scholarly journals RNase P inhibitors identified as aggregators

2021 ◽  
Author(s):  
Isabell Schencking ◽  
Eva M. Schäfer ◽  
J. H. William Scanlan ◽  
Benjamin M. Wenzel ◽  
Rolf E. Emmerich ◽  
...  
Keyword(s):  
High Throughput ◽  
Thermotoga Maritima ◽  
Rnase P ◽  
Inhibition Zone ◽  
Model Systems ◽  
Catalytic Rna ◽  
Plant Origin ◽  
Essential Enzyme ◽  
P Depletion ◽  
Specific Inhibitors

RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies reported small molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 and derivatives, iriginol hexaacetate and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg2+ ions. These findings were deduced from solubility analyses by microscopy and HPLC, RnpA-inhibitor co-pulldown experiments, detergent addition and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported non-specific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of “false positives” that are also termed Pan-assay interference compound s (PAINS).

scholarly journals A Novel Cell-Based, High-Content Assay for Phosphorylation of Lats2 by Aurora A

2011 ◽  
Vol 16 (8) ◽  
pp. 925-931 ◽  
Author(s):  
Amy Emery ◽  
David A. Sorrell ◽  
Stacy Lawrence ◽  
Emma Easthope ◽  
Mark Stockdale ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
High Content Screening ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Assay Performance ◽  
A Cell ◽  
Significant Addition ◽  
Specific Inhibitors

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration–response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.

scholarly journals Effective inhibition in animals of viral pathogenesis by a ribozyme derived from RNase P catalytic RNA

2008 ◽  
Vol 105 (31) ◽  
pp. 10919-10924 ◽  
Author(s):  
Y. Bai ◽  
P. Trang ◽  
H. Li ◽  
K. Kim ◽  
T. Zhou ◽  
...  
Keyword(s):  
Rnase P ◽  
Viral Pathogenesis ◽  
Catalytic Rna ◽  
Effective Inhibition

scholarly journals Therapeutic Drugs Targeting 2019-nCoV Main Protease by High-Throughput Screening

2020 ◽  
Author(s):  
Yan Li ◽  
Jinyong Zhang ◽  
Ning Wang ◽  
Haibo Li ◽  
Yun Shi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Binding Capacity ◽  
Effective Means ◽  
Clinical Applications ◽  
Therapeutic Drugs ◽  
Main Protease ◽  
Novel Coronavirus ◽  
Clinical Drug ◽  
Specific Inhibitors

Abstract2019 Novel Coronavirus (2019-nCoV) is a virus identified as the cause of the outbreak of pneumonia first detected in Wuhan, China. Investigations on the transmissibility, severity, and other features associated with this virus are ongoing. Currently, there is no vaccine or therapeutic antibody to prevent the infection, and more time is required to develop an effective immune strategy against the pathogen. In contrast, specific inhibitors targeting the key protease involved in replication and proliferation of the virus are the most effective means to alleviate the epidemic. The main protease of SARS-CoV is essential for the life cycle of the virus, which showed 96.1% of similarity with the main proteaseof 2019-nCoV, is considered to be an attractive target for drug development. In this study, we have identified 4 small molecular drugs with high binding capacity with SARS-CoV main protease by high-throughput screening based on the 8,000 clinical drug libraries, all these drugs have been widely used in clinical applications with guaranteed safety, which may serve as promising candidates to treat the infection of 2019-nCoV.

scholarly journals Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
Author(s):  
Upasana Singh ◽  
Vinita Panchanadikar ◽  
Dhiman Sarkar
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Coli Strain ◽  
Compound Library ◽  
Essential Enzyme ◽  
Assay Protocol

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

scholarly journals Organoid Models for Cancer Research

2019 ◽  
Vol 3 (1) ◽  
pp. 223-234 ◽  
Author(s):  
Hans Clevers ◽  
David A. Tuveson
Keyword(s):  
Cancer Research ◽  
Tumor Microenvironment ◽  
Personalized Medicine ◽  
High Throughput ◽  
Stromal Cells ◽  
Transcriptome Sequencing ◽  
Cancer Biology ◽  
Three Dimensional ◽  
Model Systems ◽  
Liver Cancers

Organoid cultures have emerged as powerful model systems accelerating discoveries in cellular and cancer biology. These three-dimensional cultures are amenable to diverse techniques, including high-throughput genome and transcriptome sequencing, as well as genetic and biochemical perturbation, making these models well suited to answer a variety of questions. Recently, organoids have been generated from diverse human cancers, including breast, colon, pancreas, prostate, bladder, and liver cancers, and studies involving these models are expanding our knowledge of the etiology and characteristics of these malignancies. Co-cultures of cancer organoids with non-neoplastic stromal cells enable investigation of the tumor microenvironment. In addition, recent studies have established that organoids have a place in personalized medicine approaches. Here, we describe the application of organoid technology to cancer discovery and treatment.

scholarly journals NMR resonance assignments of RNase P protein from Thermotoga maritima

2018 ◽  
Vol 12 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Danyun Zeng ◽  
Benjamin P. Brown ◽  
Markus W. Voehler ◽  
Sheng Cai ◽  
Nicholas J. Reiter
Keyword(s):  
Thermotoga Maritima ◽  
Resonance Assignments ◽  
Rnase P ◽  
Nmr Resonance Assignments ◽  
P Protein

In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 731-737 ◽  
Author(s):  
C. Cobaleda ◽  
I. Sánchez-Garcı́a
Keyword(s):  
Cancer Treatment ◽  
Chromosomal Abnormalities ◽  
Human Cancer ◽  
Rnase P ◽  
Catalytic Rna ◽  
Fusion Genes ◽  
Novel Approach ◽  
Chimeric Molecules

One major obstacle to the effective treatment of cancer is to distinguish between tumor cells and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities are ideal therapeutic targets because they are unique to the disease. We describe the use of a novel approach based on the catalytic RNA subunit of RNase P to destroy specifically the tumor-specific fusion genes created as a result of chromosome abnormalities. Using as a target model the abnormal BCR-ABL p190 and p210 products, we constructed M1-RNA with guide sequences that recognized the oncogenic messengers at the fusion point (M1-p190-GS and M1-p210-GS). To test the effectiveness and the specificity of M1-p190-GS and M1-p210-GS, we studied in vitro and in vivo effects of these RNA enzymes againstBCR-ABLp190 andBCR-ABLp210, bearing in mind that both fusion genes share the ABL sequence but differ in the sequence coming from the BCR gene. We showed that M1-p190-GS and M1-p210-GS can act as sequence-specific endonucleases and can exclusively cleave target RNA that forms a base pair with the guide sequence (GS). We also demonstrated that when M1-p190-GS and M1-p210-GS were expressed in proper mammalian cell models, they abolished the effect of BCR-ABL by specifically decreasing the amount of the target BCR-ABL mRNA and preventing the function of theBCR-ABL oncogenes. These data clearly demonstrate the usefulness of the catalytic activity of M1-GS RNA to cleave specifically the chimeric molecules created by chromosomal abnormalities in human cancer and to represent a novel approach to cancer treatment.

scholarly journals Receptor-Ligand Interactions Studied with Homogeneous Fluorescence-Based Assays Suitable for Miniaturized Screening

2001 ◽  
Vol 6 (1) ◽  
pp. 11-18
Author(s):  
Andreas A. Scheel ◽  
Bettina Funsch ◽  
Michael Busch ◽  
Gabriele Gradl ◽  
Johannes Pschorr ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
High Throughput Screening ◽  
Free Ligand ◽  
Membrane Vesicles ◽  
Membrane Receptors ◽  
Receptor Expression ◽  
Model Systems ◽  
Fluorescence Signal ◽  
Distribution Analysis

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and β2-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1,IL without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Sign in / Sign up

Export Citation Format

Share Document