Bioluminescence and chemiluminescence in drug screening

Drug screening, that is, the evaluation of the biological activity of candidate drug molecules, is a key step in the drug discovery and development process. In recent years, high-throughput screening assays have become indispensable for early stage drug discovery because of the developments in synthesis technologies, such as combinatorial chemistry and automated synthesis, and the discovery of an increasing number of new pharmacological targets.Bioluminescence and chemiluminescence represent suitable detection techniques for high-throughput screening because they allow rapid and sensitive detection of the analytes and can be applied to small-volume samples. In this paper we report on recent applications of bioluminescence and chemiluminescence in drug screening, both for in vitro and in vivo assays. Particular attention is devoted to the latest and most innovative bioluminescence and chemiluminescence-based technologies for drug screening, such as assays based on genetically modified cells, bioluminescence resonance energy transfer (BRET)-based assays, and in vivo imaging assays using transgenic animals or bioluminescent markers. The possible relevance of bioluminescence and chemiluminescence techniques in the future developments of high-throughput screening technologies is also discussed.     

Cell-based assays: fuelling drug discovery

It has been estimated that over a billion dollars in resources can be consumed to obtain clinical approval, and only a few new chemical entities are approved by the US Food and Drug Administration (FDA) each year. Therefore it is of utmost importance to obtain the maximum amount of information about biological activity, toxicological profile, biochemical mechanisms, and off-target interactions of drug-candidate leads in the earliest stages of drug discovery. Cell-based assays, because of their peculiar advantages of predictability, possibility of automation, multiplexing, and miniaturization, seem the most appealing tool for the high demands of the early stages of the drug-discovery process. Nevertheless, cellular screening, relying on different strategies ranging from reporter gene technology to protein fragment complementation assays, still presents a variety of challenges. This review focuses on main advantages and limitations of different cell-based approaches, and future directions and trends in this fascinating field.     

Development and application of fluorescence polarization assays in drug discovery

Fluorescence polarization technology has been used in basic research and commercial diagnostic assays for many decades, but has begun to be widely used in drug discovery only in the past six years. Originally, FP assays for drug discovery were developed for single-tube analytical instruments, but the technology was rapidly converted to high-throughput screening assays when commercial plate readers with equivalent sensitivity became available. This review will discuss fluorescence polarization assays in current use in drug discovery research as well as those in development that will likely be used in the near future. These assays include targets such as kinases, phosphatases, proteases, G-protein coupled receptors, and nuclear receptors.     

High content screening for G protein-coupled receptors using cell-based protein translocation assays

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.     

Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.     

Parallel and multiplexed bead-based assays and encoding strategies

Advances in high throughput screening (HTS), together with the rapid progress in combinatorial chemistry, genomic and proteomic sciences have dramatically stimulated the development of a variety tools to enable the drug discovery process to become more efficient. Major future challenges in HTS include obtaining high density and good quality data based on assays that are rapid, reliable, inexpensive, sensitive, simple and miniaturised. This paper reviews the development and role of bead-based assays for HTS including DNA and single nucleotide polymorphism (SNP) assays, particularly from a multiplex perspective and evaluating the recent advances in bead-based arrays. The encoding strategies that are commonly used in bead-based assays are highlighted, while the importance of magnetic beads in genomic and proteomic purifications is discussed. In conclusion, bead-based assays offer a powerful promising approach for many aspects of drug discovery.     

High-throughput enzyme assay on a multichannel microchip using optically gated sample introduction

To meet the requirements for high-throughput screening for drug discovery research, it is very important to develop techniques with the ability of performing multiple enzyme assays simultaneously. Using optically gated sample introduction on a multichannel microchip, multiple enzyme assays have been demonstrated in four parallel channels. The hydrolysis of fluorescein mono-beta-D-galactopyranoside by beta-galactosidase and the inhibition of this reaction by the competitive inhibitor phenylethyl beta-D-thiogalactoside were initially studied to determine the effect of system movement using the voice coil actuator on the enzyme assay reaction. The results from these two studies are consistent with the results from the assay using a single-channel microchip, and they demonstrate that the system using optically gated sample introduction on multichannel microchip can be used to perform multiple enzyme assays. Three unique enzyme assays were also performed in different channels, which show this technique could be competitive for high-throughput screening in drug discovery with other traditional techniques.     

An overview of drug screening using primary and embryonic stem cells

Cellular technologies are widely used in drug discovery to treat human diseases. Most studies involve the expression of recombinant targets in immortalized cells and measure drug interactions using simple, quantifiable responses. Such cells are also amenable to high throughput screening (HTS) methods. However, the cell phenotype employed in HTS is often determined by the assay technology available, rather than the physiological relevance of the cell background. They are, therefore, suboptimal surrogates for cells that accurately reflect human diseases. Consequently, there is growing interest in adopting primary and embryonic stem cells in drug discovery. Primary cells are already used in secondary screening assays in conjunction with confocal imaging techniques, as well as in target validation studies employing, for example, gene silencing approaches. Stem cells can be grown in unlimited quantities and can be derived from transgenic animals engineered to express disease causing proteins better coupling the molecular target with function in vivo. Human stem cells also offer unique opportunities for drug discovery in that they can be directed to specific phenotypes thus providing a framework to identify tissue-selective agents. Organizing stem cells into networks resembling those in native tissues, potentially returns drug discovery back to the highly successful pharmacological methods of the past, in which organ and tissue based systems were used, but with the advantage that they can be utilized using modern HTS technologies. This emerging area will lead to discovery of compounds whose effect in vivo is more predictable thereby increasing the efficiency of drugs that ameliorate human disease.     

Data visualization during the early stages of drug discovery

Multidimensional compound optimization is a new paradigm in the drug discovery process, yielding efficiencies during early stages and reducing attrition in the later stages of drug development. The success of this strategy relies heavily on understanding this multidimensional data and extracting useful information from it. This paper demonstrates how principled visualization algorithms can be used to understand and explore a large data set created in the early stages of drug discovery. The experiments presented are performed on a real-world data set comprising biological activity data and some whole-molecular physicochemical properties. Data visualization is a popular way of presenting complex data in a simpler form. We have applied powerful principled visualization methods, such as generative topographic mapping (GTM) and hierarchical GTM (HGTM), to help the domain experts (screening scientists, chemists, biologists, etc.) understand and draw meaningful decisions. We also benchmark these principled methods against relatively better known visualization approaches, principal component analysis (PCA), Sammon's mapping, and self-organizing maps (SOMs), to demonstrate their enhanced power to help the user visualize the large multidimensional data sets one has to deal with during the early stages of the drug discovery process. The results reported clearly show that the GTM and HGTM algorithms allow the user to cluster active compounds for different targets and understand them better than the benchmarks. An interactive software tool supporting these visualization algorithms was provided to the domain experts. The tool facilitates the domain experts by exploration of the projection obtained from the visualization algorithms providing facilities such as parallel coordinate plots, magnification factors, directional curvatures, and integration with industry standard software.     

Modern drug discovery technologies: opportunities and challenges in lead discovery

The identification of promising hits and the generation of high quality leads are crucial steps in the early stages of drug discovery projects. The definition and assessment of both chemical and biological space have revitalized the screening process model and emphasized the importance of exploring the intrinsic complementary nature of classical and modern methods in drug research. In this context, the widespread use of combinatorial chemistry and sophisticated screening methods for the discovery of lead compounds has created a large demand for small organic molecules that act on specific drug targets. Modern drug discovery involves the employment of a wide variety of technologies and expertise in multidisciplinary research teams. The synergistic effects between experimental and computational approaches on the selection and optimization of bioactive compounds emphasize the importance of the integration of advanced technologies in drug discovery programs. These technologies (VS, HTS, SBDD, LBDD, QSAR, and so on) are complementary in the sense that they have mutual goals, thereby the combination of both empirical and in silico efforts is feasible at many different levels of lead optimization and new chemical entity (NCE) discovery. This paper provides a brief perspective on the evolution and use of key drug design technologies, highlighting opportunities and challenges.     

Monoclonal antibody proteomics: discovery and prevalidation of chronic obstructive pulmonary disease biomarkers in a single step

We define mAb proteomics as the global generation of disease specific antibodies that permit mass screening of biomarkers. An integrated, high-throughput, disease-specific mAb-based biomarker discovery platform has been developed. The approach readily provided new biomarker leads with the focus on large-scale discovery and production of mAb-based, disease-specific clinical assay candidates. The outcome of the biomarker discovery process was a highly specific and sensitive assay, applicable for testing of clinical validation paradigms, like response to treatment or correlation with other clinical parameters. In contrast to MS-based or systems biology-based strategies, our process produced prevalidated clinical assays as the outcome of the discovery process. By re-engineering the biomarker discovery paradigm, the encouraging results presented in this paper clearly demonstrate the efficiency of the mAb proteomics approach, and set the grounds for the next steps of studies, namely, the hunt for candidate biomarkers that respond to drug treatment.     

High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation

Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.     

The beautiful cell: high-content screening in drug discovery

The term “high-content screening” has become synonymous with imaging screens using automated microscopes and automated image analysis. The term was coined a little over 10 years ago. Since then the technology has evolved considerably and has established itself firmly in the drug discovery and development industry. Both the instruments and the software controlling the instruments and analyzing the data have come to maturity, so the full benefits of high-content screening can now be realized. Those benefits are the capability of carrying out phenotypic multiparametric cellular assays in an unbiased, fully automated, and quantitative fashion. Automated microscopes and automated image analysis are being applied at all stages of the drug discovery and development pipeline. All major pharmaceutical companies have adopted the technology and it is in the process of being embraced broadly by the academic community. This review aims at describing the current capabilities and limits of the technology as well as highlighting necessary developments that are required to exploit fully the potential of high-content screening and analysis.     

Chemogenomics and parasitology: small molecules and cell-based assays to study infectious processes

Infectious diseases caused by protozoan parasites--malaria, sleeping sickness, leishmaniasis, Chagas' disease, toxoplasmosis--remain chronic problems for humanity. We lack vaccines and have limited drug options effective against protozoa. Research into anti-protozoan drugs has accelerated with improved in vitro cultivation methods, enhanced genetic accessibility, completed genome sequences for key protozoa, and increased prominence of protozoan diseases on the agendas of well-resourced public figures and foundations. Concurrent advances in high-throughput screening (HTS) technologies and availability of diverse small molecule libraries offer the promise of accelerated discovery of new drug targets and new drugs that will reduce disease burdens imposed on humanity by parasitic protozoa. We provide a status report on HTS technologies in hand and cell-based assays under development for biological investigations and drug discovery directed toward the three best-characterized parasitic protozoa: Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. We emphasize cell growth assays and new insights into parasite cell biology speeding development of better cell-based assays, useful in primary screens for anti-protozoan drug leads and secondary screens to decipher mechanisms of action of leads identified in growth assays. Small molecules that interfere with specific aspects of protozoan biology, identified in such screens, will be valuable tools for dissecting parasite cell biology and developing anti-protozoan drugs. We discuss potential impacts on drug development of new consortia among academic, corporate, and public partners committed to discovery of new, effective anti-protozoan drugs.     

Modern Approaches in the Discovery and Development of Plant-Based Natural Products and Their Analogues as Potential Therapeutic Agents

Natural products represents an important source of new lead compounds in drug discovery research. Several drugs currently used as therapeutic agents have been developed from natural sources; plant sources are specifically important. In the past few decades, pharmaceutical companies demonstrated insignificant attention towards natural product drug discovery, mainly due to its intrinsic complexity. Recently, technological advancements greatly helped to address the challenges and resulted in the revived scientific interest in drug discovery from natural sources. This review provides a comprehensive overview of various approaches used in the selection, authentication, extraction/isolation, biological screening, and analogue development through the application of modern drug-development principles of plant-based natural products. Main focus is given to the bioactivity-guided fractionation approach along with associated challenges and major advancements. A brief outline of historical development in natural product drug discovery and a snapshot of the prominent natural drugs developed in the last few decades are also presented. The researcher’s opinions indicated that an integrated interdisciplinary approach utilizing technological advances is necessary for the successful development of natural products. These involve the application of efficient selection method, well-designed extraction/isolation procedure, advanced structure elucidation techniques, and bioassays with a high-throughput capacity to establish druggability and patentability of phyto-compounds. A number of modern approaches including molecular modeling, virtual screening, natural product library, and database mining are being used for improving natural product drug discovery research. Renewed scientific interest and recent research trends in natural product drug discovery clearly indicated that natural products will play important role in the future development of new therapeutic drugs and it is also anticipated that efficient application of new approaches will further improve the drug discovery campaign.     

Quality, not quantity: the role of natural products and chemical proteomics in modern drug discovery

Chemical genetics and reverse chemical genetics parallel classical genetics but target genes at the protein level and have proven useful in recent years for screening combinatorial libraries for compounds of biological interest. However, the performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide may be turning back to Nature in the search for new drug candidates. Even though diversity oriented synthesis is now producing molecules that are natural product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules. Natural products, on the other hand, have evolved to interact with biomolecules, which is why so many can be found in pharmacopoeias. However, the cellular targets and modes of action of these fascinating compounds are seldom known, hindering the drug development process. This review focuses on the emergence of chemical proteomics and reverse chemical proteomics as tools for the discovery of cellular receptors for natural products, thereby generating protein/ligand pairs that will prove useful in identifying new drug targets and new biologically active small molecule scaffolds. Such a system-wide approach to identifying new drugable targets and their small molecule ligands will help unblock the pharmaceutical product pipelines by speeding the process of target and lead identification.     

Rapid identification of substrates for novel proteases using a combinatorial peptide library

Fluorogenic substrates for assaying novel proteolytic enzymes could be rapidly identified using an easy, solid-phase combinatorial assay technology. The methodology was validated with leader peptidase of Escherichia coli using a subset of an intramolecularly quenched fluorogenic peptide library. The technique was extended toward the discovery of substrates for a new aspartic protease of pharmaceutical relevance (human napsin A). We demonstrated for the first time known to us that potent fluorogenic substrates can be discovered using extracts of cells expressing recombinant enzyme to screen the peptide library. The straightforward and rapid optimization of protease substrates greatly facilitates the drug discovery process by speeding up the development of high throughput screening assays and thus helps more effective exploitation of the enormous body of information and chemical structures emerging from genomics and combinatorial chemistry technologies.     

In‐depth insight into the methods of plasma protein‐drug interaction studies: Comparison of capillary electrophoresis‐frontal analysis,isothermal titration calorimetry,circular dichroism and equilibrium dialysis

Plasma protein‐drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high‐throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico‐chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis‐frontal analysis being the best candidate for such studies.     

Incurred sample reanalysis in drug discovery bioanalysis

Bioanalysis plays a key role during the drug discovery process to generate the pharmacokinetic data to facilitate unbiased evaluation of leads, optimized leads and drug candidates. Such pharmacokinetic data are used to enable key decisions in the drug discovery process. The aim of the work is to put forward a new strategy of performing the incurred sample reanalysis for select small molecule novel chemical entities at different stages of drug discovery prior to candidate selection. Three discovery programs representing hits, leads and optimized lead candidates were selected for the incurred sample reanalysis (ISR) analysis. From each discovery program, two novel chemical entities were selected for the ISR analysis. The time points considered for ISR generally varied among the programs; however, samples coinciding with drug absorption, distribution and elimination were considered in the ISR assessment. With the exception of a single ISR value that gave a high deviation (about 63%), the observed ISR values supported the discovery bioanalytical assays. While the individual bioanalytical laboratory can draw an algorithm for selecting novel chemical entities and fixing the acceptance criteria for the ISR data, it is proposed that the percentage difference between ISR vs. original concentration for 67% of the repeat samples is contained within ±30% for discovery bioanalysis.