Michael P. Valley

Bioluminescent assays for ADMET.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.

Self-Cleavable Bioluminogenic Luciferin Phosphates as Alkaline Phosphatase Reporters

Alkaline phosphatase (AP)—a stable enzyme with high specific activity for the hydrolysis of phosphate esters—is widely used as a conjugated enzyme label in enzyme-linked immunosorbent assays (ELISA) and DNA hybridization assays. It is also used as an in situ probe to monitor the expression and translocation of fusion proteins from the cytoplasm and for visualization of the spatial distribution of target biomolecules, such as cognate ligands or receptors in cells, tissues, and embryos. Among the many methods for detecting AP activity, there are various phosphate substrates, such as the colorimetric p-nitrophenyl phosphate, the fluorescent AttoPhos<, and the chemiluminescent adamantyl 1,2-diACHTUNGTRENNUNGoxetane AMPPD derivatives (Scheme 1). It is the ultrasensitivity of chemiluminescence, specifically with 1,2-dioxetane AMPPD derivatives, that has made this the overwhelming choice for monitoring AP activity. Although a luciferase-coupled bioluminescent assay is not only generically similar to the chemiluminescent assay and could show similar sensitivity, it also has the additional potential of creating recombinant luciferase to AP protein fusions, which might be preferable for the detection of AP activity in situ. The development of a suitable substrate to reach this ultrasensitivity is needed in order to promote the bioluminescent AP assay for practical applications. Chemical modification of the 6-hydroxyl group of luciferin (or the 6-amino group of aminoluciferin) is an effective means to approach bioluminescent assays for enzymes of interest, and 6-luciferin phosphate (Scheme 1) has been previously shown to detect AP activity. However, the detection limit of 10 19 mol of AP was 2–3 orders of magnitude lower than that for the AMPPD assay. Since the hydrolysis of phosphate monoesters is highly dependent on the pKa of the leaving group and the lower pKa 8.5 [11] of the luciferin phenol compared to a pKa ~9.0 of the adamantyl dioxetane phenol favors both nucleophilic attack and P O bond fission Scheme 1. Chemical structures of substrates for AP enzyme. A) Known chemiluminescent substrate AMPPD derivatives and bioluminescent substrate 6-luciferin phosphate; B) proposed self-cleavable luciferin phosphates, aminoluciferin trimethyl lock phosphate 1, and luciferin p-hydroxymethylphenyl phosphate 2.

Abstract 3731: Luminescent cell health assays for tumor spheroid evaluation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Microtissues produced in 3D cell culture are much more representative of actual living tissue compared to monolayers produced in 2D cell culture. In fact, in the area of oncology research, multicellular tumor spheroids are considered an excellent platform for testing drug delivery and efficacy. As the necessity for established 3D cell culture models rises, there is also a need for convenient assays that have been specifically demonstrated to be effective for use with 3D microtissues. The more complex architecture of 3D microtissues demands increased lytic effectiveness and reagent penetration, characteristics that are often only minor considerations for reagents designed for 2D cell culture. Here we report on a variety of bioluminescent and fluorescent cell-based assays applied to hanging-drop spheroids produced from HCT116 colon cancer cells. The first assay to be described is an ATP detection reagent for measuring cell viability. This reagent has both an improved formulation and an optimized assay protocol and has clear advantages over other viability assays. Other cell health assays will also be described, including reagents that measure cell death, apoptosis, mechanistic cytotoxicity, or reporter gene expression. These additional assays do not require a change in formulation, but do require new protocols in order to optimize their effectiveness when applied to 3D microtissues. As with their application to cells in 2D culture, these “add-mix-measure” reagents are robust and amenable to both low- and high-throughput applications. Citation Format: Michael P. Valley, Kevin R. Kupcho, Chad A. Zimprich, Andrew L. Niles, James J. Cali, Jens M. Kelm, Wolfgang Moritz, Dan F. Lazar. Luminescent cell health assays for tumor spheroid evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2014-3731

Abstract 5435: A bioluminescent assay for measuring glucose uptake

Glucose management is a key biological process. In adipose tissue and skeletal muscle, decreased rates of glucose uptake commonly associated with diabetes mellitus lead to chronic hyperglycemia and a wide array of physiological damage. In contrast, the overexpression of glucose transporters in cancer cells results in increased rates of glucose uptake to support their high rates of proliferation. Thus, activators and inhibitors of glucose uptake are useful for both diabetes management and anticancer therapies. The standard method of assaying glucose uptake involves addition of a radioactive glucose analog (2-deoxyglucose) and measurement of the accumulation of the stable and impermeable phosphorylated derivative, 2-deoxyglucose-6-phosphate (2DG6P). However, radioactive assays are laborious, costly, and require special handling of radioactive materials and waste. A simpler assay can be made by measuring the production of NADPH through the oxidation of 2DG6P by glucose-6-phosphate dehydrogenase. We have developed a bioluminescent glucose uptake assay that is both rapid and convenient and exhibits a larger signal window than comparable fluorescent or colorimetric approaches. The utility of this assay will be demonstrated with both cancer cells and insulin-sensitive 3T3L1 adipocytes and L6 myotubes. Citation Format: Michael P. Valley, Mary Sobol, Jolanta Vidugiriene, James J. Cali. A bioluminescent assay for measuring glucose uptake. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5435. doi:10.1158/1538-7445.AM2015-5435

Abstract 5531: A bioluminescent cell viability assay optimized for 3D microtissues.

It has been well demonstrated that the morphology, gene expression, and overall biological response of cells assessed in 3-dimensional (3D) culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats. In spite of rapidly growing interest, one hindrance to the use of 3D models for drug discovery and other research efforts is the lack of convenient and effective assays explicitly validated for application to 3D microtissues. Here we report on a bioluminescent ATP-detection assay comprised of both an improved formulation and an optimized protocol specifically designed to measure the viability of cells grown in 3D culture. The ability of the 3D-optimized reagent to effectively lyse cells from throughout the full thickness of 3D microtissues, including cancer cell spheroids, was clearly demonstrated by use of confocal laser fluorescent microscopy in conjunction with a fluorogenic, membrane-impermeant DNA-binding dye. Importantly, the assay9s ability to accurately report the ATP content, and therefore viability, of various 3D microtissues (including a range of cancer cell line spheroids) was demonstrated by comparison to a trichloroacetic acid ATP extraction method. In scaffold-free as well as scaffold-dependent 3D models, recovery and detection of greater than 80% of ATP present was routinely observed. In contrast, another frequently used ATP-detection assay was found to have greatly reduced lytic and ATP detection properties ( Citation Format: Michael P. Valley, Chad Zimprich, James J. Cali, Dan F. Lazar. A bioluminescent cell viability assay optimized for 3D microtissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2013-5531