A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors.
The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibition. The generic nanoparticle donors further promote versatility and cost-efficiency of the method. The performance evaluated as the inhibitor (Z-DEVD-FMK) dose-response curve (IC(50) value of approximately 12 nM) was in good agreement with that of the recent methods found in literature. This assay serves as a model application proving the feasibility of the europium-chelate-doped nanoparticle labels in a homogeneous assay for proteolytic activity.
Valanne
A
A
Department of Biotechnology, University of Turku, 20520 Turku, Finland. antti.valanne@utu.fi
Malmi
P
P
Appelblom
H
H
Niemelä
P
P
Soukka
T
T
D019921:Biotinylation; D061945:Caspase Inhibitors; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D053758:Nanoparticles; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors