Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

O’Brien, Martha A.; Daily, William J.; Hesselberth, P. Eric; Moravec, Richard A.; Scurria, Michael A.; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.

doi:10.1177/1087057104271865

Cited by 109 publications

(95 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Z-DEVD-aminoluciferin, a proluminescent caspase-3/7 substrate, has been successfully used for in vitro assessment of caspase-3 or caspase-7 activity. [18][19][20] Specific cleavage of the DEVD peptide by caspase-3/7 liberates free aminoluciferin, which is consumed by the luciferase, generating a luminescent signal that is proportional to activity. Docetaxel is a microtubule-disruptive apoptotic agent commonly used as chemotherapy medication to treat various cancer indications, and has previously been shown to be a potent activator of caspase-3/7 in human mammary (MDA-MB-231) and ovarian (SKOV3) adenocarcinoma cell lines.…”

Section: Resultsmentioning

confidence: 99%

“…This substrate has been previously characterized and is currently widely used as an in vitro reporter for apoptosis. [18][19][20] However, experiments to date have failed to transition this probe in vivo without acute toxicity to the animal. 21 Using chemical material purified for in vivo use, we report a successful formulation of Z-DEVD-aminoluciferin that is well tolerated in vivo after multiple doses and enables quantification of apoptosis noninvasively in two oncology models.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin

et al. 2010

View full text Add to dashboard Cite

Apoptosis is a highly regulated process of programmed cell death essential for normal physiology. Dysregulation of apoptosis contributes to the development and progression of various diseases, including cancer, neurodegenerative disorders, and chronic heart failure. Quantitative noninvasive imaging of apoptosis in preclinical models would allow for dynamic longitudinal screening of compounds and facilitates a more rapid determination of therapeutic efficacy. In this study, we report the in vivo characterization of Z-DEVD-aminoluciferin, a modified firefly luciferase substrate that in apoptotic cells is cleaved by caspase-3 to liberate aminoluciferin, which can be consumed by luciferase to generate a luminescent signal. In two oncology models, namely SKOV3-luc and MDA-MB-231-luc-LN, at 24, 48, and 72 h after treatment with docetaxel, animals were injected with Z-DEVD-aminoluciferin and bioluminescent images were acquired. Significantly more light was detected at 24 (Po0.05), 48 (Po0.01), and 72 h (Po0.01) in the docetaxel-treated group compared with the vehicle-treated group, with caspase-3 activation at these time points confirmed using immunohistochemistry. Importantly, whereas significant differences between groups were detected as early as 24 h after treatment by molecular imaging, caliper measurements were unable to detect a difference for 4-5 additional days. Taken together, these data show that in vivo imaging of apoptosis using Z-DEVD-aminoluciferin could provide a sensitive and rapid method for early detection of drug efficacy, which could potentially be used by numerous therapeutic programs.

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin

et al. 2010

View full text Add to dashboard Cite

show abstract

“…This proteolytic activity, released into culture medium upon cell death, can be measured with a luminogenic, cell-impermeant peptide substrate. This technology utilizes an amino-derivatized luciferin and a luciferase-based detection reagent that generates luminescence proportional to proteolytic activity (O'Brien et al, 2005). This cell-based protease-release assay measures cell membrane integrity homogenously, which has only one step of reagent addition, with luminescent readout.…”

Section: Discussionmentioning

confidence: 99%

A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker

Cho

Niles

Huang

et al. 2008

Toxicology in Vitro

View full text Add to dashboard Cite

Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic "release" assays which measure the extracellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known toxins from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.

show abstract

“…The bioluminescent product of the reaction of interest can then be detected as a luminescent signal in a second reaction with firefly luciferase. Assays based on bioluminogenic substrates have been reported for proteases (O'Brien et al, 2005), oxidases (Zhou et al, 2006b), phosphatases (Zhou et al, 2008), glutathione transferases (Zhou et al, 2006a), and glycosidases . In the majority of these cases, the bioluminogenic substrates have been modified at the 6Ј-carbon atom of luciferin with a recognition moiety for the enzyme of interest (see Scheme 1 for luciferin numbering).…”

Section: Introductionmentioning

confidence: 99%

Proluciferin Acetals as Bioluminogenic Substrates for Cytochrome P450 Activity and Probes for CYP3A Inhibition

Meisenheimer¹,

Uyeda²,

Ma³

et al. 2011

Drug Metab Dispos

Self Cite

View full text Add to dashboard Cite

show abstract

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

Cited by 109 publications

References 25 publications

Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin

Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin

A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker

Proluciferin Acetals as Bioluminogenic Substrates for Cytochrome P450 Activity and Probes for CYP3A Inhibition

Contact Info

Product

Resources

About