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HTS and Lead Optimization Using FLIPR

12/98

Presentation:  HTS and Lead Optimization Using FLIPR

Joseph Gunnet, Ph.D.

Principal Scientist, Endocrine Therapeutics

The R.W. Johnson Pharmaceutical Research Institute, Rt. 202, Raritan, NJ 08869

The identification of functional agonists and antagonists for some G-protein coupled
receptors (GPCRs) can be done by monitoring intracellular calcium mobilization. FLIPR
(Fluorometric Imaging Plate Reader; Molecular Devices Corp.) allows GPCR-induced calcium
responses to be accurately and reliably quantitated in an entire 96 well plate. With its
CCD camera, FLIPR collects data at rate sufficient to follow the magnitude and time course
of GPCR activation in each well. The large amount of information gathered from each well
may be analyzed to simply identify hits in HTS or may be analyzed in more detail to
optimize leads and ensure receptor-mediated activity. While most of the varibles in using
FLIPR are the same as for any 96 well liquid handling system and fluorescent-based assay,
performing HTS with FLIPR poses some unique issues and opportunities. We have worked
through some of these biological, mechanical and data analysis issues and have
successfully run HTS with FLIPR. Improvements in FLIPR hardware and data processing will
soon be available and will expand the utility of an already useful instrument.

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Table of Contents

HTS and Lead Optimization Using FLIPR

Intracellular Signaling by G-Protein Coupled
Receptors

FLIPR – Fluorometric Imaging Plate Reader

FLIPR 1 System

Schematic Diagram of FLIPR System

FLIPR Performance

FLIPR Program Window

HTS Protocol for Detecting GPCR Agonists &
Antagonists

HTS Protocol for Detecting GPCR Agonists &
Antagonists

HTS Protocol for Detecting GPCR Agonists &
Antagonists

Response to Sequential Fluid Additions

Issues in Assay Validation

Effect of Ionomycin on Calcium Mobilization in
Different Cell Types

FLIPR Specific Issues in Assay Validation

Export of FLIPR Data

Analysis of FLIPR Response

HTS Performance

Lead Identification & Characterization

Identification of “True” Receptor Agonists

Agonist Dose Response

Agonist Dose Response

Antagonist Dose Response

Antagonist Dose Response

The New FLIPR – FLIPR384

FLIPR384 Overview

FLIPR384 Overview

Summary

Author: Ortho-McNeil Pharmaceutical

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