): Genetics indicate that polymorphisms in the α7 nachr gene are associated with schizophrenia Schizophrenic patients have diminished α7 nachr expression in hippocampus Impaired auditory gating, an endophenotype of schizophrenia, showed close relationship to α7 nachr expression transiently restores sensory gating; More than 8% of schizophrenics are heavy smokers 15 5

6 Main steps in drug discovery Cycle time is approximately 11 years from concept to launch Discovery Research Exploratory Development Development Filing Launch Growth Maintenance Discovery Team Exploratory Development Team Development Team Brand Development Team Drug Attri Target Product Profile Early Commercial Team Global Commercial Team Oversight 16 Comm Profile Exp Comm Profile Comm Plan Comm Plan Life Cycle All start with a discovery phase Discovery Development Discovery research: years -5 Disease Target Hypothesis 1 s Protein, Assay, Screen, Hit Pre-Clinical + Safety: years s 1 s Lead, Med Chem, Pharmacology, ADMET, Candidate, Tox, FTIH Clinical: years 7-12 Drug to public: years s PoC (Phase 2), Phase 3, File DRUG Disease/Mol/Assay Expertise Med Chem/Drug Met/Safety/Ph Sci Clinical/Reg/ Commercial 17...with a discovery/lead optimization funnel Creation of an α7 nachr HTS drug target for discovering agonists α7 nachr Ligand binding H 2 domain COOH H 2 5HT 3 Ligand binding domain COOH Calcium pore H 2 Monovalent cation pore α7-5ht 3 COOH 18 6

7 High fidelity cell-based HTS assay HTS assay for agonists, antagonists and modulators all in one well Screen at 1 μm 19 α7 nachr lead optimization funnel 2 Directed chemical synthesis Functional potency and selectivity Binding affinity, selectivity, in vitro PK In vivo PK, ephysiology, BDA, early safety In vivo P5 gating In depth safety Selection Criteria α7 EC 5 >1-fold functional selectivity over α3 (ganglion), α1 (neuromuscular junction), muscarinic, and 5HT 3 receptors Binding affinity for α7 receptor Selectivity over other CS receptors Favorable patent position Adequate exposure w/cs penetration Improve P5 sensory gating in an animal model Acceptable drug: Drug interaction profile 21 Characterization of α7 nachr agonists using cultured rat hippocampal neurons Patch clamp assay: Confirm activity at native receptor Assess activity at non-α7 nachrs Evaluate desensitization potential Control Solution Motor Test Solution Activity (% 1 μm nic.) 1 μm Cl.3 μm 3 μm 3 μm Tropisetron 1 s AR-R Concentration (μm) O H 1 pa 7

8 Low concentrations of enhance GABAergic synaptic transmission CA1 stratum pyramidale Baseline (3 nm) GABAergic interneuron Pyramidal cell * 22 Presynaptic α7 nachr Hajos et al., 25 Activation and desensitization are not tightly coupled Cl Ki = 26 nm IC 5 = 15 nm 23 O H CH 3 Ki = 92 nm IC 5 = 662 nm H 3 CO S S O H PHA Ki = 4 nm IC 5 =.6 nm O CH 3 GTS-21 Ki = 22 nm IC 5 = 4 nm Current (% control) % 18 Activation Desensitization Concentration (nm) PHA produces a long-lasting desensitized state O (1 μm).3 μm (1 μm) 24 Cl S H Ki = 26 nm S O H PHA Ki = 4 nm (1 μm) 3 μm 3 μm PHA μm 3 μm 3 μm (1 μm) c9d162 2 pa 2 s e pa 2 s 8

9 Endophenotypes: Reflecting pathophysiology underlying or contributing to the disease Auditory gating deficit in schizophrenia 25 Ward et al., Psychiatric Res, 1996 Modeling auditory gating impairment of schizophrenia for in vivo efficacy assay Amphetamine disrupts auditory gating in healthy humans and rats Light et al., Biol. Psychiatry, Hajós, Trends Pharmacol Sci. 26 Krause et al., Biol. Psychiatry, 23 Many but not all α7 nachr agonists restore auditory gating Auditory gating K i = 26 nm * * PHA K i = 9 nm * * PHA K i = 4 nm Hajos et al., JPET 25 * Significant reversal of amphetamine deficit (P <.5) 27 9

10 Positive allosteric modulators (PAMs) of α7 nachrs Why develop PAMs? Avoiding toxicology associated with previously developed agonists Avoiding agonist s induced receptor desensitization Instead of direct stimulation of the receptor, PAMs enhance response to endogenously released transmitter 28 Prototype of α7 nachr PAMs, PU enhances and prolongs ACh-evoked increases of GABA synaptic activity Rat hippocampal slice preparation 29 ACh (1 μm) PU (3 nm) ACh (1 μm) + PU (3 nm) 2 min 25 pa kr.b249 Jl.d243 Jl.d249 Synaptic Activity (change from baseline) ACh (1 μm) PU (3 nm) ACh (1 μm) + PU (3 nm) Addition Time (min) Hurst et al., 25 α7 nachrs can be activated by different mechanisms: Type-I and Type-II PAMs Agonist-induced activation and desensitization Possible mechanisms to enhance agonist activity with a PAM Closed Open Desensitized 3 + PU PU Hajós & Rogers, 21 1

11 PU-12596, a Type-II PAM improves auditory gating deficits induced by amphetamine in anesthetized rats 31 Hurst et al., J. eurosci. 25 PHA , a Type-I PAM reverses amphetamine-induced auditory gating deficit in rats 32 Temperature-dependent allosteric modulation of α7 nachr by PU ormalised to 3 mm Ach+1 μm PU peak RT ormalised to 3 mm Ach+1 μm PU area at RT 26ºC 31ºC 37ºC 26ºC 31ºC 37ºC 33 Sitzia et al.,

12 When an agonist turns out to be a PAM: PHA , a mixed agonist/pam on α7 nachrs In auditory gating assay PHA active at plasma concentrations lower than the Ki value % Reversal * p<.5 vs. amphetamine (n=38) 1 ormal * * * * * PHA (mg/kg) * 1 PHA K i = 44 nm (rat) Dose (mg/kg FBE) [Brain] (nm, 3 ).7 ot detected ± ± ± ± ± ± 98 Hurst et al., Sf 27 PHA can both enhance and inhibit receptor activity at low concentrations 3 nm 3 nm PHA PHA nm 3 nm 1 s 5 pa Current (% control) Inhibition Potentiation GTS s 5 pa Concentration (μm) 35 = (1 μm, 1s) Hurst et al., Sf 27 Complexity of receptor activation α7 nachr functions both as ionotropic and metabotropic receptor Impact on neuronal network: changes in profile of neuronal network activity/oscillation Gap in connecting receptor/channel pharmacology to: Receptor kinetics Downstream cellular processes euronal network responses Impacts on disease pathology and clinical response 36 Hajós and Rogers, 21 12

13 Academia Pharma collaboration in drug discovery Main obstacles: Decreasing number of FDA-approved new drugs, especially in CS drugs Leaving high-risk drug discovery projects by industry Reduction in effort at early-stage discovery by big pharma (e.g., preference for in-licensing) Virtual neuroscience department at big pharma 37 Academia Pharma collaboration in drug discovery (2) Critical steps: Better evaluation of drug targets, closer interaction between clinical, academic labs and pharma Pharma: ending continuous, mainly brainless, re-organizations Re-evaluation of research areas/activities both at pharma and academia, redistribute roles ew level and style of collaboration between academia and pharma: end of so-called window collaborations 38 Biomarker discovery Tool/legacy compounds 39 13

14 European consortium to address shortfall in innovative medicines 4 ovel methods leading to new medications in depression and schizophrenia Acknowledgements Many outstanding scientists at Pharmacia and Pfizer - present and past and external academic collaborators contributed to and enabled our work on α7 nachr ligands Pharmacia/Pfizer europhysiology Lab: William Hoffmann, MS Tamas Kiss, PhD Chester Siok, MS Michael Krause, PhD Brian Harvey, MS James Rogers, PhD Liam Scott, MS Christopher L. Shaffer, PhD (PDM) Jianlin Feng, MS Elena Morozova, MS Yale Translational europharmacology Lab: David agy, PhD Elizabeth Arnold, BSc David Tingley, MSc Milan Stoiljkovic, MD, PhD