Assay development for screening on purified or partially purified targets with fluorescent and bioluminescent readouts

Paradigms in Drug Discovery Physiology-based Target-based Target is unknownKnown target Physiological/phenotypic read-outsRead-outs are based on activity or expression of target Cell-based assaysBiochemical or Cell-based assays

Types of Assays Assays in Drug Discovery Biochemical assays Cell-based assays Target-based Phenotype-based - Enzymes (e.g. kinases, proteases)- Transcriptional read-outs - Receptors (e.g. Nuclear receptors, - Second messenger levels Kinase receptors, ion channels, GPCRs)- Protein interactions - Hormones- Protein expression and localization - Cell viability (cell death/apoptosis) - Proliferation

Fluorescence-based assays Based on excitation of a fluorophore Variety of assays using fluorescence protein of interest is conjugated to fluorophore, protein of interest generates a fluorescent product Advantages: - High sensitivity - relative easy to handle - safer than radioactivity - Simultaneously detection of more than one molecule Disadvantage: - inner filter effects - other fluorescent compounds in cell

FRET: Fluorescence Resonance Energy Transfer Principle: Two fluorophores - Donor and Acceptor Based on transfer of energy between donor and acceptor Distance is critical Far: No energy transfer -> no FRET Close: Energy transfer from donor to acceptor -> FRET Use: Protein-protein, antigen-antibody, DNA-DNA, DNA-protein

TR-FRET Time Resolved-Florescence Resonance Energy Transfer “Improved” version of FRET Uses long lived fluorophores and timeresolved detection to reduce background Rare earth elements (Lanthanides):Samarium (Sm), Europium (Eu),Terbium (Tb), and Dysprosium (Dy) fluorescence lifetime: period of time in which the fluorophore is in an excited state Advantage: Low background; better signal to noise Disadvantage:Lanthanides have poor ability to absorb light, -> have to be complexed with organic moieties that can harvest light and transfer it to them.

Bioluminescence-based assays Bioluminescence production and emission of light by a living organism (e.g luciferase by firefly) detects the ATP depletion over time by using the phosphorescence of luciferase and luciferin

Examples

Promega: Kinase/ADP-Glo™ assay test selectivity and potency of the inhibitor on multiple kinases from different classes measures kinase activity by quantifying the amount of ADP produced during the enzymatic reaction positive detection assay for product formation can also be used for other reaction that produce ADP measurement of ATP remaining after kinase-reaction

Promega: Kinase/ADP-Glo™ assay

PerkinElmer: LANCE Ultra PKC Kinase Assay Time-resolved fluorescence resonance energy transfer (TR-FRET) assay Uses a proprietary europium chelate donor dye and a small molecular weight acceptor dye Measures the phosphorylation of a PKC peptide substrate at Ser25 Intensity of the light emission is proportional to the level of substrate phosphorylation - Binding of Eu-labeled antibody directed against phosphorylation of PKC peptide substrate - Eu donor and ULight acceptor dye in close proximity - Causes red-shifted fluorescent emission

PerkinElmer: LANCE Ultra PKC Kinase Assay

ThermoFisher: Amplex® Red Enzyme Assays Quantitative enzyme activity detection Fluorescence emission outside the range of compound autofluorescence -> Resorufin (excitation/emission maxima=570/585 nm) High-throughput compatibility Detection of H 2 O 2

Thanks for your attention Questions?