Multiparametric microsensor chips for screening applications

The identification of drug targets for pharmaceutical screening can be greatly accelerated by gene databases and expression studies. The identification of leading compounds from growing libraries is realized by high throughput screening platforms. Subsequently, for optimization and validation of identified leading compounds studies of their functionality have to be carried out, and just these functionality tests are a limiting factor. A rigorous preselection of identified compounds by in vitro cellular screening is necessary prior to using the drug candidates for the further time consuming and expensive stage, e.g. in animal models. Our efforts are focused to the parallel development, adaptation and integration of different microelectronic sensors into miniaturized biochips for a multiparametric, functional on-line analysis of living cells in physiologically environments. Parallel and on-line acquisition of data related to different cellular targets is required for advanced stages of drug screening and for economizing animal tests.     

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications

BACKGROUND: Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. RESULTS: We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. CONCLUSIONS: We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.     

Tetrazole‐Based Probes for Integrated Phenotypic Screening,Affinity‐Based Proteome Profiling,and Sensitive Detection of a Cancer Biomarker

Target‐identification phenotypic screening has been a powerful approach in drug discovery; however, it is hindered by difficulties in identifying the underlying cellular targets. To address this challenge, we have combined phenotypic screening of a fully functionalized small‐molecule library with competitive affinity‐based proteome profiling to map and functionally characterize the targets of screening hits. Using this approach, we identified ANXA2, PDIA3/4, FLAD1, and NOS2 as primary cellular targets of two bioactive molecules that inhibit cancer cell proliferation. We further demonstrated that a panel of probes can label and/or image annexin A2 (a cancer biomarker) from different cancer cell lines, thus providing opportunities for potential cancer diagnosis and therapy.     

Tetrazole‐Based Probes for Integrated Phenotypic Screening,Affinity‐Based Proteome Profiling,and Sensitive Detection of a Cancer Biomarker

Target‐identification phenotypic screening has been a powerful approach in drug discovery; however, it is hindered by difficulties in identifying the underlying cellular targets. To address this challenge, we have combined phenotypic screening of a fully functionalized small‐molecule library with competitive affinity‐based proteome profiling to map and functionally characterize the targets of screening hits. Using this approach, we identified ANXA2, PDIA3/4, FLAD1, and NOS2 as primary cellular targets of two bioactive molecules that inhibit cancer cell proliferation. We further demonstrated that a panel of probes can label and/or image annexin A2 (a cancer biomarker) from different cancer cell lines, thus providing opportunities for potential cancer diagnosis and therapy.     

Micro-patterned porous substrates for cell-based assays

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (μPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. μPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.     

In vitro bioassays for the study of endocrine-disrupting food additives and contaminants

Endocrine-disrupting chemicals (EDCs) are capable of interfering with normal hormone homeostasis by acting on several targets and through a wide variety of mechanisms. Unwanted exposure to EDCs can lead to a wide spectrum of adverse health effects, especially when exposure is during critical windows of development. Feed and food are considered to be among the main routes of inadvertent exposure to EDCs, so there is an important need for efficient detection of EDCs in these matrices.We describe in vitro bioassays that can complement current analytical chemistry in order to detect unwanted EDCs and describe their action, emphasizing assays that can measure effects on nuclear receptor signaling or hormone production. We outline both validated and unvalidated in vitro assays currently available in the scientific community for detecting and studying the effects of EDCs, and discuss their possible role in the food-safety context. We conclude by identifying gaps in the current battery of in vitro assays available for EDCs and suggest future possibilities for development and validation.     

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.     

An integrated "4-phase" approach for setting endocrine disruption screening priorities--phase I and II predictions of estrogen receptor binding affinity

Recent legislation mandates the US Environmental Protection Agency (EPA) to develop a screening and testing program for potential endocrine disrupting chemicals (EDCs), of which xenoestrogens figure prominently. Under the legislation, a large number of chemicals will undergo various in vitro and in vivo assays for their potential estrogenicity, as well as other hormonal activities. There is a crucial need for priority setting before this strategy can be effectively implemented. Here we report an integrated computational approach to priority setting using estrogen receptor (ER) binding as an example. This approach rationally integrates different predictive computational models into a "Four-Phase" scheme so that it can effectively identify potential estrogenic EDCs based on their predicted ER relative binding affinity (RBA). The system has been validated using an in-house ER binding assay dataset for 232 chemicals that was designed to have both broad structural diversity and a wide range of binding affinities. When applied to 58,000 chemicals identified by Walker et al. as candidates for endocrine disruption screening, some 9100 chemicals were predicted to bind to ER. Of these, only 3600 were expected to bind to ER at RBA values up to 100,000-fold less than that of 17beta-estradiol. The method ruled out 83% of the chemicals as non-binders with a very low rate of false negatives. We believe that the same integrated scheme will be equally applicable to endpoints of other endocrine disrupting mechanisms, e.g. androgen receptor binding.     

An integrated "4-phase" approach for setting endocrine disruption screening priorities--phase I and II predictions of estrogen receptor binding affinity

Recent legislation mandates the US Environmental Protection Agency (EPA) to develop a screening and testing program for potential endocrine disrupting chemicals (EDCs), of which xenoestrogens figure prominently. Under the legislation, a large number of chemicals will undergo various in vitro and in vivo assays for their potential estrogenicity, as well as other hormonal activities. There is a crucial need for priority setting before this strategy can be effectively implemented. Here we report an integrated computational approach to priority setting using estrogen receptor (ER) binding as an example. This approach rationally integrates different predictive computational models into a "Four-Phase" scheme so that it can effectively identify potential estrogenic EDCs based on their predicted ER relative binding affinity (RBA). The system has been validated using an in-house ER binding assay dataset for 232 chemicals that was designed to have both broad structural diversity and a wide range of binding affinities. When applied to 58,000 chemicals identified by Walker et al. as candidates for endocrine disruption screening, some 9100 chemicals were predicted to bind to ER. Of these, only 3600 were expected to bind to ER at RBA values up to 100,000-fold less than that of 17 g -estradiol. The method ruled out 83% of the chemicals as non-binders with a very low rate of false negatives. We believe that the same integrated scheme will be equally applicable to endpoints of other endocrine disrupting mechanisms, e.g. androgen receptor binding.     

High-throughput screening for modulators of protein-protein interactions: use of photonic crystal biosensors and complementary technologies

High-throughput screening (HTS) has played an integral role in the development of small molecule modulators of biological processes. These screens are typically developed for enzymes (such as kinases or proteases) or extracellular receptors, two classes of targets with well-established colorimetric or fluorimetric activity assays. In contrast, methods for detection of protein-protein interactions lack the simplicity inherent to enzyme and receptor assays. Technologies that facilitate the discovery of small molecule modulators of protein-protein interactions are essential to the exploitation of this important class of drug targets. As described in this critical review, photonic crystal (PC) biosensors and other emerging technologies can now be utilized in high-throughput screens for the identification of compounds that disrupt or enhance protein-protein interactions (167 references).     

Label free binding assay with spectroscopic detection for pharmaceutical screening

Many therapeutic drugs exert their effects by interaction with well defined molecular targets. Increasing knowledge in molecular biology allows identification of more and more molecular key compounds and in consequence a molecular approach to disease and therapy. As binding of drugs to their target compounds is a key event, binding assays with an appropriate target molecule are useful means for primary screening of novel substances. We have investigated the potential of thin film interference spectroscopy (RIFS) as a label free detection method for pharmaceutical screening in a binding inhibition assay. To meet the throughput requirements in pharmaceutical screening a parallel detection system based on imaging spectroscopy was constructed. Thrombin/thrombin inhibitor interaction was investigated as a model system. The thin film transducer was covalently modified with a thrombin inhibitor. Specific binding of thrombin and binding inhibition by inhibitor compounds could be observed. A test cycle of less than 10 min could be reached. The parallel setup allows the simultaneous detection of 96 binding curves and can reach a throughput of more than 10⁶ samples per year.     

In-silico screening of high production volume chemicals for mutagenicity using the MCASE QSAR expert system

Computational screening is suggested as a way to set priorities for further testing of high production volume (HPV) chemicals for mutagenicity and other toxic endpoints. Results are presented for batch screening of 2484 HPV chemicals to predict their mutagenicity in Salmonella typhimurium (Ames test). The chemicals were tested against 15 databases for Salmonella strains TA100, TA1535, TA1537, TA97 and TA98, both with metabolic activation (using rat liver and hamster liver S9 mix test) and without metabolic activation. Of the 2484 chemicals, 1868 are predicted to be completely nonmutagenic in all of the 15 data modules and 39 chemicals were found to contain structural fragments outside the knowledge of the expert system and therefore suggested for further evaluation. The remaining 616 chemicals were found to contain different biophores (structural alerts) believed to be linked to mutagenicity. The chemicals were ranked indescending order according to their predicted mutagenic potential and the first 100 chemicals with highest mutagenicity scores are presented. The screening result offers hope that rapid and inexpensive computational methods can aid in prioritizing the testing of HPV chemicals, save time and animals and help to avoid needless expense.     

“Stainomics”: Identification of mitotracker labeled proteins in mammalian cells

Organelle‐specific cell‐permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR‐labeled proteins by 2DE. The MTR‐specific fluorescent signals on the gel revealed the spots that contained MTR‐conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock‐like responses. The identification of the molecular targets of biological dyes, or “stainomics,” can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.     

Plant pathogen recognition as a natural, original and simple model for chemogenomics: a brief overview of cell-based assays to screen for peptides acting as plant defense activators

As plants lack a circulatory system and adaptive immune system, they have evolved their own defense systems distinct from animals, in which each plant cell is capable of defending itself from pathogens. Plants induce a number of defense responses, which are triggered by a variety of molecules derived from pathogenic microorganisms, referred to as microbe-associated molecular patterns (MAMPs), including peptides, proteins, lipopolysaccharide, beta-glucan, chitin, and ergosterol. The interaction between plants and chemicals in the context of plant defense represents a "natural" and simple model for chemogenomics, at the intersection between chemical and biological diversities. For protection of crop plants from diseases, it has been shown to be effective to stimulate the plant immunity by chemical compounds, the so-called "plant defense activators". Combinatorial chemistry techniques can be applied to the search for novel plant defense activators, but it is essential to establish an efficient and reliable screening system suitable for library screening. For studies of the plant immune system, it is difficult to use isolated proteins as biological targets because the receptors for MAMP recognition are largely unknown and even the receptors identified so far are transmembrane proteins. Therefore, screening for novel peptides acting on MAMP receptors from combinatorial libraries must rely on a solution-phase assay using cells as the biological targets. In this review, we introduce the cell-based lawn format assay for identification of peptides acting as plant defense activators from combinatorial peptide libraries. The requirements and limitations in constructing the screening system using combinatorial libraries in the studies of plant sciences are also discussed.     

In-Silico Screening of High Production Volume Chemicals for Mutagenicity using the mcase QSAR Expert System

Computational screening is suggested as a way to set priorities for further testing of high production volume (HPV) chemicals for mutagenicity and other toxic endpoints. Results are presented for batch screening of 2484 HPV chemicals to predict their mutagenicity in Salmonella typhimurium (Ames test). The chemicals were tested against 15 databases for Salmonella strains TA100, TA1535, TA1537, TA97 and TA98, both with metabolic activation (using rat liver and hamster liver S9 mix test) and without metabolic activation. Of the 2484 chemicals, 1868 are predicted to be completely nonmutagenic in all of the 15 data modules and 39 chemicals were found to contain structural fragments outside the knowledge of the expert system and therefore suggested for further evaluation. The remaining 616 chemicals were found to contain different biophores (structural alerts) believed to be linked to mutagenicity. The chemicals were ranked in descending order according to their predicted mutagenic potential and the first 100 chemicals with highest mutagenicity scores are presented. The screening result offers hope that rapid and inexpensive computational methods can aid in prioritizing the testing of HPV chemicals, save time and animals and help to avoid needless expense.     

Ligand fishing as a tool to screen natural products with anticancer potential

Cancer is the second leading cause of death in the world and its incidence is expected to increase with the aging of the world's population and globalization of risk factors. Natural products and their derivatives have provided a significant number of approved anticancer drugs and the development of robust and selective screening assays for the identification of lead anticancer natural products are essential in the challenge of developing personalized targeted therapies tailored to the genetic and molecular characteristics of tumors. To this end, a ligand fishing assay is a remarkable tool to rapidly and rigorously screen complex matrices, such as plant extracts, for the isolation and identification of specific ligands that bind to relevant pharmacological targets. In this paper, we review the application of ligand fishing with cancer-related targets to screen natural product extracts for the isolation and identification of selective ligands. We provide critical analysis of the system configurations, targets, and key phytochemical classes related to the field of anticancer research. Based on the data collected, ligand fishing emerges as a robust and powerful screening system for the rapid discovery of new anticancer drugs from natural resources. It is currently an underexplored strategy according to its considerable potential.     

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.     

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1a were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1a were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.