Development of Screening Tools to Identify Nicotinic Subtype-Selective Compounds Glenn Kirsch, PhD Discovery Services Charles River Cleveland, Ohio USA Aurora Ion Channel Retreat July 7-9, 2015
Tobacco Control Act US FDA Signed into law 2009 Authorized the FDA to regulate tobacco products to protect public health The Center for Tobacco Products (CTP) established to oversee implementation - including research CTP contracted with ChanTest to run the NicScreen project in August 2013, as part of an initiative to provide scientific support for developing tobacco product regulations 2
NicScreen Project Objectives Primary NicSreen Objective: Develop and validate bioassay screening tools for rapid identification of tobacco product constituents with nachr subtype selective activity. Phase I (9/2013 8/2014) - Develop cell lines and highthroughput assays Phase II (9/2015 8/2016) Pilot testing to determine the pharmacological profiles of different tobacco products Phase III (9/2016 8/2017) Proof of concept study for high-throughput screening to evaluate tobacco product constituents for subtype selectivity. Develop methods for evaluating similarities and differences in the pharmacologic activity of marketed tobacco products and constituents 3
Nicotinic Acetylcholine Receptors Are Therapeutic Targets Neuronal nicotinic receptors - pentameric ligand-gated cationselective channels Subtypes defined by α and β subunit composition, and functional properties (agonistsensitivity, pharmacologic profile, Ca 2+ permeability, and desensitization kinetics) Cognition impairment in schizophrenia and Alzheimer s - α7 Agonists and PAMs in clinical trials (e.g., DMXB-A, galantamine, EVP-6124). Parkinson s disease transdermal nicotine and varenicline in clinical trials. Depression α4β2 antagonist to augment SSRIs in clinical trials (e.g., mecamylamine). Tobacco addiction - α4β2 partial agonist (varenicline), marketed. Varenicline for treatment of alcohol, cocaine and methamphetamine addiction in clinical trials. Inflammatory diseases (e.g. inflammatory bowel disease) - α7 receptor activation is anti-inflammatory in animal models. 4
Ion Channel HTS Screening & Profiling Platforms for Nicotinic Receptor Assays Screening/Profiling Assays Ligand Gated Channels Ion Works Barracuda (IWB): automated patch clamp FLIPR: ion- or voltage-sensitive dyes Voltage Gated Channels IWB: automated patch clamp FLIPR: ion-sensitive dyes (Ca 2+, Tl + ) Assay Modes agonist antagonist positive or negative allosteric modulator IonWorks Barracuda FLIPR TETRA 5
Cell Line and Assay Validation Workflow Cell Line Development & Validation ~3 months Assay Development & Optimization ~3 weeks Assay Validation ~2 weeks Validated Assay ~2 weeks Cell line production: cdna cloning & transfection Clone selection Expression confirmation Functional characterization Stability and Scale-up Optimize recording conditions: Adjust stimulus parameters Adjust intra- and extracellular solutions, holding potential Conduct stimulus/response testing Optimize cell density Increase signal window and minimize variability (Z factor optimization) Evaluate DMSO tolerance Functional Validation: Signal stability/uniformity 2-Day plate uniformity tests (intra- and inter-day variability) 2-Day reference compound testing: 3 10 cpds, 8 and 16 pt CRC Reporting: Data analysis Data QC Report writing Format Report QC 6
Assay Acceptance Criteria Plate Level Parameters (FLIPR & IWB) Value Z factor 0.5 CV for MAX Control 20% CV for MIN Control 20% Signal Window (SW) 3 EC 50 for reference agonists 0.5 log* IC 50 for reference antagonists 0.5 log* *from historical mean Well Level Parameter (IWB only) Value Rseal > 100 MΩ Current Amplitude > 0.2 na Rseal Stability < 50% decrease Current Stability < 50% decrease Success rate (% valid wells) 90% Validation data are obtained in independent experiments conducted on separate days 7
Ligand-Gated Assays in IonWorks Barracuda (IWB) pipette electrode IonWorks Barracuda Population Patch Clamp (PPC) and single hole recording modes (384-well patch plate) Controls single cell membrane potential and measures ionic currents in single-cell or cell population ( 64 cells/well). 384-channel pipettor, integrated 384-channel electronic head Validated assays for multiple subtypes in ligandgated 5-HT3R, GABA A R, nachr, NMDAR and TRP channels cells Continuous voltage-clamp current measurement with rapid solution addition for fast-desensitizing, ligand-gated channels Throughput (manual plate handling) 10 plates/day/instrument 3200 compounds/day 16,000 compounds/week Timeline for screening 100,000 cpd library ~7 weeks 8
Nicotinic Cell Lines and Assays nachr Subtype Parental Cell Assays α3β4 CHO IonWorks Barracuda, FLIPR α4β2 CHO IonWorks Barracuda, FLIPR α3β4α5 CHO IonWorks Barracuda, FLIPR α7 CHO IonWorks Barracuda, QPatch, FLIPR α6/3β2β3 V273S HEK293 IonWorks Barracuda, FLIPR 9
α4β2: Ionic Current Distribution in IonWorks Barracuda (Single Cell Mode) Untransfected CHO Cells a4b2-cho Cells Clone #26 333 mm ACh 333 mm ACh Stimulation 0.3 mm ACh Mean peak ±SD = 1.21 ± 0.82 na
C u r r e n t (n A ) α4β2: Signal Uniformity IonWorks Barracuda (PPC) Io n W o r k s B a r r a c u d a U n ifo r m ity - D a y s 1 a n d 2 0 2 /1 7 /2 0 1 4 FLIPR 1.2 5 S ig n a l L e v e l M IN 1.0 0 0.7 5 M ID M A X 1 0 0 m M (-)-N ic o tin e 0.5 0 0.2 5 0.0 0 V e h ic le C o n tro l 5 m M (-)-N ic o tin e D a y 1 D a y 2 Z ` = 0.5 2 0.5 7 S W = 3.4 4 4.0 8 P la t e 1 P la t e 4 P la t e 2 P la t e 5 P la t e 3 P la t e 6 Mean Std. Deviation Std. Error of Mean Plate 1-0.001421 0.002791 0.0006977 Plate 4-0.002157 0.0009560 0.0002390 Plate 2 0.6130 0.03558 0.008896 Plate 5 0.5803 0.04661 0.01165 Plate 3 1.061 0.03884 0.009709 Plate 6 0.9834 0.04179 0.01045
Activation, % Area Under Curve Fluorescence (RLU) α4β2: Agonist Assay 16 point CRC IonWorks Barracuda FLIPR MP-Assay 180 Acetylcholine 160 140 120 Nicotine Epibatidine 1.2 10 06 1,000,000 nachr a4/b 2 Response to Agonists Curve Fit Min and Max Constrained Acetylcholine chloride EC 50 = 150 nm 100 800,000 (-)-Nicotine ditartrate EC 50 = 122 nm 80 60 600,000 400,000 ( )-Epibatidine EC 50 = 2.35 nm 40 20 0 200,000 0-20 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 1000 [Agonist], mm -200,000 10E-6 10E-5 0.0001 0.001 0.01 0.1 1 10 100 1,000 [Agonist] (mm) Reference Agonist EC 50, μm IWB FLIPR Xenopus* Acetylcholine 9.4 0.15 4 Nicotine 5.7 0.12 1 Epibatidine 0.17 0.0024 ND *Moroni et al., 2006 12
Normalized Peak Current, % Normalized Peak Current, % IWB: α7-cho Agonist Assay Max Response, no PAM Max Response, PNU120596 (6 μm) 140 120 100 80 60 40 20 0 Acetylcholine, EC 50 = 95.3 mm Nicotine, EC 50 = 86.7 mm Epibatidine, EC 50 = 9.1 mm 0.01 0.1 1 10 100 1000 [Concentration], μm EC 50, μm 120 Nicotine Cytisine 1.3 1.4 100 Epibatidine 0.03 Acetylcholine 2.7 80 PNU282987 282987 0.07 60 40 20 0 1E-4 1E-3 0.01 0.1 1 10 100 [Concentration], μm 13
Activation, % α7 Positive Allosteric Modulators IonWorks Barracuda FLIPR 120 100 NS 1738 PNU 120596 SB-206553 80 60 40 20 0 1E-3 0.01 0.1 1 10 100 [Concentration], mm Reference EC 50, µm Compound IWB FLIPR Published NS 1738 21.7 ND 12.5* PNU 120596 0.53 3.6 0.216 SB-206553 >5 µm ND 45 PNU 120596 was the most effective PAM *Friis et al. 2008. QPatch ionic current Hurst et al. 2005. FLIPR Ca 2+ flux Roncarati et al., 2008. FLIPR Ca 2+ flux 14
EC 50, μm EC 50, μm Agonist Profiles 100 IWB Agonist 100 FLIPR Agonist 10 10 α3β4 α3β4α5 1 α3β4 α3β4α5 1 α4β2 α7 0.1 α4β2 α7 α6* 0.01 α6* 0.1 Acetylcholine Nicotine 0.001 Acetylcholine Nicotine 15
IWB: Subtype Selectivity Pilot Study: ENZO Library Agonist Mode SP Screen, 786 Drugs @ 60 µm 16
FLIPR: Subtype Selectivity Pilot Study: ENZO Library Agonist Mode SP Screen, 786 Drugs @ 12 µm 110 90 70 50 30 10-10 α3β4 %Activation 110 90 70 50 30 10-10 α3β4α5 %Activation 110 90 70 50 30 10-10 α4β2 %Activation 110 α7 %Activation 90 70 50 30 10-10 17
IWB: Enzo Lib. Screen (60 µm) PAM Mode α7-selective PAMs α3β4-selective PAM PM1 PM2 antineoplastic, EGFR inhibitor antineoplastic, kinase inhibitor PM6 antibacterial, KCNQ1 channel activator PM3 phosphodiesterase inhibitor PM4 microtubule disruptor PM5 corticosteroid 18
% Activation α7 PAM Confirmation CRC: EGFR Inhibitor 0.01 μm 1 μm 550 PM1 450 350 250 AUC (5 s) 150 50 Peak 0.3 μm 30 μm -50 0.01 1 Concentration, µm 19
% Inhibition @10 μm FLIPR vs. IWB Antagonist Screen 100 α7: FLIPR vs. IWB 50 IWB FLIPR 0 20
IC 50, μm IWB: Antagonist Evaluation 100.0 10.0 IWB: nachr Subtype Selectivity α3β4 α4β2 α7 1.0 0.1
% Inhibition % Inhibition % Inhibition IWB: Evidence for Pore Blocking by Nondepolarizing Neuromuscular Blocking Agents in α7 Receptors Concentration-Response Curves: Peak current (blue) vs. AUC (green) Pancuronium Vercuronium Rocuronium 100 100 100 70 70 70 40 40 40 10 10 10-20 -20-20 -50 0.01 1 Conc (um) -50 0.01 1 Conc (um) -50 0.01 1 Conc (um) Drug IC 50, μm (AUC) IC 50, μm (Peak Current) Pancuronium 0.17 0.57 Rocuronium 0.21 0.59 Vecuronium 0.19 0.50 22
nachr Summary IWB and FLIPR platforms are suitable for high-throughput nachr assays, but have different agonist/antagonist sensitivities IWB Agonist & antagonist sensitivity/selectivity of the α3β4, α3β4α5 and α4β2 IWB assays were similar to that of conventional e-phys. IWB Sensitivity of α4β2 and α7 subtypes positive allosteric modulators was similar to conventional e-phys. α6/3 β2 β3 V273S Cell line showed the expected high sensitivity to agonists. EC 50 values were similar to conventional e-phys. α7 Subtype-selective, novel agonists and PAMs were identified in the Enzo Library. 23
Acknowledgements Cell and Molecular Biology Luke Armstrong, Head Zhiqi Liu Abby Sewell Amy Wright Discovery Services Jessica Brimecombe, Head Yuri Kuryshev Nikolai Fedorov Caiyun Wu Xiaoyi Du Peter Hawryluk Support US FDA, Center for Tobacco Products 24