New Approach for High-Throughput Screening of Drug Activity on Plasmodium Liver Stages

ABSTRACT Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrared fluorescence scanning system. This method allowed us to count automatically and rapidly Plasmodium-infected hepatocytes, using different hepatic cells and different Plasmodium species, including Plasmodium falciparum. This new technique is well adapted for high-throughput drug screening and should facilitate the identification of new antimalarial compounds active on Plasmodium liver stages.

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Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

Biochemical Journal ◽

10.1042/bj20070692 ◽

2007 ◽

Vol 408 (2) ◽

pp. 173-180 ◽

Cited By ~ 45

Author(s):

Paul W. Bowyer ◽

Ruwani S. Gunaratne ◽

Munira Grainger ◽

Chrislaine Withers-Martinez ◽

Sasala R. Wickramsinghe ◽

...

Keyword(s):

Escherichia Coli ◽

Plasmodium Falciparum ◽

High Throughput ◽

High Throughput Screening ◽

Small Scale ◽

Expression And Purification ◽

Purification System ◽

Pure Protein ◽

Ic50 Values

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 μg·l−1 to >400 μg·l−1 by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale ‘piggyback’ inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 μM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 μM.

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Faculty Opinions recommendation of High throughput immuno-screening of cDNA expression libraries produced by in vitro recombination; exploring the Plasmodium falciparum proteome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1012421.201876 ◽

2004 ◽

Author(s):

Vishvanath Nene

Keyword(s):

Plasmodium Falciparum ◽

High Throughput ◽

In Vitro Recombination ◽

Cdna Expression ◽

Expression Libraries

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A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

Nucleic Acids Research ◽

10.1093/nar/gkaa1213 ◽

2020 ◽

Author(s):

Yuru Wang ◽

Christopher D Katanski ◽

Christopher Watkins ◽

Jessica N Pan ◽

Qing Dai ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Targeted Mutagenesis ◽

Rna Repair ◽

Dna And Rna ◽

Modified Dna ◽

Dna Substrates ◽

Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

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Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18073332 ◽

2021 ◽

Vol 18 (7) ◽

pp. 3332

Author(s):

Olga V. Naidenko ◽

David Q. Andrews ◽

Alexis M. Temkin ◽

Tasha Stoiber ◽

Uloma Igara Uche ◽

...

Keyword(s):

Immune System ◽

High Throughput ◽

Environmental Protection Agency ◽

High Throughput Screening ◽

Molecular Mechanisms ◽

Specific Activity ◽

Laboratory Animals ◽

In Vitro Assays ◽

Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

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High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

Nature Communications ◽

10.1038/s41467-021-23954-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhou Fang ◽

Junjian Chen ◽

Ye Zhu ◽

Guansong Hu ◽

Haoqian Xin ◽

...

Keyword(s):

Antimicrobial Activity ◽

High Throughput ◽

High Throughput Screening ◽

Rational Design ◽

Click Reaction ◽

Reaction Times ◽

Great Promise ◽

Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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