John Shultz

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.

A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.

A high-throughput 1,536-well luminescence assay for glutathione S-transferase activity.

Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.

Self-Immolative Bioluminogenic Quinone Luciferins for NAD(P)H Assays and Reducing Capacity-Based Cell Viability Assays

Highly sensitive self‐cleavable trimethyl lock quinone‐luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H‐dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

Abstract 3505: A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis

The selective elimination of malignant cells via the apoptotic process continues to be the cornerstone of modern anti-cancer therapy regimens. Therefore, in vitro screening approaches aimed at identifying clinically useful apoptosis inducers remain critically important. Recently, phenotypic screening methods have enjoyed a resurgence due to more biologically complex and relevant cell models as well as advances in chemical proteomics which have allowed for more successful target identification. As a consequence, novel probes and tools with enabling attributes are required to fully realize this discovery potential. In an effort to address this unmet need, we have developed a bioluminescent and homogeneous annexin V binding assay for the assessment of apoptosis. Unlike traditional fluorescent annexin V methodology, the “no-wash” reagent employed in this new assay utilizes binary components of a novel luciferase separately fused to annexin V. The annexin V-luciferase subunit fusion pairs have low intrinsic affinity for each other and thus produce no or low luminescence until phosphatidylserine (PtdSer) exposure drives annexin-fusion pair oligimerization. Ultimately, this protein:protein interaction on or near the cell surface reconstitutes full luciferase activity causing an increase in luminescence in the presence of a luciferase substrate. A separate, pro-fluorescent, multiplexed component of the reagent further delineates differences in annexin positivity based on maintenance or loss of membrane integrity corresponding to apoptosis or necrosis, respectively. We validated this method using a panel of diverse cancer cell lines (U2-OS, DLD-1, HeLa, Jurkat, K562, A549, and PC-3), representing both attachment-dependent and -independent morphologies after dose-dependent challenge with intrinsic (bortezomib, panobinostat, staurosporine, and paclitaxel) and extrinsic (rhTRAIL) inducers of apoptosis as well as agents known to produce primary necrosis (ionomycin and digitonin). Caspase activation data was also collected in parallel plates at endpoint as a well-validated and sensitive orthogonal comparator. The bioluminescent annexin V method proved sufficiently robust in 384 well microplate formats to routinely produce Z’ > 0.7 and rank-order potencies in good agreement with caspase activation values. In addition to this microplate functionality, the reagent allowed for sensitive, facile imaging of apoptotic induction in living cells using different imaging platforms. Taken together, the method and reagent should provide unparalleled flexibility with regard to live cell apoptosis detection in both conventional microplate and high content-like imaging formats and advance the pace of new chemical entity discovery. Citation Format: Kevin Kupcho, John Shultz, Andrew Niles, Wenhui Zhou, Robin Hurst, Jim Hartnett, Thomas Machleidt, Terry Riss, Dan Lazar, Jim Cali. A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3505.

Abstract 1135: Bioluminescent methods to investigate cellular metabolic status: NAD(P)/NAD(P)H levels, redox potential, and hydrogen peroxide concentration

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cancer is a disease defined by uncontrolled cell growth where cellular energy metabolism pathways must evolve for tumors to survive and proliferate. NAD(P) and NAD(P)H play a major role in oxidative phosphorylation and their role in aerobic glycolysis is of great interest. Additionally, NAD(P)/NAD(P)H act as co-factors for enzymes involved in cancer pathogenesis through regulation of chromatin structure, DNA repair, and transcription (e.g. sirtuins and poly(ADP-ribose) polymerase). The study of how NAD(P)/NAD(P)H are generated and utilized during adaptive cancer cell energy metabolism would benefit from the development of a rapid, sensitive, and homogeneous assay to measure the level of these nucleotides. We developed a bioluminescent assay for measuring NAD(P)/NAD(P)H that can detect ≤ 0.1 µM NADH and has a 1000-fold dynamic range. The assay is well suited for high throughput screening (Z’ = 0.92, S/B = 95) and has been used to screen the LOPAC library. By coupling other reactions with NAD(P)/NAD(P)H measurement, we analyzed the levels of metabolites and the activity of enzymes, including isocitrate dehydrogenase and pyruvate dehydrogenase, in enzyme preparations and crude cell lysates. This method is applicable for measuring cellular NAD and NADH levels directly from cell culture without further sample handling. This assay is based on a novel proluciferin derivative that is processed in vitro through an enzymatic reaction. The released luciferin is detected in a coupled luciferin/luciferase reaction and the luminescent signal is correlated with the amount of NAD(P)/NAD(P)H present in the sample. As an extension of this approach, similar proluciferin chemistries were developed to detect the reducing potential of metabolically active cells. This robust luminescent assay can detect the reducing potential of less than 100 cells/well in 96-well format and distinguish small changes in cell number. Multiple cell lines were treated with cancer therapeutic compounds and the performance of the proluciferin approach was compared to commonly used colorimetric (e.g. MTT, MTS, and XTT) and fluorogenic (e.g. resazurin) methods. The proluciferin assay resulted in comparable pharmacological responses, significantly increased signal window, and superior sensitivity. Additionally, the proluciferin-based approach has been extended to other metabolic readouts. For example, novel proluciferin substrates were developed and applied to the detection of hydrogen peroxide in cells and in biological samples through a HRP-independent process. Bioluminescent assays provide greater sensitivity and dynamic range than most fluorescent or colorimetric assays, and are better suited for high throughput screening. Applying these assays to the study of cancer cell energy metabolism will provide a significant advantage over existing methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1135. doi:1538-7445.AM2012-1135

Reversed enzyme activity DNA interrogation test: a new method for mutation detection

The Reversed Enzyme Activity DNA Interrogation Test (READITTM) System provides a new, rapid method for genotype determination. The system can be used for the analysis of single nucleotide polymorphisms as well as insertions, deletions and chromosomal rearrangements. The READITTM System is based upon driving various enzymatic reactions in the reverse direction from the direction they are normally considered. The method is based on a pyrophosphorylation reaction that is the reverse of DNA polymerization. Interrogation probes annealed to DNA fragments with which they have a 3- mismatch are not a substrate for the reaction and do not generate significant signals compared with probes that match the interrogation sequence perfectly. By interrogation of specific DNA sequences with carefully designed probes, mutant genes can be identified with speed and precision.