Guided Analytics for FLIPR fluorescence processing in biotech R&D Tod Flak, Ph.D. Head, Lab Informatics & Automation Axxam srl San Raffaele Biomedical Science Park Milan, Italy

About Axxam AXXAM is located at the San Raffaele Science Park, and has more than 1,400 square meters of fully equipped laboratories and office space November 2001 entered a five year research agreement (worth € 30 million) with Bayer AG AXXAM srl was founded May 2001 in Milan Our team consists of 50 people

Axxam: From Genes to Leads Business model Industrial collaborations and services Internal R&D for technologies and know-how / IP position Currently several pharma/biotech partners Activities Target identification and validation Development of screening assays for lead identification (HTS, uHTS, etc...) Screening and lead profiling Industrial areas Pharmaceutical Diagnostic Agro-chemical

AXXAM: The Assay Factory Over 20 clean benches, 30 cell incubators Multiple Pipetting robots: Matrix and CyBio workstations (96/384/1536 well formats); Packard Multiprobes 2 FACS stations 6 Luminescence readers (3x 96 and 3x 384 well format) 3 Fluorescence readers (1x 96, 1x 384 and 1x 1536) FLIPR 384 well workstation Assay development and validation Screening services More than 100 screening assays are already available

FLIPR based Assays for HTS FLIPR® (Fluorometric Imaging Plate Reader) system from Molecular Devices is an industry-standard platform CCD-based plate reader with integrated plate handling and pipetting (384 channel) Axxam has configured FLIPR assays for multiple gene classes: GPCRs coupled to Gq GPCRs artificially coupled to Gq Ligand gated ion channels Voltage gated Ca 2+ and K + channels Electrogenic transporters Ion exchangers All assays are optimized for the use in 384 well format Instrument used for internal development and also high-throughput screening for partners

Requirements for FLIPR data analysis Specific import of FLIPR data format Curve-fitting Adapt easily to research & development phase :: single plates, never the same plate layout twice! Easy to use Customizable

Spotfire FLIPR Guide Walks users through analysis steps Reorganization and summarization of data Visualize data at all steps Mark bad data Integrated Curve fitting – using Excel (no need to purchase specialized curve-fitting software) Open code -- easily customizable All the other benefits of Spotfire

Demo of basic FLIPR Guide

FLIPR Guide – Demo Slide 1

FLIPR Guide – Demo Slide 2

FLIPR Guide – Demo Slide

Customization of FLIPR Guide If only used in processing HTS data, no customization needed – Plate layout fixed – Typically stimulate with known agonist, experimental compounds are potential inhibitors In assay development, need to change things such as: – Plate layouts – Filenames – Variable of interest – optimize agonist concentrations (agonist alone), incubation times, etc.

Plate Layout Tool Users can define layout on-the-fly Integrated into Spotfire guide workflow Built in Excel

DEMO I will do demonstration here of Spotfire FLIPR guide Read in FLIPR files Define plate layout (using my tool) Walk through guided analytic Demonstrate marking bad data Do curve fitting Point out customized options for other assay modes (variable agonist conc, with or without antagonist, etc.) Time: about 5-7 minutes

ATP-sensitive Potassium Channel K ATP channels are formed as a complex of : Z sulfonylurea receptor (SURx): SUR1 (pancreas) SUR2A (heart) SUR2B (smooth muscle) & Z inward rectifier (Kir6.x) subunit: Kir6.1 Kir6.2 Family of weak inward rectifiers Distinguishing feature: intracellular ATP inhibits the channel, increase in intracellular MgADP activates the channel C N SUR1 Kir6.2 K+K+ OUT IN ATP glibenclamide Openers MgADP + +

Complex Voltage-sensitive experiment 100 uM Diazoxide (induces K+ outflow) Glibenclamide (blocks channel, restores intracellular potential) High Glibenclamide Low Glibenclamide Voltage sensitive dye Monitoring depolarization and restoration by inhibitor

Complex Voltage-sensitive experiment 100 uM Diazoxide (induces K+ outflow) Glibenclamide (blocks channel, restores intracellular potential) High Glibenclamide Low Glibenclamide Voltage sensitive dye Monitoring depolarization and restoration by inhibitor

Voltage-sensitive channel assay testing First injection: 100uM diazoxide Second injection: glibenclamide 0.01nM -- dose curve  30uM

ATP-sensitive Potassium Channel, Kir6.2/SUR1, in CHO-K1 Blocker Analysis Literature IC 50 : 1.6 nM Calculated IC 50 : 31 nM Tyrode 0.01 uM 0.05 uM 0.1 uM 0.5 uM 1 uM 5 uM 10 uM 50 uM Time(minutes) Dose-inhibition curves to glyburide Fluorescence Change (Counts) Diazoxide 200 uM Gliburide 30’’ 4’30’’ 8’30’’ 200 uM DiazoxideGlibenclamide Assay Protocol

Time (minutes) Fluorescence Change (Counts) Diazoxide:  M - 10  M  M  M Diazoxide Glibenclamide 10  M ATP-sensitive Potassium Channel, Kir6.2/SUR1 in CHO 30’’ 4’30’’ 8’30’’ 100 uM Diazoxide10 uM Glibenclamide Assay Protocol

Summary For Axxam, Spotfire provided a good solution for our needs in analysis of FLIPR data Customization and extensibility of the tool are crucial We added options for – Quickly defining plate layouts – Non-standard experimental design processing In future we will add – More processing options – Mechanism to retrieve data using LIMS database links – Mechanism to save results into LIMS database