Statistical considerations for high throughput screening data

High throughput screening (HTS) is a widely used effective approach in genome-wide association and large scale protein expression studies, drug discovery, and biomedical imaging research. How to accurately identify candidate ‘targets’ or biologically meaningful features with a high degree of confidence has led to extensive statistical research in an effort to minimize both false-positive and false-negative rates. A large body of literature on this topic with in-depth statistical contents is available. We examine currently available statistical methods on HTS and aim to summarize some selected methods into a concise, easy-tofollow introduction for experimental biologists.     

Novel fluorescence sensing methods for high throughput screening

We describe two new methods of fluorescence sensing for use in high throughput screening (HTS). Modulation sensing transforms analyte-dependent intensity changes into a change in the low-frequency modulation signal. Polarization sensing transforms an intensity change into a change in polarization. Both methods are internally calibrated by using a reference film immediately adjacent to the sample, which can be readily located on the HTS plate or on a nearby optical component and provides an intensity or polarization reference. Modulation sensing and polarization sensing were both shown useful for measurements of fluorophore concentrations, pH, or calcium concentrations in the wells of HTS plates. Sensing with a reference film provides the opportunity to internally reference HTS measurements without the need for additions to the sample. This approach can provide standardization for assays performed at different times.     

药物筛选新方法--高通量筛选

介绍了高通量筛选的概念、原理及其各个组成部分,着重阐明了高通量筛选的筛选模式,检测方法和应用实例,并简单介绍了国内在这方面的研究进展。     

A high throughput mechanical screening device for cartilage tissue engineering

Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying ‘hits’, or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput.     

Micro-colony array based high throughput platform for enzyme library screening

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.     

An image-based assay for high throughput screening of Giardia lamblia

Giardia lamblia is a protozoan parasite that causes widespread gastrointestinal illness. Drugs to treat giardiasis are limited, but efforts to discover new anti-giardial compounds are constrained by the lack of a facile system for cell culture and inhibitor testing. We achieved robust and reproducible growth of G. lamblia in 384-well tissue culture plates in a modified TYI-S-33 medium. A high throughput assay for the screening of potential anti-giardial compounds was developed utilizing the WB strain of G. lamblia and automated optical detection of parasites after growth with tested inhibitors. We screened a library of 1600 known bioactive molecules and identified 12 compounds that inhibited growth of G. lamblia at low- or sub-micromolar concentrations. Our high throughput assay should facilitate evaluation of available chemical libraries for novel drugs to treat giardiasis.     

A set of UV-inducible autolytic vectors for high throughput screening

A high throughput screening scheme is often a prerequisite for directed evolution of enzymes or metagenomic analysis of DNA samples. For assaying intracellular enzymes of interest (e.g. when Escherichia coli is used), it requires cell lysis in many cases, chemical or enzymatic, which can be tedious and cost-consuming. In this study, a set of UV-inducible autolytic vectors was constructed to offer a simpler means of cell lysis that is free of additional liquid handling. The SRRz lysis gene cassette from bacteriophage Lambda was cloned downstream of a UV-inducible promoter, the recA promoter or the umuDC promoter, and further inserted into the backbone of pUC18, and transformed into E. coli BL21 cells. The SRRz expression and cell lysis was induced by UV irradiation. For both the recA and umuDC promoters, at 30 degrees C the lysis efficiency was found to be consistent and above 60% as measured using beta-galactosidase as the reporter. However, at 37 degrees C the lysis profiles were found to be erratic. UV lysis in 96-well plates also produced consistent lysis results that were comparable to those obtained by lysozyme treatment, demonstrating the utility of these autolytic vectors in high throughput screening. This set of artificial SRRz autolysis units should be transferable to other vectors. Surprisingly, it was found that the E. coli BL21(DE3) was also partially disrupted under UV irradiation, with a lysis efficiency of 44.5% at 30 degrees C, and 22.5% at 37 degrees C.     

Comparison of high throughput screening technologies for luminescence cell-based reporter screens

As higher density formats become more and more common in HTS labs, the expectations for maintaining faster, lower cost screens puts great pressure on traditional 96-well screens. In some cases higher density formats are not compatible with the assay. This seems especially true in cell-based assays. In our case, the nature of the cells' response forced us to remain in 96-well plates. In this paper, we describe the development of a luminescence reporter assay and its performance in two detection modes, flash and glow. The advantages in cost and throughput for each technique are explored, along with automation considerations. An additional new technology, the use of pins for low-volume transfers, is also briefly described because of its dramatic effect on our screen's throughput. However, it will be more thoroughly presented in a future publication. Comparing the technologies available for HTS aids in designing automated systems that meet the unique needs of each assay.     

Application of QuantiGene nucleic acid quantification technology for high throughput screening

To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the QuantiGene nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-1alpha (IL-1alpha) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.     

A yeast assay for high throughput screening of natural anti-viral agents

Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions. Many variations of these basic systems have originated over the years. Besides these vast and varied applications of the yeast expression system, S. cerevisiae has also been used extensively in fundamental research as a model simple eukaryote. We have used the S. cerevisiae system to design a high throughput screen for anti-viral agents from natural sources. The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting. A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate. Using such a process, we have initiated a high throughput screening process for natural anti-viral agents.     

Fluorescence polarization assays for high throughput screening of G protein-coupled receptors

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to [(125)I]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 microM. These attributes suggest that accurate assessment of drug binding can be obtained.     

Fluorescent substrates for ADAM15 useful for assaying and high throughput screening

A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs −2, –9, and −13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M⁻¹ s⁻¹ and 4300 M⁻¹ s⁻¹, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (k_cat/K_m) of 5200 M⁻¹ s⁻¹. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 μM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.     

Substrate independent ATPase activity may complicate high throughput screening

Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening.     

Microbiological high throughput screening: an opportunity for the lead discovery process

Microbial HTS has been implemented at Rh?ne-Poulenc Rorer through the development of a dedicated robotic platform. This robot (Turbo) has been designed with the aim of fully integrating microbial HTS into the lead discovery processes. Innovative solutions have been found to reach high throughput as well as flexibility. This opens up new prospects for solid-phase microbial screening, taking advantage of the easy implementation and the very low costs of such screens. The different types of microbial screens done in our laboratory, as well as the throughputs and outputs obtained, are described. Some of the specific aspects of microbial HTS, as compared to biochemical and cell-based assays, are also discussed.     

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae

ABSTRACT: We describe a new selection method based on BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about 3 weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to K_V2.1 channels is inhibited by another K_V2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel Na_V1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-K_V2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl K_V channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of K_V channels can be selectively targeted by small molecules to modify channel function.     

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae

Background

Termites are highly effective at degrading lignocelluloses, and thus can be used as a model for studying plant cell-wall degradation in biological systems. However, the process of lignin deconstruction and/or degradation in termites is still not well understood.

Methods

We investigated the associated structural modification caused by termites in the lignin biomolecular assembly in softwood tissues crucial for cell-wall degradation. We conducted comparative studies on the termite-digested (i.e. termite feces) and native (control) softwood tissues with the aid of advanced analytical techniques: ¹³C crosspolarization magic angle spinning and nuclear magnetic resonance (CP-MAS-NMR) spectroscopy, flash pyrolysis with gas chromatography mass spectrometry (Py-GC/MS), and Py-GC-MS in the presence of tetramethylammonium hydroxide (Py-TMAH)-GC/MS.

Results

The ¹³C CP/MAS NMR spectroscopic analysis revealed an increased level of guaiacyl-derived (G unit) polymeric framework in the termite-digested softwood (feces), while providing specific evidence of cellulose degradation. The Py-GC/MS data were in agreement with the ¹³C CP/MAS NMR spectroscopic studies, thus indicating dehydroxylation and modification of selective intermonomer side-chain linkages in the lignin in the termite feces. Moreover, Py-TMAH-GC/MS analysis showed significant differences in the product distribution between control and termite feces. This strongly suggests that the structural modification in lignin could be associated with the formation of additional condensed interunit linkages.

Conclusion

Collectively, these data further establish: 1) that the major β-O-4' (β-aryl ether) was conserved, albeit with substructure degeneracy, and 2) that the nature of the resulting polymer in termite feces retained most of its original aromatic moieties (G unit-derived). Overall, these results provide insight into lignin-unlocking mechanisms for understanding plant cell-wall deconstruction, which could be useful in development of new enzymatic pretreatment processes mimicking the termite system for biochemical conversion of lignocellulosic biomass to fuels and chemicals.