High-throughput Screening Strategy Research Articles

A strategy for high-throughput screening of ligands suitable for molecular imprinting of proteins

For facilitating the identification of appropriate functionalities that may serve as a binding motif of functional monomers, a selection strategy based on high-throughput screening of the binding properties of readily available sorbent materials has been developed. Thereby, the affinity of such ligands to the protein of interest may be rapidly determined. From these studies, it is anticipated that ligand functionalities will be derived, which may lead to advanced selection and design of dedicated functional monomers suitable for decorating the surface of a scavenger material. Thus, specific binding of the target protein of interest should be enabled even in complex solutions such as e.g., biotechnologically relevant cell lysates.In the present contribution, an automated screening method for studying ligand interactions of selected sorbent materials with pepsin – a protein of the protease family – was developed. Aqueous buffer solutions containing pepsin at known constant concentration were pipetted through an array of miniaturized chromatographic solid phase extraction (SPE) columns containing a variety of sorbent materials, and the eluted solutions were analyzed by UV/vis spectroscopy. The established screening protocol was validated against resin materials of known interaction with pepsin. Finally, the developed screening strategy was adapted for a robot system enabling high-throughput screening for a wide variety of sorbent materials and ligand functionalities in a fully automated approach.The obtained results clearly indicate that the established screening routine provides valuable data for characterizing resin-immobilized ligands, and their affinity toward pepsin.

Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy

Herein we present the outcome of a high throughput screening (HTS) campaign-based strategy for the rapid identification and optimization of selective and general chemotypes for both kappa (κ) opioid receptor (KOR) activation and inhibition. In this program, we have developed potent antagonists (IC(50) < 120 nM) or agonists of high binding affinity (K(i) < 3 nM). In contrast to many important KOR ligands, the compounds presented here are highly modular, readily synthesized and, in most cases, achiral. The four new chemotypes hold promise for further development into chemical tools for studying the KOR or as potential therapeutic lead candidates.

A High Throughput Screen for Inhibitors of RTK Post-translational Processing

Receptor tyrosine kinase (RTK) signaling promotes tumor cell proliferation and resistance to ionizing radiation. Inhibition of RTK post-translational modifications such as N-linked glycosylation (NLG) reduce levels of over-expressed RTKs and sensitize tumor cells to radiation therapy. We therefore developed a high throughput screening (HTS) strategy to identify compounds that inhibit endoplasmic reticulum (ER) protein processing and thus block RTK function.

An Integrated In Vitro and In Vivo High-Throughput Screen Identifies Treatment Leads for Ependymoma

Using a mouse model of ependymoma-a chemoresistant brain tumor-we combined multicell high-throughput screening (HTS), kinome-wide binding assays, and invivo efficacy studies, to identify potential treatments with predicted toxicity against neural stem cells (NSC). We identified kinases within the insulin signaling pathway and centrosome cycle as regulators of ependymoma cell proliferation, and their corresponding inhibitors as potential therapies. FDA approved drugs not currently used to treat ependymoma were also identified that posses selective toxicity against ependymoma cells relative to normal NSCs both invitro and invivo, e.g., 5-fluorouracil. Our comprehensive approach advances understanding of the biology and treatment of ependymoma including the discovery of several treatment leads for immediate clinical translation.

(−)-Epigallocatechin gallate inhibits hepatitis C virus (HCV) viral protein NS5B

In this study, we elucidated a small molecule inhibitor on viral protein NS5B identified through a high-throughput screening strategy using optical nanoparticle-based RNA oligonucleotide. We have previously shown that quantum dots (QDs)–RNA oligonucleotide can specifically recognize the HCV viral proteins. We have also demonstrated that conjugated QDs–RNA oligonucleotide can specifically and sensitively interact with designed biochips [1,2]. Among the flavonoids examined, (−)-epigallocatechin gallate (EGCG) demonstrated a remarkable inhibition activity on HCV viral protein, NS5B. (−)-Epigallocatechin gallate, at 0.005μgmL−1 or more, concentration-dependently attenuated the binding affinity on a designed biochip as evidenced by QDs–RNA oligonucleotide. At a concentration of 0.1μgmL−1, (−)-epigallocatechin gallate showed a 50% inhibition activity on QDs–RNA oligonucleotide biochip assay. We screened a small molecule inhibitor on the viral protein, NS5B, identified through a high-throughput screening strategy using on-chip optical nanoparticle-based RNA oligonucleotide on chip. In this designed strategy, the convenient and efficient screening and development of an on-chip viral protein inhibitor using a QDs–RNA oligonucleotide assay is achievable with high sensitivity and simplicity. In addition, this platform is expected to be applicable toward the inhibitor screening of other types of diseases.

Noncanonical amino acids in the interrogation of cellular protein synthesis.

Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a "pulse" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology.

Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UC(CF)-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UC(CF)-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o(-) and Fischer rat thyroid cells, expressing various CFTR forms. UC(CF)-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC(CF)-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.

A rapid, novel and high-throughput identification of putative bell pepper transformants generated through in planta transformation approach

The recent progress on bell pepper transformation through in planta approach is incredibly simple with high transformation efficiencies in indigenous varieties. This method produces chimeric primary transformants (T 0) hence, a large number of T 0 plants and an efficient high throughput screening strategy is necessary to be developed. In the present investigation, we have standardized a rapid and highly efficient method of screening putative bell pepper transformants. Bell pepper transformants harbouring gfp genes was developed using in planta approach and 1050 etiolated T 1 seedlings were screened using fluorescent microscope to select GFP positive plants. The molecular analysis such as, gene specific PCRs, semiquantitative RT-PCR and Southern blots analysis corroborated the transgene integration in selected T 1 transformants. Based on progressive selection strategy, the transformation efficiency was 27.4% in gfp screened plants. The fluorescence based screening is viable, rapid, requires minimal labour, expense, and expertise with less percent of non-transformant escapes; further, this method can be extended from bell pepper to other crops to identify putative transformants developed through in planta approach.

High throughput screening strategy to identify vaccine epitopes for intracellular pathogens

High throughput screening strategy to identify vaccine epitopes for intracellular pathogens

Abstract 587: B-Raf status predicts response to pharmacological activation of p53 in melanoma

Abstract The formulation of highly-potent and specific inhibitors of the MAPK pathway has yielded several promising candidates and these are currently under advanced clinical development; however, early data suggests that inhibition of the MAPK pathway will not be sufficient to elicit sustained tumor regression in malignant melanoma. Thus, other pathways likely need to be therapeutically exploited to achieve sustained responses in melanoma patients. Because the tumor suppressor protein p53 is functional, yet inactive, in nearly 100% of melanomas, we sought to identify novel pharmacological activators of p53 in melanoma. To accomplish this goal, we employed a high-throughput screening strategy that identified hycanthone, a compound previously characterized for anti-schistosomal activity, as a specific activator of p53 function. In vitro characterization of hycanthone demonstrated that it activates the p53-transcriptional response leading to cell cycle arrest and subsequent apoptosis in a panel of melanoma cell lines harboring wild-type p53; these effects are largely absent in isogenic cells expressing p53 shRNA or alternative lines expressing mutant p53. Screening of a substantial panel of cell lines revealed that activation of p53 initiated anti-melanoma effects exclusively in cells that expressed mutant Braf, indicating that activated MAPK signaling may serve as a correlate to predict response to pharmacological activators of p53 in melanoma. However, additional experimentation has demonstrated that, instead, it is the presence of mutations in Braf, rather than the resultant MAPK activity, that predicts response to p53 activation. Studies investigating the mechanistic details governing this biological phenomenon are ongoing. Collectively, our data indicate that reactivation of p53 in melanoma, like multiple other tumors, can elicit an anti-tumor phenotype; however, this response is negatively modulated by the expression of mutant Braf, which would suggest that patients should be prospectively genotyped to identify potential responders to p53 reactivators. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 587. doi:10.1158/1538-7445.AM2011-587

Molecular Diagnostics of Cardiomyopathies

Major advances have been made in defining the genetic basis of inherited cardiac diseases. There are now >40 cardiovascular diseases in which a genetic cause has been identified, ranging from the inherited cardiomyopathies, such as hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), to primary arrhythmogenic disorders, such as familial long QT syndrome.1–3 The successful identification of causative genes in these diseases has resulted from a combination of dedicated efforts by researchers around the world as well as from rapidly emerging genetic technologies to analyze DNA. Genetic analysis, which previously took months or years to undertake, can now be performed in days or weeks. These exciting developments are now facilitating a surge in genetic information related to a range of human diseases and enhancing our understanding of how genetic differences influence health and disease. Article see p 110 In this issue of Circulation: Cardiovascular Genetics , Meder et al4 present an exciting insight into the future of how the latest genetic technologies may be used to improve the effectiveness and efficiency of comprehensive genetic screening of patients with HCM and DCM. The authors devised a rapid, high-throughput mutation screening strategy using a microarray-based target enrichment covering the coding regions of 29 known and 18 novel potential cardiomyopathy genes followed by next-generation sequencing (NGS). NGS represents a collection of newer technologies that use a combination of new strategies related to how genetic samples are prepared, advanced methods of DNA sequencing and imaging, and novel approaches to genome alignment and assembly.5 Although essentially a proof-of-principle study looking at a small selection of …

Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase

Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.

Novel sul I binary vectors enable an inexpensive foliar selection method in Arabidopsis

BackgroundSulfonamide resistance is conferred by the sulI gene found on many Enterobacteriaceae R plasmids and Tn21 type transposons. The sulI gene encodes a sulfonamide insensitive dihydropteroate synthase enzyme required for folate biosynthesis. Transformation of tobacco, potato or Arabidopsis using sulI as a selectable marker generates sulfadiazine-resistant plants. Typically sulI-based selection of transgenic plants is performed on tissue culture media under sterile conditions.FindingsA set of novel binary vectors containing a sulI selectable marker expression cassette were constructed and used to generate transgenic Arabidopsis. We demonstrate that the sulI selectable marker can be utilized for direct selection of plants grown in soil with a simple foliar spray application procedure. A highly effective and inexpensive high throughput screening strategy to identify transgenic Arabidopsis without use of tissue culture was developed.ConclusionNovel sulI-containing Agrobacterium binary vectors designed to over-express a gene of interest or to characterize a test promoter in transgenic plants have been constructed. These new vector tools combined with the various beneficial attributes of sulfonamide selection and the simple foliar screening strategy provide an advantageous alternative for plant biotechnology researchers. The set of binary vectors is freely available upon request.

Editorial: A new year and a new Associate Editor

Biomicrofluidics continued to experience another year of growth in 2010, both in terms of the number of papers published and, more importantly, their quality. We are particularly encouraged by the strong jump in the journal’s impact factor from 2.318 in 2008 to 2.895 in 2009, and are working hard to maintain a steady upward trend. There is no doubt in our minds that this is largely due to the continued support we have received from the microfluidics and nanofluidics community and we would like to thank our readers, authors, and reviewers for your contribution in various forms to the journal. As we write, we have just returned from the Second Advances for Microfluidics and Nanofluidics and Asia-Pacific Lab-on-Chip Conference (AMN-APLOC) in Singapore, which was sponsored by Biomicrofluidics. We are pleased to have seen many of our readers and authors at the conference, which has certainly grown in stature from the first AMN meeting in Hong Kong in January 2009, and we hope to see many more of you at the next meeting, which will be held in Dalian, China in mid-2012. The AMN-APLOC conference in Singapore also saw the launch of the Small Matters video contest and we will be announcing the opening of the next round soon, the winners of which will be announced in Dalian in 2012. In a field where visualization of various phenomena at small length scales and short time scales is crucial, multimedia plays an important role in the ability for authors to communicate results effectively and in a manner that creates impact. Therefore, we would like to encourage our readers and authors to include multimedia as part of your submission and invite you to participate in the contest. All multimedia that constitute part of the submission to the journal are included in our video gallery, and this collection is already freely available online athttp://bmf.aip.org/features/video_gallery. It is our aim that such a resource be made freely available to the public, with the hope of promoting and advancing the field, as well as constituting a pedagogical tool for use by instructors in undergraduate and graduate classes. The Methods and Fabrication section continues to grow since its launch in June 2010 with 13 articles published following its inception. Again, we see this as a valuable resource to students and researchers, particularly in building upon existing protocols in furthering micro∕nanofluidic science and technology as well as advancing micro∕nanofabrication techniques themselves. Thus, we would like to urge more researchers to share your fabrication and laboratory protocols with the wider community in the interest of advancing science, reducing duplication of effort, and lowering research costs. In recognition of our continued readership and authorship growth in the Asia-Pacific region and our ongoing strive to further improve the journal, we have appointed Professor Jianhua Qin as a new Associate Editor to the journal. Professor Qin, who has previously served on our Editorial Board, is currently the Director of the Microfluidics Research Center at the Dalian Institute of Chemical Physics (Chinese Academy of Sciences) and plays a prominent leading role in microfluidics in the region. Her research which interfaces technology, biology, and medicine is widely respected within the community and currently encompasses the development of miniaturized tools to aid the study of cancer biology, tissue vascularization, and stem cells in addition to the design of new diagnostic, therapeutic, and high throughput screening strategies at the cellular and organism level. Of particular significance is her effort to use microfluidics to understand cell interactions and the interplay between different cell types within the tumor milieu by recreating its microenvironment in vitro. In addition, Professor Qin’s work is also aimed at investigating the effect of mechanical forces and the role of various combinatorial factors in the regulation of stem cell differentiation and lineage commitment. Finally, we welcome the addition of the following to our Editorial Board: Professor Justin Cooper-White (University of Queensland, Australia), Professor Kwan Hyoung Kwan (Pohang University of Science and Technology, Korea), Professor Frieder Mugele (University of Twente, The Netherlands), Professor Alexander Revzin (University of California, San Diego, USA), Professor Patrick Tabeling (Ecole Superieure de Physique et Chimie Industrielles de Paris, France), and Professor Hongkai Wu (Hong Kong University of Science and Technology, Hong Kong). We are honored to have such a distinguished group of researchers on the board and look forward to working with them as well as our readers, authors, and reviewers to takeBiomicrofluidics to new heights.

An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability

Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.

Rapid evolution of arginine deiminase for improved anti-tumor activity

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K(m) value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100-120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime-thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K(m) under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K(m) value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers.

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.

Alternative splicing variability: exactly how similar are two identical cells?

Alternative splicing variability: exactly how similar are two identical cells?

A Noncompetitive Small Molecule Inhibitor of Estrogen-regulated Gene Expression and Breast Cancer Cell Growth That Enhances Proteasome-dependent Degradation of Estrogen Receptor α

The mechanisms responsible for 17β-estradiol (E(2))-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor α (ERα) antagonists are not fully understood. We describe a new tool for dissecting ERα action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor α that does not compete with estrogen for binding to ERα. TPSF noncompetitively inhibits estrogen-dependent ERα-mediated gene expression with little inhibition of transcriptional activity by NF-κB or the androgen or glucocorticoid receptor. TPSF inhibits E(2)-ERα-mediated induction of the proteinase inhibitor 9 gene, which is activated by ERα binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ERα to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E(2)-ERα-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ERα-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ERαHA cells that overexpress ERα, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ERα protein levels in MCF-7 cells and several other cell lines without altering ERα mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ERα by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ERα. TPSF represents a novel class of ER inhibitor with significant clinical potential.

How can high-throughput screening deliver drugs to treat atherosclerosis?

Importance of the field: Atherosclerosis is a progressive disease that is characterized by the accumulation of lipid-rich plaques within the artery walls. Despite the past 3 decades witnessing the most significant advances in the pharmacotherapy of atherosclerosis with statins, atherosclerosis is still one of the leading causes of mortality in industrialized and developing nations. The applications of high-throughput screening (HTS) have retrieved hits and lead compounds which may be further developed to new promising therapeutics to achieve more effective reductions in the risk of cardiovascular morbidity and mortality.Areas covered in this review: The review provides a summary of potential drug targets other than HMG-CoA reductase (primary target of statins) and their application in biochemical or cell-based HTS assays used by pharmaceutical companies and academic laboratories for anti-atherosclerotic drug discovery.What the reader will gain: The reader will gain an overview of the HTS strategies currently used in the development of anti-atherosclerotic agents. The reader is also provided with some abortive examples in anti-atherosclerotic drug discovery as well as the associated limitations and challenges of the process that HTS delivers new drugs to treat atherosclerosis.Take home message: HTS can assist in the efficient discovery of new drugs towards the potential targets involved in the progress of atherosclerosis.