An offline-addition format for identifying GPCR modulators by screening 384-well mixed compounds in the FLIPR

Although fluorescence imaging plate reader (FLIPR)-based assays have been widely used in high-throughput screening, improved efficiencies in throughput and fidelity continue to be investigated. This study presents an offline compound addition protocol coupled with a testing strategy using mixtures of compounds in a 384-well format to identify antagonists of the neurokinin-1 receptor expressed in the human astrocytoma cell line (U373 MG). Substance P evoked a concentration-dependent increase in intracellular cellular Ca(2+) with an EC(50) value of 0.30 +/- 0.17 nM, which was inhibited by neurokinin-1 (NK1) antagonists L-733,060 and L-703,606. Test compounds, as mixtures of 10 compounds/well, were added to the cells offline using an automated dispensing unit and incubated prior to performing the assay in the FLIPR. Using the offline protocol, a higher through put of ~200,000 compounds was achieved in an 8-h working day, and several novel structural classes of compounds were identified as antagonists for the NK1 receptor. These studies demonstrate that the offline compound addition format using a mixture of compounds in a 384-well FLIPR assay provides an efficient platform for screening and identifying modulators for G-protein-coupled receptors.     

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.     

新型冠状病毒主蛋白酶抑制剂的筛选方法研究进展

新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)席卷全球,具有较高的传染性和死亡率,但目前尚缺乏安全有效的COVID-19疫苗与治疗药物.新型冠状病毒主蛋白酶(main protease,Mpro)的进化高度保守,在调控新冠病毒RNA复制中具有重要的生物学功能,已成为新型广谱抗冠状...     

Profiling of toxicity and identification of distinct apoptosis profiles using a 384-well high-throughput flow cytometry screening platform

Methods and techniques used to detect apoptosis have benefited from advances in technologies such as flow cytometry. With a large arsenal of lasers, fluorescent labels, and readily accessible biological targets, it is possible to detect multiple targets with unique combinations of fluorescent spectral signatures from a single sample. Traditional flow cytometry has been limited as a screening tool as the sample throughput has been low, whereas the data analysis and generation of screening relevant results have been complex. The HTFC Screening System running ForeCyt software is an instrument platform designed to perform high-throughput, multiplexed screening with seamless transformation of flow cytometry data into screening hits. We report the results of a screen that simultaneously quantified caspase 3/7 activation, annexin V binding, cell viability, and mitochondrial integrity. Assay performance over 5 days demonstrated robustness, reliability, and performance of the assay. This system is high throughput in that a 384-well plate can be read and fully analyzed within 30 min and is sensitive with an assay window of at least 10-fold for all parameters and a Z' factor of ≥0.75 for all endpoints and time points. From a screen of 231 compounds, 11 representative toxicity profiles highlighting differential activation of apoptotic pathways were identified.     

Development of a high-content high-throughput screening assay for the discovery of ATM signaling inhibitors

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.     

Environmental determinants of Vibrio cholerae biofilm development

Vibrio cholerae is a versatile bacterium that flourishes in diverse environments, including the human intestine, rivers, lakes, estuaries, and the ocean. Surface attachment is believed to be essential for colonization of all of these natural environments. Previous studies have demonstrated that the vps genes, which encode proteins required for exopolysaccharide synthesis and transport, are required for V. cholerae biofilm development in Luria-Bertani broth. In this work, we showed that V. cholerae forms vps-dependent biofilms and vps-independent biofilms. The vps-dependent and -independent biofilms differ in their environmental activators and in architecture. Our results suggest that environmental activators of vps-dependent biofilm development are present in freshwater, while environmental activators of vps-independent biofilm development are present in seawater. The distinct environmental requirements for the two modes of biofilm development suggest that vps-dependent biofilm development and vps-independent biofilm development may play distinct roles in the natural environment.     

A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology

To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen trade mark technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation.     

A comparative study of cell classifiers for image-based high-throughput screening

Background

Millions of cells are present in thousands of images created in high-throughput screening (HTS). Biologists could classify each of these cells into a phenotype by visual inspection. But in the presence of millions of cells this visual classification task becomes infeasible. Biologists train classification models on a few thousand visually classified example cells and iteratively improve the training data by visual inspection of the important misclassified phenotypes. Classification methods differ in performance and performance evaluation time. We present a comparative study of computational performance of gentle boosting, joint boosting CellProfiler Analyst (CPA), support vector machines (linear and radial basis function) and linear discriminant analysis (LDA) on two data sets of HT29 and HeLa cancer cells.

Results

For the HT29 data set we find that gentle boosting, SVM (linear) and SVM (RBF) are close in performance but SVM (linear) is faster than gentle boosting and SVM (RBF). For the HT29 data set the average performance difference between SVM (RBF) and SVM (linear) is 0.42 %. For the HeLa data set we find that SVM (RBF) outperforms other classification methods and is on average 1.41 % better in performance than SVM (linear).

Conclusions

Our study proposes SVM (linear) for iterative improvement of the training data and SVM (RBF) for the final classifier to classify all unlabeled cells in the whole data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-342) contains supplementary material, which is available to authorized users.     

Development of homogeneous 384-well high-throughput screening assays for Abeta1-40 and Abeta1-42 using AlphaScreen technology

Amyloid beta (Abeta) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimer's disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimer's disease research. The current most established assay for analysis of Abeta peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Abeta1-42 and Abeta1-40 using AlphaScreen technology. The assays are capable of detecting Abeta peptides at concentrations <2 pg/mL and, using a final assay volume of 20 microL, routinely generate Z' values >0.85. The AlphaScreen format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen technology permits robust, efficient, and cost-effective quantitation of Abeta peptides.     

Hypothesis testing in high-throughput screening for drug discovery

Following the success of small-molecule high-throughput screening (HTS) in drug discovery, other large-scale screening techniques are currently revolutionizing the biological sciences. Powerful new statistical tools have been developed to analyze the vast amounts of data in DNA chip studies, but have not yet found their way into compound screening. In HTS, characterization of single-point hit lists is often done only in retrospect after the results of confirmation experiments are available. However, for prioritization, for optimal use of resources, for quality control, and for comparison of screens it would be extremely valuable to predict the rates of false positives and false negatives directly from the primary screening results. Making full use of the available information about compounds and controls contained in HTS results and replicated pilot runs, the Z score and from it the p value can be estimated for each measurement. Based on this consideration, we have applied the concept of p-value distribution analysis (PVDA), which was originally developed for gene expression studies, to HTS data. PVDA allowed prediction of all relevant error rates as well as the rate of true inactives, and excellent agreement with confirmation experiments was found.     

Steps in the development of a Vibrio cholerae El Tor biofilm

We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture.     

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.     

Combinatorial chemistry and high-throughput screening for the discovery of organocatalysts

In recent years, interest in organocatalysis, the catalysis with small organic molecules, has been revitalized. Independently, high-throughput-screening and combinatorial chemistry became practical methodologies in the discovery of novel catalytic entities. The logical extension of these methodologies to organocatalysis has led to several interesting results, which are highlighted in this review.     

An image-based, high-throughput screening assay for molecules that induce excess DNA replication in human cancer cells

Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer.