Combinatorial Chemistry & High Throughput Screening - Volume 14, Issue 8, 2011

A fundamental challenge in bioscience research is to quantify changes in the concentration of key signaling molecules within the context of biologically relevant cellular model systems. Cell-based assays represent a useful midpoint between whole animals and purified molecules for understanding complex biological processes in a statistically valid way. In particular, they enable a reductionist approach to understanding biochemical interactions within the context of the native cytoplasmic milieu. The inverse is not always true however, in that cell based assays do not necessarily describe the native tissue based environment behavior of cells in a whole organism. While a number of technologies have been developed to quantify biochemical changes within cells, bioluminescent methods have evolved as a dominant technique for monitoring changes in cellular bioanalytes. The physics of bioluminescence provides inherent advantages for cell based assays because the photonic excitation energy is generated by a chemical reaction. In fluorescent methods the excitation energy is derived from an excitation photon that can overlap with the the emission spectra or cause off target fluorescence from cellular components or the exogenously added small molecule being studied. The chemistry of the luciferase based bioluminescence reaction has been exploited to quantify small molecules such as cellular ATP, the enzymatic activity of proteins such as Caspase-3, or cell signaling pathways with transcriptional endpoints such as NF-kappaB (Fig. 2). Recently, a circularly permuted luciferase has been developed that allows for real time monitoring of changes in intracellular cAMP concentrations (Fig. 3). The combination of this platform of bioluminescent chemistries with add-and-read Glo methodologies provides highly accurate tools for quantifying cellular changes due to pharmacological, developmental, oncological, or immunological perturbations. This issue of Combinatorial Chemistry and High Throughput Screening is designed to provide a partial survey of methods that have proved useful in describing different biological processes. Miraglia et al., present an overview of luminescent reporter assays and technical issues that impact assay robustness; Larson et al., leverage liquid handling instrumentation to multiplex bioluminescent ADMETox assays; Takagi et al., used biochemical luminescent assays to compare radioactive assays with bioluminescent methods for screening kinase inhibitors; Simmons reviews the use of luminescent genetic reporters as toxicity biomarkers; and Kokatam et al., used a creative reporter gene design to help understand the complex angiogenic signaling pathway....

The luminescent reporter gene assay (LRGA) is arguably the most prominent type of reporter gene assay used in biomolecular and pharmaceutical development laboratories. Part of this popularity is due to the high signal associated with luciferases, the foundation of this technology. This feature makes them ideally suited for high throughput screening applications where potentially millions of chemical compounds can be analyzed in a given assay. Recent technical advancements that enhance signal stability of the luciferases along with development and commercialization of multiple forms of luciferases, their respective substrates, and improvements in expression vectors for reporter gene assay (RGA) applications have broadened their use. While the practical challenges related to the application of luminescent technology in a laboratory setting have been overcome, there remains much to do in laying a systematic approach towards the construction of RGAs, which are essential to the elucidation of the basic biology for genes of interest. This mini-review aims at giving a birds-eye view of the available luciferases, substrates and other luminescent technologies available and provides a general blueprint as well as practical considerations for constructing and interfacing RGAs with chemical biology and functional genomics for the elucidation of fundamental biological questions and for biomedical research.

ADME-Tox testing examines the effects of an organism, tissue or cell on a compound, as well as the effects that the compound has on an organism, tissue or cell, and has gained in importance in the overall drug discovery process over the past twenty years. This is due to the rising percentage of drug candidate attrition in the 1990s and early 2000s due to adverse ADME/Tox profiles. The increased importance placed upon ADME/Tox testing has brought about new types of in-vitro assay technologies utilizing microplates to deliver the most pharmacologically relevant data. These tests, however, have typically been performed sequentially, where multiple assays over multiple microplates are used. This typically leads to increased time and cost required to generate the required information, and can sacrifice data quality. Multiplexed assays, however, where more than one piece of data can be attained from a single well or a single microplate, performed using appropriate liquid handling and detection instrumentation, can improve data quality and reduce the time and expense required to attain this information.

Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [33P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [33P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.

Whole-animal studies have been the mainstay of toxicity testing for decades. These approaches are too expensive and laborious to effectively characterize all of the chemicals currently in commercial use. In addition, there are social and ethical pressures to reduce, refine and replace animal testing in toxicology. The National Research Council (NRC) has outlined a new strategy to transition from animal-based tests to high throughput, cell-based assays and computational modeling approaches to characterize chemical toxicants. Critical to this vision, assays that measure toxicity pathways associated with adverse health effects must be developed. Bioluminescent assays are particularly well suited to the demands of next-generation toxicity testing because they measure a wide range of biological activities in a quantitative and high throughput manner. This review describes the limitations of traditional, animal-based toxicity testing and discusses the current and developing uses of bioluminescent technologies in next-generation testing based on three general assay formats: luciferase-limited assays, ATP-limited assays and luciferin-limited assays.

Angiogenesis is a promising area of research that targets key therapeutic areas like cancer; wound healing, inflammatory diseases, etc. There is an increasing demand for screening of potential angiogenic and anti-angiogenic agents using sensitive, robust cell-based assays. We have developed a reporter vector containing cis-acting elements that respond to growth factors/angiogenic ligands for use in a cell-based luciferase reporter assay. We performed transient transfection of our reporter gene vector in MCF-7 cells to establish its application for screening of potential pro/antiangiogenic agents. Reporter gene transactivation studies with different concentrations of fetal bovine serum clearly indicated that the vector is functionally responsive to the angiogenic signals mediated by serum growth factors. We also used endostatin to inhibit transactivation and prove responsiveness to the anti-angiogenic agent. This vector is a promising tool for studying angiogenesis using cell-based reporter gene assays.

Dr. Watson has a B.S. in Genetics and Cell Biology from the University of Minnesota, an M.S. in Bacteriology; and a Ph.D. in Biomolecular Chemistry from the University of Wisconsin. After a clinical postdoctoral fellowship in Clinical Chemistry and Molecular Diagnostics he became Director of Molecular Diagnostics at Epicentre Technologies where he led R&D and Marketing efforts for their clinical research products. Subsequent positions have included Director of Cellular Analysis at Promega Corporation, and Director of Marketing for the PanVera division Vertex Pharmaceuticals (becoming Director of Marketing for Drug Screening at Invitrogen after the acquisition). He also managed R&D, applications and Marketing for the FTIR instrument division of Thermo Electron Corporation. He currently is the Director of Cellular Analysis and Pharma/Biotech at Promega.

Cell surfaces, especially mammalian cell surfaces, are heavily coated with complex poly- and oligosaccharides, and these glycans have been implicated in many functions, such as cell-to-cell communication, host-pathogen interactions and cell matrix interactions. Not surprisingly then, the aberrations of glycosylation are usually indicative of the onset of specific diseases, such as cancer. Therefore, glycans are expected to serve as important biomarkers for disease diagnosis and/or prognosis. Recent development of the lectin microarray technology has allowed researchers to profile the glycans in complex biological samples in a high throughput fashion. This relatively new tool is highly suitable for both live cell and cell lysate analyses and has the potential for rapid discovery of glycan-based biomarkers. In this review, we will focus on the basic concepts and the latest advances of lectin microarray technology. We will also emphasize the application of lectin microarrays for biomarker discovery, and then discuss the challenges faced by this technology and potential future directions. Based on the tremendous progress already achieved, it seems apparent that lectin microarrays will soon become an indispensible tool for glycosylation biomarker discovery.

The application of Isodon species in Chinese folk medicine has a long histroy, especially the ones called ‘Xihuangcao’ in Chinese. ‘Xihuangcao’ has been successfully applied to treat acute hepatitis, cholecystitis, enteritis, dysentery and trauma. The original species of ‘Xihuangcao’ is Isodon lophanthoides (Buch.-Ham.ex D.Don). However, there are five sources of Chinese medicinal herb ‘Xihuangcao’ due to their similar morphology and close pharmaceutical activity. Each source belongs to Isodon. However, their chemical composition and bioactivities are significantly different. In order to differentiate these sources of ‘Xihuangcao’ and to know their pharmaceutical effects, this review summarizes the chemical constituents, bioactive properties of ‘Xihuangcao’ and their available application.

β-Fructofuranosidases can divert their hydrolytic activity towards transglycosylation for the synthesis of high value-added products, including prebiotic fructooligosaccharides (FOS). A directed evolution strategy has been employed to enhance the transferase rate of the β-fructofuranosidase (SoINV) from the Schwanniomyces occidentalis yeast for the production of β-(2→6)-linked FOS. To screen for transferase activity of the SoINV functionally expressed in Saccharomyces cerevisiae, a high-throughput screening protocol based on two colorimetric assays was validated (with coefficient of variance below 11%). Mutagenic libraries were constructed by error-prone PCR and clones showing higher glucose:fructose ratio with respect to the parental type were identified. Further analysis by anion-exchange chromatography coupled with pulsed amperometric detection helped to identify mutants with improved yields for the synthesis of β-(2→6) fructooligosaccharides. Selected mutants displayed transferase initial rates enhanced ∼2-fold over parent type, reaching production levels up to 47 g/L after 48 h of reaction for the formation of 6-kestose.

The section on patent review will be focused in the areas of interest to the readers of CCHTS. The search was conducted using the following key words: combinatorial chemistry, high throughput screening, drug repurposing, chemical library, high content screening, drug discovery and natural products. All patents highlighted here are identified by the patent number issued either by the World Intellectual Property Organization or by a regional patent office.