scholarly journals Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

2013 ◽  
Vol 58 (1) ◽  
pp. 527-535 ◽  
Author(s):  
Erika van den Bogaart ◽  
Gerard J. Schoone ◽  
Paul England ◽  
Dorien Faber ◽  
Kristina M. Orrling ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Genetic Recombination ◽  
Colorimetric Assay ◽  
Reference Method ◽  
Published Data ◽  
Content Type ◽  
Intracellular Amastigotes ◽  
Efficient Test ◽  
Enzymatic Reduction

ABSTRACTCritical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity againstLeishmaniaclinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of aLeishmanianative enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth ofLeishmaniaparasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.

scholarly journals Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

2018 ◽  
Vol 84 (16) ◽  
Author(s):  
Wei Zhou ◽  
Rui Huang ◽  
Zhiguang Zhu ◽  
Yi-Heng P. Job Zhang
Keyword(s):  
High Activity ◽  
Double Layer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Specific Activity ◽  
Colorimetric Assay ◽  
Petri Dish ◽  
Rapid Identification ◽  
Thermobifida Fusca ◽  
Content Type

ABSTRACT Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

Phospholipid/Polydiacetylene Vesicle-Based Colorimetric Assay for High-Throughput Screening of Bacteriocins and Halocins

2016 ◽  
Vol 182 (1) ◽  
pp. 142-154 ◽  
Author(s):  
Manoj Kumar Yadav ◽  
Vijay Kumar ◽  
Bijender Singh ◽  
Santosh Kumar Tiwari
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Assay

scholarly journals Imaging-Based High-Throughput Screening Assay To Identify New Molecules with Transmission-Blocking Potential against Plasmodium falciparum Female Gamete Formation

2015 ◽  
Vol 59 (6) ◽  
pp. 3298-3305 ◽  
Author(s):  
Celia Miguel-Blanco ◽  
Joël Lelièvre ◽  
Michael J. Delves ◽  
Ana I. Bardera ◽  
Jesús L. Presa ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Female Gamete ◽  
Transmission Blocking ◽  
Content Type ◽  
Female Gametocyte ◽  
Gamete Formation ◽  
Selection Of

ABSTRACTIn response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated byPlasmodium falciparumstage V gametocytes. Lately, metabolic activity has been usedin vitroas a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated theP. falciparumfemale gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z′ factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 μM concentration and 48 h of incubation with gametocytes. TheP. falciparumFGAA has been proven to be a robust HTS assay faithful toPlasmodiumtransmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential.

scholarly journals nBioChip, a Lab-on-a-Chip Platform of Mono- and Polymicrobial Biofilms for High-Throughput Downstream Applications

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Anand Srinivasan ◽  
Nelson S. Torres ◽  
Kai P. Leung ◽  
Jose L. Lopez-Ribot ◽  
Anand K. Ramasubramanian
Keyword(s):  
Antibiotic Treatment ◽  
High Throughput ◽  
High Throughput Screening ◽  
Susceptibility Testing ◽  
Unmet Need ◽  
Mortality Rates ◽  
Content Type ◽  
Bacteria And Fungi ◽  
And Control ◽  
Nanoscale Level

ABSTRACT With an estimated 80% of infections being associated with a biofilm mode of growth and the ensuing recalcitrance of these biofilms with respect to conventional antibiotic treatment leading to high mortality rates, there is a dire and unmet need for the development of novel approaches to prevent, treat, and control these infections. Both bacteria and fungi are capable of forming biofilms that are inherently fragile and often polymicrobial in nature, which further complicates treatment. In this work, we showcase a nanobiofilm chip as a convenient platform for culturing several hundreds of mono- or polymicrobial biofilms and for susceptibility testing. This platform enables true ultra-high-throughput screening for antimicrobial drug discovery or diagnostics or for addressing fundamental issues in microbiology. Current in vitro techniques for the culture of microorganisms, and particularly of delicate microbial biofilms, are still mostly limited to low-density plates and manual labor and are not amenable to automation and true high-throughput (HT) applications. We have developed a novel fully automated platform for the formation of mono- and polymicrobial biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans at the nanoscale level. The nBioChip is robotically printed, robustly handled, and scanned using a standard microarray reader. Using this technique, hundreds to thousands of identical nanobiofilms encapsulated in hydrogel spots were cultured on microscope slides. The spots can withstand the washing steps involved in screening assays. The miniaturized biofilms demonstrated characteristics similar to those displayed by conventionally formed macroscopic biofilms, including (i) three-dimensional architectural features, (ii) synthesis of exopolymeric matrix material, and (iii) elevated resistance to antibiotic treatment. On the basis of our results, the nBioChip can generate reliable high-throughput antimicrobial susceptibility testing (HT-AST) in 12 to 18 h. The chip serves as a proof-of-concept universal platform for high-throughput drug screening and other downstream applications and furthers understanding of microbial interactions in mixed-species communities at the nanoscale level. IMPORTANCE With an estimated 80% of infections being associated with a biofilm mode of growth and the ensuing recalcitrance of these biofilms with respect to conventional antibiotic treatment leading to high mortality rates, there is a dire and unmet need for the development of novel approaches to prevent, treat, and control these infections. Both bacteria and fungi are capable of forming biofilms that are inherently fragile and often polymicrobial in nature, which further complicates treatment. In this work, we showcase a nanobiofilm chip as a convenient platform for culturing several hundreds of mono- or polymicrobial biofilms and for susceptibility testing. This platform enables true ultra-high-throughput screening for antimicrobial drug discovery or diagnostics or for addressing fundamental issues in microbiology.

scholarly journals Corifungin, a New Drug Lead against Naegleria, Identified from a High-Throughput Screen

2012 ◽  
Vol 56 (11) ◽  
pp. 5450-5457 ◽  
Author(s):  
Anjan Debnath ◽  
Josefino B. Tunac ◽  
Silvia Galindo-Gómez ◽  
Angélica Silva-Olivares ◽  
Mineko Shibayama ◽  
...  
Keyword(s):  
Amphotericin B ◽  
High Throughput ◽  
High Throughput Screening ◽  
Plasma Membranes ◽  
Kinase Inhibitors ◽  
Water Soluble ◽  
Drug Status ◽  
Content Type ◽  
Compound Libraries

ABSTRACTPrimary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amebaNaegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related speciesNaegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity againstNaegleriathan that of amphotericin B. Transmission electron microscopy ofN. fowleritrophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae.In vivoefficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

ChemInform Abstract: A Rapid, Sensitive Colorimetric Assay for the High-Throughput Screening of Transaminases in Liquid or Solid-Phase.

ChemInform ◽  
2016 ◽  
Vol 47 (15) ◽  
pp. no-no
Author(s):  
D. Baud ◽  
N. Ladkau ◽  
T. S. Moody ◽  
J. M. Ward ◽  
H. C. Hailes
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Colorimetric Assay

scholarly journals Novel Yeast Bioassay for High-Throughput Screening of Matrix Metalloproteinase Inhibitors

2011 ◽  
Vol 77 (24) ◽  
pp. 8573-8577 ◽  
Author(s):  
Bjoern Diehl ◽  
Thorsten M. Hoffmann ◽  
Nina C. Mueller ◽  
Jens L. Burkhart ◽  
Uli Kazmaier ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Biologically Active ◽  
Yeast Cells ◽  
Mmp Inhibitors ◽  
Content Type ◽  
Yeast Cell Surface ◽  
Mmp Inhibitor ◽  
Wide Range ◽  
Anticancer Treatment

ABSTRACTDiverse malfunctions in the expression and regulation of matrix metalloproteinases (MMPs) are often the cause of severe human diseases, bringing the identification of specific MMP inhibitors into major focus, particularly in anticancer treatment. Here, we describe a novel bioassay based on recombinant yeast cells (Pichia pastoris) that express, deliver, and incorporate biologically active human MMP-2 and MMP-9 at the yeast cell surface. Using Sed1p for cell wall targeting and covalent anchorage, a highly efficient bioassay was established that allows high-throughput screening and subsequent validation of novel MMP inhibitors as potential anticancer drugs. In addition, we developed a straightforward synthesis of a new aspartate-derived MMP inhibitor active in the nM range and bearing an amino functionality that should allow the introduction of a wide range of side chains to modify the properties of these compounds.

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