Use of high-content analysis for compound screening and target selection

High-content analysis (HCA) has rapidly established itself as a core technology in drug discovery for secondary cell-based screening. When combined with our knowledge of genetics, HCA can provide a powerful tool for target validation, but excitingly, HCA may also enable the increased use of cellular assays in high-throughput screening for novel drug leads.     

Functional genomic and high-content screening for target discovery and deconvolution

Introduction: Functional genomic screens apply knowledge gained from the sequencing of the human genome toward rapid methods of identifying genes involved in cellular function based on a specific phenotype. This approach has been made possible through advances in both molecular biology and automation. The utility of this approach has been further enhanced through the application of image-based high-content screening: an automated microscopy and quantitative image analysis platform. These approaches can significantly enhance the acquisition of novel targets for drug discovery.

Areas covered: Both the utility and potential issues associated with functional genomic screening approaches are discussed in this review, along with examples that illustrate both. The considerations for high-content screening applied to functional genomics are also presented.

Expert opinion: Functional genomic screening and high-content screening are extremely useful in the identification of new drug targets. However, the technical, experimental, and computational parameters have an enormous influence on the results. Thus, although new targets are identified, caution should be applied to the interpretation of screening data in isolation. Genomic screens should be viewed as an integral component of a target identification campaign that requires both the acquisition of orthogonal data, as well as a rigorous validation strategy.     

Using high-content screening technology for studying drug-induced hepatotoxicity in preclinical studies

Introduction: The need for alternatives to animal experimentation and traditional testing methods has been widely discussed in recent years. This has led scientists and regulatory authorities to investigate alternative methods for toxicity testing. High-content screening (HCS) has emerged as a powerful tool in predictive toxicology since it permits molecular, cellular and tissue-based toxicity assessments. HCS allows automated image acquisition and analysis, and provides information on multiple properties of individual cells loaded simultaneously with fluorescent dyes, which is used for drug safety evaluations.

Areas covered: Herein, the authors review the principles of HCS technology and some of the most widely used HCS assays for studying drug-induced hepatotoxicity in preclinical studies in general and in the pharmaceutical industry in particular.

Expert opinion: The widespread acceptation of HCS by pharmaceutical companies and academic researchers highlights the potential usefulness of this technology as a prioritization tool in drug development. The improvement of different key points such as fluorescent probes or bioinformatics tools will consolidate HCS in drug discovery.     

Novel technologies for virtual screening

There are several methods for virtual screening of databases of small organic compounds to find tight binders to a given protein target. Recent reviews in Drug Discovery Today have concentrated on screening by docking and by pharmacophore searching. Here, we complement these reviews by focusing on virtual screening methods that are based on analyzing ligand similarity on a structural level. Specifically, we concentrate on methods that exploit structural properties of the complete ligand molecules, as opposed to using just partial structural templates, such as pharmacophores. The in silico procedure of virtual screening (VS) and its relationship to the experimental procedure, HTS, is discussed, new developments in the field are summarized and perspectives on future research are offered.     

Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators

Aim： To develop a robust, cell-based, high-content screening （HCS） assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor （EGFR）. Methods： Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor （EGF） was used to visualize the internalized receptor- ligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated, optimized, and applied to a large-scale screening of a library containing 48 000 synthetic compounds. Results： The competition between fluorescent EGF and unlabeled EGF reveals the IC50 of unlabeled EGF is approximately 0.2 nmol/L, which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified. One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase （ERK）. Conclusion： This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.     

Validation of a high-content screening assay using whole-well imaging of transformed phenotypes

Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFRα) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z' value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFRα inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRα inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries.     

A quantitative cell-based high-content screening assay for the epidermal growth factor receptor-specific activation of mitogen-activated protein kinase

The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.     

基于FP法的EPOR配体高通量筛选模型

目的寻找疗效好、副作用小的EPO功能类似物。方法用FITC标记EPO,从FVA细胞中提取含EPO受体的细胞膜,建立了用FP法筛选EPOR配体的模型,并对实验条件进行了优化和可能影响模型的因素(DMSO)进行了分析,此外用Z′因子对此模型进行了评估。结果样品中含0%~5%DMSO不会对实验结果产生影响,Z′因子的值为0.78。结论建立的筛选模型稳定性好和准确率高。     

The development of high-content screening (HCS) technology and its importance to drug discovery

ABSTRACT

Introduction: High-content screening (HCS) was introduced about twenty years ago as a promising analytical approach to facilitate some critical aspects of drug discovery. Its application has spread progressively within the pharmaceutical industry and academia to the point that it today represents a fundamental tool in supporting drug discovery and development.

Areas covered: Here, the authors review some of significant progress in the HCS field in terms of biological models and assay readouts. They highlight the importance of high-content screening in drug discovery, as testified by its numerous applications in a variety of therapeutic areas: oncology, infective diseases, cardiovascular and neurodegenerative diseases. They also dissect the role of HCS technology in different phases of the drug discovery pipeline: target identification, primary compound screening, secondary assays, mechanism of action studies and in vitro toxicology.

Expert opinion: Recent advances in cellular assay technologies, such as the introduction of three-dimensional (3D) cultures, induced pluripotent stem cells (iPSCs) and genome editing technologies (e.g., CRISPR/Cas9), have tremendously expanded the potential of high-content assays to contribute to the drug discovery process. Increasingly predictive cellular models and readouts, together with the development of more sophisticated and affordable HCS readers, will further consolidate the role of HCS technology in drug discovery.     

Orthotopic mouse models expressing fluorescent proteins for cancer drug discovery

Importance of the field: Currently used rodent tumor models, including transgenic tumor models, or subcutaneously growing human tumors in immunodeficient mice, do not sufficiently represent clinical cancer, especially with regard to metastasis and drug sensitivity.

Areas covered in this review: To obtain clinically accurate models, we have developed the technique of surgical orthotopic implantation (SOI) to transplant histologically intact fragments of human cancer, including tumors taken directly from the patient, to the corresponding organ of immunodeficient rodents. SOI allows the growth and metastatic potential of the transplanted tumors to be expressed and reflects clinical cancer of all types. Effective drugs can be discovered and evaluated in the SOI models utilizing human tumor cell lines and patient tumors. Visualization of many aspects of cancer initiation and progression in vivo has been achieved with fluorescent proteins. Tumors and metastases in the SOI models that express fluorescent proteins can be visualized noninvasively in intact animals, greatly facilitating drug discovery.

What the reader will gain: This review will provide information on the imageable mouse models of cancer that are clinically relevant, especially regarding metastasis and their use for drug discovery and evaluation.

Take home message: SOI mouse models of cancer reproduce the features of clinical cancer.     

Orthotopic mouse models expressing fluorescent proteins for cancer drug discovery

Importance of the field: Currently used rodent tumor models, including transgenic tumor models, or subcutaneously growing human tumors in immunodeficient mice, do not sufficiently represent clinical cancer, especially with regard to metastasis and drug sensitivity. Areas covered in this review: To obtain clinically accurate models, we have developed the technique of surgical orthotopic implantation (SOI) to transplant histologically intact fragments of human cancer, including tumors taken directly from the patient, to the corresponding organ of immunodeficient rodents. SOI allows the growth and metastatic potential of the transplanted tumors to be expressed and reflects clinical cancer of all types. Effective drugs can be discovered and evaluated in the SOI models utilizing human tumor cell lines and patient tumors. Visualization of many aspects of cancer initiation and progression in vivo has been achieved with fluorescent proteins. Tumors and metastases in the SOI models that express fluorescent proteins can be visualized noninvasively in intact animals, greatly facilitating drug discovery. What the reader will gain: This review will provide information on the imageable mouse models of cancer that are clinically relevant, especially regarding metastasis and their use for drug discovery and evaluation. Take home message: SOI mouse models of cancer reproduce the features of clinical cancer.     

NMR screening for lead compounds using tryptophan-mutated proteins

NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. They are, however, one of the least effective methods in terms of the amount of protein required and the time needed for acquiring an NMR experiment. We show here that the introduction of tryptophan to proteins permits rapid screening by monitoring a simple 1D proton NMR signal of the NH side chain ((N)H(epsilon)) of the tryptophan. The method could also provide quantitative characterization of the antagonist-protein and antagonist-protein-protein interactions in the form of KDs and fractions of the released proteins from their mutual binding. We illustrate the method with the lead compounds that block the Mdm2-p53 interaction and by studying inhibitors that bind to cyclin-dependent kinase 2 (CDK2).     

In vitro evaluation of the hepatic lipid accumulation of bisphenol analogs: A high-content screening assay

Bisphenol A (BPA) has a variety of adverse effects on human health; therefore, BPA analogs are increasingly used as replacements. Notably, recent studies have revealed that BPA exposure induced hepatic lipid accumulation, but few studies are available regarding the similar effects of other bisphenol analogues (BPs). Thus, in the present study, a high-content screening (HCS) assay was performed to simultaneously evaluate the hepatic lipid accumulation of 13 BPs in vitro. The BPs induced lipid deposition in HepG2 cells ranking as below: 4,4′-thiodiphenol (TDP) < bisphenol S (BPS) < 4,4′-dihydroxybenzophenone (DHBP) < tetrabromobisphenol A (TBBPA) < tetrachlorobisphenol A (TCBPA) < bisphenol E (BPE) < bisphenol F (BPF) < bisphenol B (BPB) < bisphenol AF (BPAF) < bisphenol A (BPA) < bisphenol C (BPC) < tetramethylbisphenol A (TMBPA) < bisphenol AP (BPAP). Meanwhile, Oil Red O staining and triacylglycerol detection further validated the lipid accumulation elicited by the latter 8 BPs, which exhibited the more significant effects on lipid deposition. Mechanistically, significantly increased expressions of genes involved in fatty acid synthesis and nuclear receptors and decreased levels of genes associated with fatty acid β-oxidation were observed under BPs treatment. Therefore, the present work is the first to systematically provide direct evidence for BPs-induced hepatic lipid accumulation in vitro via HCS, which can be helpful for safety assessments of BPs.     

Novel trends in high-throughput screening

Lead discovery by high-throughput screening (HTS) has evolved into a mature scientific discipline in modern drug discovery since its beginning about 10–15 years ago. Owing to the strong efforts in automation and miniaturization, even relatively large compound collections of over one million compounds or more can be screened against a large number of biological targets in relatively short time and at relatively low cost compared to the efforts of just 5 or 10 years ago. This was only possible with the concomitant development of high-quality readout technologies for highly miniaturized screening. Whereas most of the conventional drug targets can be approached via current HTS-readout technologies, the challenge goes toward the hitherto non-tractable families of drug targets. Future trends will focus strongly toward these novel target classes such as ion channels, transporters, protein–protein interactions, among many others. It will be essential to make proper readout technologies and adequate chemical libraries available for these target classes. Chemical libraries derived from natural products, but also derived from combinatorial chemistry and automated synthesis will be a key prerequisite for success in the field, as long as enough diversity and drug-like properties are included in these chemical libraries [25]. The proper readout technologies for screening of large chemical libraries have seen strong advances in recent years [^{[2], [7] and [22••]}], nevertheless none of these technologies is void of artifacts, in particular artifacts derived from the inherent physical nature of chemical compounds in aqueous buffer [11^••]. We therefore propose that future lead discovery should pay more attention toward unambiguous identification of these compound related artifacts and toward efficient removal of these false-positive compounds from the HTS hit-lists. We strongly recommend the use of biophysical and enzymological studies in the HTS hit-list follow-up phase (‘hit validation’) in order to deliver information of the highest possible quality for subsequent hit-to-lead studies. Finally, the science and art of HTS has evolved in various phases from its beginning in the early 1990s toward today's state-of-the-art operation in lead discovery. During these 15 years, one can distinguish three phases (‘generations’) of HTS operations: during the first phase, HTS has been just the same as laboratory screening, albeit at much larger capacity; in the second phase (‘second generation HTS’), HTS has evolved toward more sophisticated assay development/adaptation, more toward dedicated tool production, but also more toward counter-screening and hit-list follow-up; in the current phase (‘third generation HTS’) we see much more flexibility with regards to the applied processes for lead discovery, a stronger focus on quality and validation of the obtained results and a better awareness for choosing a proper lead finding strategy in a target-by-target specific manner.Taken together, we can conclude that better flexibility and creativity, more quality and the use of project-related, tailor-made lead finding strategies in the discovery process will become the key drivers for the successful application of high-throughput screening in the Pharmaceutical, Biotech, and Academic drug discovery programs of the future.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest