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Special Issue "High-throughput Screening"

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Molecular Diversity".

Deadline for manuscript submissions: closed (15 January 2010)

Special Issue Editor

Guest Editor
Dr. Florian Hollfelder (Website)

Department of Biochemistry, 80 Tennis Court Road, 3.84 Sanger Building, GB, Cambridge CB2 1GA, United Kingdom
Fax: +44 1223 766002

Special Issue Information

Dear Colleagues,

In addition to being the workhorse of the pharmaceutical industry, high-throughput screening is becoming a more and more central experimental approach in diverse research areas ranging from genomics, proteomics to systems and synthetic biology. The idea of characterizing entire ensembles of genes, proteins, small molecule or organismal repertoires is attractive when the complexity of a system defies straightforward understanding and rational manipulation.

In contrast to selection approaches that obey a ‘first-past-the-post’ logic, screening quantitatively measures one or more experimental parameters for the entire library. The knowledge about an entire library may give rise to structure-activity relationships and allows a more informed choice about which molecules to select e.g. for further rounds of evolutionary improvements.

Accessibility of these approaches to a wider circle of experimentalists has been driven by technological advances, specifically miniaturization and advances in detection systems. This issue will focus on the technical advances that give insight into property spaces and combine it with an outlook towards the potential of such massively parallel experimentation in Chemistry and Biology.

Florian Hollfelder, Ph.D.
Guest Editor

Keywords

  • DNA library
  • Encoding /decoding
  • In vitro compartmentalisation
  • Systems biology
  • Synthetic biology

Published Papers (8 papers)

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Research

Jump to: Review

Open AccessArticle High-Throughput Behavioral Screens: the First Step towards Finding Genes Involved in Vertebrate Brain Function Using Zebrafish
Molecules 2010, 15(4), 2609-2622; doi:10.3390/molecules15042609
Received: 9 February 2010 / Revised: 26 March 2010 / Accepted: 7 April 2010 / Published: 12 April 2010
Cited by 89 | PDF Full-text (199 KB)
Abstract
The zebrafish has been in the forefront of developmental biology for three decades and has become a favorite of geneticists. Due to the accumulated genetic knowledge and tools developed for the zebrafish it is gaining popularity in other disciplines, including neuroscience. The [...] Read more.
The zebrafish has been in the forefront of developmental biology for three decades and has become a favorite of geneticists. Due to the accumulated genetic knowledge and tools developed for the zebrafish it is gaining popularity in other disciplines, including neuroscience. The zebrafish offers a compromise between system complexity (it is a vertebrate similar in many ways to our own species) and practical simplicity (it is small, easy to keep, and prolific). Such features make zebrafish an excellent choice for high throughput mutation and drug screening. For the identification of mutation or drug induced alteration of brain function arguably the best methods are behavioral test paradigms. This review does not present experimental examples for the identification of particular genes or drugs. Instead it describes how behavioral screening methods may enable one to find functional alterations in the vertebrate brain. Furthermore, the review is not comprehensive. The behavioral test examples presented are biased according to the personal interests of the author. They will cover research areas including learning and memory, fear and anxiety, and social behavior. Nevertheless, the general principles will apply to other functional domains and should represent a snapshot of the rapidly evolving behavioral screening field with zebrafish. Full article
(This article belongs to the Special Issue High-throughput Screening)
Open AccessArticle HTS-Driven Discovery of New Chemotypes with West Nile Virus Inhibitory Activity
Molecules 2010, 15(3), 1690-1704; doi:10.3390/molecules15031690
Received: 25 January 2010 / Revised: 20 February 2010 / Accepted: 8 March 2010 / Published: 12 March 2010
Cited by 7 | PDF Full-text (697 KB)
Abstract
West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a [...] Read more.
West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a high throughput screen to discover small molecule probes that inhibit virus infection of Vero E6 cells. A primary screen of a 13,001 compound library at a 10 µM final concentration was conducted using the 384-well format. Z′ values ranged from 0.54–0.83 with a median of 0.74. Average S/B was 17 and S/N for each plate ranged from 10.8 to 23.9. Twenty-six compounds showed a dose response in the HT screen and were further evaluated in a time of addition assay and in a titer reduction assay. Seven compounds showed potential as small molecule probes directed at WNV. The hit rate from the primary screen was 0.185% (24 compounds out of 13,001 compounds) and from the secondary screens was 0.053% (7 out of 13,001 compounds) respectively. Full article
(This article belongs to the Special Issue High-throughput Screening)
Open AccessArticle Hitherto Unrecognized Fluorescence Properties of Coniferyl Alcohol
Molecules 2010, 15(3), 1645-1667; doi:10.3390/molecules15031645
Received: 14 January 2010 / Revised: 23 February 2010 / Accepted: 8 March 2010 / Published: 11 March 2010
PDF Full-text (596 KB)
Abstract
We instituted a quasi-quality assurance program for demonstrating coniferyl alcohol’s fluorescence and fluorescence diminishment following enzymatic oxidation. The magnitude of diminishment was a measure of catalysis. High throughput screening was performed in pseudo-kinetic and endpoint modes by measuring the fluorescence at [...] Read more.
We instituted a quasi-quality assurance program for demonstrating coniferyl alcohol’s fluorescence and fluorescence diminishment following enzymatic oxidation. The magnitude of diminishment was a measure of catalysis. High throughput screening was performed in pseudo-kinetic and endpoint modes by measuring the fluorescence at 416 nm following excitation at 290, 310 or 340 nm. Dose-response tracings were linear between two and three orders of magnitude with average limits of detection and quantitation of 1.8 and 6.9 mM coniferyl alcohol, respectively. Oxidation was evident with 0.025 mg/mL laccase or 0.003 mg/mL peroxidase or inside 5 min using 0.5 mg/mL laccase or 5 mM substrate. Sodium chloride inhibited (IC50, 25 mM) laccase oxidation of coniferyl alcohol. Fluorescence from 10 concentrations (1 to 1000 mM) of coniferyl alcohol was stable for 24 hours over 14 excitation/emission cycles at 3 different combinations of excitation and emission wavelengths. In conclusion, coniferyl alcohol absorption and fluorescence assays should facilitate biomass lignin analyses and improve delignification. Full article
(This article belongs to the Special Issue High-throughput Screening)
Figures

Open AccessArticle Screening and Improvement of an Anti-VEGF DNA Aptamer
Molecules 2010, 15(1), 215-225; doi:10.3390/molecules15010215
Received: 6 November 2009 / Revised: 17 December 2009 / Accepted: 31 December 2009 / Published: 7 January 2010
Cited by 51 | PDF Full-text (595 KB) | Supplementary Files
Abstract
To obtain an aptamer with a high affinity for vascular endothelial growth factor (VEGF), we focused on the receptor-binding domain (RBD) of VEGF as a target epitope. Three rounds of screening gave Vap7, which bound to the VEGF isoforms VEGF121 and [...] Read more.
To obtain an aptamer with a high affinity for vascular endothelial growth factor (VEGF), we focused on the receptor-binding domain (RBD) of VEGF as a target epitope. Three rounds of screening gave Vap7, which bound to the VEGF isoforms VEGF121 and VEGF165 with KD values of 1.0 nM and 20 nM, respectively. Moreover, Vap7 showed specificity within the VEGF family. Secondary structure predictions and circular dicrhoism suggested that Vap7 folds into a G-quadruplex structure. We obtained a mutant aptamer that contains only this region of the aptamer sequence. This truncated mutant (V7t1) bound to both VEGF121 and VEGF165 with KD values of 1.1 nM and 1.4 nM, respectively. Its sequence was 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3', and it appeared to form a G-quadruplex structure. We also produced an aptamer heterodimer consisting of our previously derived aptamer (del5-1), which binds to the heparin-binding domain of VEGF, linked to V7t1. The resulting heterodimer bound strongly to VEGF165 with a KD value of 4.7 × 102 pM. Full article
(This article belongs to the Special Issue High-throughput Screening)
Open AccessArticle Spectroscopic Analyses of the Biofuels-Critical Phytochemical Coniferyl Alcohol and Its Enzyme-Catalyzed Oxidation Products
Molecules 2009, 14(11), 4758-4778; doi:10.3390/molecules14114758
Received: 21 October 2006 / Revised: 16 November 2006 / Accepted: 19 November 2009 / Published: 23 November 2009
PDF Full-text (564 KB)
Abstract
Lignin composition (monolignol types of coniferyl, sinapyl or p-coumaryl alcohol) is causally related to biomass recalcitrance. We describe multiwavelength (220, 228, 240, 250, 260, 290, 295, 300, 310 or 320 nm) absorption spectroscopy of coniferyl alcohol and its laccase- or peroxidase-catalyzed [...] Read more.
Lignin composition (monolignol types of coniferyl, sinapyl or p-coumaryl alcohol) is causally related to biomass recalcitrance. We describe multiwavelength (220, 228, 240, 250, 260, 290, 295, 300, 310 or 320 nm) absorption spectroscopy of coniferyl alcohol and its laccase- or peroxidase-catalyzed products during real time kinetic, pseudokinetic and endpoint analyses, in optical turn on or turn off modes, under acidic or basic conditions. Reactions in microwell plates and 100 μL volumes demonstrated assay miniaturization and high throughput screening capabilities. Bathochromic and hypsochromic shifts along with hyperchromicity or hypochromicity accompanied enzymatic oxidations by laccase or peroxidase. The limits of detection and quantitation of coniferyl alcohol averaged 2.4 and 7.1 μM respectively, with linear trend lines over 3 to 4 orders of magnitude. Coniferyl alcohol oxidation was evident within 10 minutes or with 0.01 μg/mL laccase and 2 minutes or 0.001 μg/mL peroxidase. Detection limit improved to 1.0 μM coniferyl alcohol with Km of 978.7 ± 150.7 μM when examined at 260 nm following 30 minutes oxidation with 1.0 μg/mL laccase. Our assays utilized the intrinsic spectroscopic properties of coniferyl alcohol or its oxidation products for enabling detection, without requiring chemical synthesis or modification of the substrate or product(s). These studies facilitate lignin compositional analyses and augment pretreatment strategies for reducing biomass recalcitrance. Full article
(This article belongs to the Special Issue High-throughput Screening)

Review

Jump to: Research

Open AccessReview Nanofabrication of Nonfouling Surfaces for Micropatterning of Cell and Microtissue
Molecules 2010, 15(8), 5525-5546; doi:10.3390/molecules15085525
Received: 11 June 2010 / Revised: 20 July 2010 / Accepted: 4 August 2010 / Published: 10 August 2010
Cited by 19 | PDF Full-text (846 KB)
Abstract
Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behavior, as cells usually integrate and respond the microscale environment, such as chemical and mechanical properties of the surrounding fluid and extracellular [...] Read more.
Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behavior, as cells usually integrate and respond the microscale environment, such as chemical and mechanical properties of the surrounding fluid and extracellular matrix, soluble protein factors, small signal molecules, and contacts with neighboring cells. Furthermore, recent progress in cellular micropatterning has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. In this regards, the ability to control shape and spreading of attached cells and cell-cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering. To develop this kind of cellular microarray composed of a cell-resistant surface and cell attachment region, micropatterning a protein-repellent surface is important because cellular adhesion and proliferation are regulated by protein adsorption. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional surfaces with the aim to provide an introductory overview described in the literature. In particular, the importance of non-fouling surface chemistries is discussed. Full article
(This article belongs to the Special Issue High-throughput Screening)
Figures

Open AccessReview Assay Format as a Critical Success Factor for Identification of Novel Inhibitor Chemotypes of Tissue-Nonspecific Alkaline Phosphatase from High-Throughput Screening
Molecules 2010, 15(5), 3010-3037; doi:10.3390/molecules15053010
Received: 20 January 2010 / Revised: 18 March 2010 / Accepted: 22 April 2010 / Published: 27 April 2010
Cited by 14 | PDF Full-text (965 KB)
Abstract
The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) [...] Read more.
The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats. Full article
(This article belongs to the Special Issue High-throughput Screening)
Open AccessReview Automation in the High-throughput Selection of Random Combinatorial Libraries—Different Approaches for Select Applications
Molecules 2010, 15(4), 2478-2490; doi:10.3390/molecules15042478
Received: 10 February 2010 / Revised: 30 March 2010 / Accepted: 6 April 2010 / Published: 8 April 2010
Cited by 12 | PDF Full-text (279 KB)
Abstract
Automation in combination with high throughput screening methods has revolutionised molecular biology in the last two decades. Today, many combinatorial libraries as well as several systems for automation are available. Depending on scope, budget and time, a different combination of library and [...] Read more.
Automation in combination with high throughput screening methods has revolutionised molecular biology in the last two decades. Today, many combinatorial libraries as well as several systems for automation are available. Depending on scope, budget and time, a different combination of library and experimental handling might be most effective. In this review we will discuss several concepts of combinatorial libraries and provide information as what to expect from these depending on the given context. Full article
(This article belongs to the Special Issue High-throughput Screening)

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