scholarly journals High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria

Processes ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 629
Author(s):  
José Rodrigues ◽  
Vanessa T. Almeida ◽  
Ana L. Rosário ◽  
Yong Zi Tan ◽  
Brian Kloss ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Structural Biology ◽  
High Throughput ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Expression Screening ◽  
E Coli ◽  
Metal Affinity ◽  
Mycobacterial Cell Wall

Studies on membrane proteins can help to develop new drug targets and treatments for a variety of diseases. However, membrane proteins continue to be among the most challenging targets in structural biology. This uphill endeavor can be even harder for membrane proteins from Mycobacterium species, which are notoriously difficult to express in heterologous systems. Arabinofuranosyltransferases are involved in mycobacterial cell wall synthesis and thus potential targets for antituberculosis drugs. A set of 96 mycobacterial genes coding for Arabinofuranosyltransferases was selected, of which 17 were successfully expressed in E. coli and purified by metal-affinity chromatography. We herein present an efficient high-throughput strategy to screen in microplates a large number of targets from Mycobacteria and select the best conditions for large-scale protein production to pursue functional and structural studies. This methodology can be applied to other targets, is cost and time effective and can be implemented in common laboratories.

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

2020 ◽  
Vol 17 (5) ◽  
pp. 716-724
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Antibacterial Agents ◽  
Sos Response ◽  
Luciferase Assay ◽  
Translational Machinery ◽  
E Coli ◽  
New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

Comparison of Small- and Large-scale Expression of Selected Pyrococcus furiosus Genes as an Aid to High-throughput Protein Production

2005 ◽  
Vol 6 (2-3) ◽  
pp. 149-158 ◽  
Author(s):  
Frank J. Sugar ◽  
Francis E. Jenney ◽  
Farris L. Poole ◽  
Phillip S. Brereton ◽  
Michi Izumi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Protein Production ◽  
Pyrococcus Furiosus

Structural biology and structure–function relationships of membrane proteins

2018 ◽  
Vol 47 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Rosana Reis ◽  
Isabel Moraes
Keyword(s):  
Membrane Proteins ◽  
Structure Function ◽  
Structural Biology ◽  
Drug Targets ◽  
Three Dimensional ◽  
Data Bank ◽  
Technical Advances ◽  
Medical Importance ◽  
G Protein Coupled ◽  
Important Drug

Abstract The study of structure–function relationships of membrane proteins (MPs) has been one of the major goals in the field of structural biology. Many Noble Prizes regarding remarkable accomplishments in MP structure determination and biochemistry have been awarded over the last few decades. Mutations or improper folding of these proteins are associated with numerous serious illnesses. Therefore, as important drug targets, the study of their primary sequence and three-dimensional fold, combined with cell-based assays, provides vital information about their structure–function relationships. Today, this information is vital to drug discovery and medicine. In the last two decades, many have been the technical advances and breakthroughs in the field of MP structural biology that have contributed to an exponential growth in the number of unique MP structures in the Protein Data Bank. Nevertheless, given the medical importance and many unanswered questions, it will never be an excess of MP structures, regardless of the method used. Owing to the extension of the field, in this brief review, we will only focus on structure–function relationships of the three most significant pharmaceutical classes: G protein-coupled receptors, ion channels and transporters.

scholarly journals The E. coli single protein production system for production and structural analysis of membrane proteins

2009 ◽  
Vol 11 (1) ◽  
pp. 81-84 ◽  
Author(s):  
Lili Mao ◽  
S. Thangminlal Vaiphei ◽  
Tsutomu Shimazu ◽  
William M. Schneider ◽  
Yuefeng Tang ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Membrane Proteins ◽  
Production System ◽  
Protein Production ◽  
E Coli ◽  
Single Protein

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2018 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

AbstractCell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed cell-based cytotoxicity of nearly 10,000 compounds in NCATS annotated libraries, and over 100,000 compounds in a diversity library, against four ‘normal’ cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitutes a valuable resource for the scientific community and provides insight on the extent of cytotoxic compounds in screening libraries, identifying and avoiding compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals Membrane-active Polymers: NCMNP13-x, NCMNP21-x and NCMNP21b-x for Membrane Protein Structural Biology

2022 ◽  
Author(s):  
Thi Kim Hoang Trinh ◽  
Claudio Catalano ◽  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Maleic Acid ◽  
Drug Targets ◽  
Design Synthesis ◽  
Native Cell ◽  
Wide Range ◽  
Lipid Particles ◽  
High Resolution Structure

Membrane proteins are a ubiquitous group of bio-macromolecules responsible for many crucial biological processes and serve as drug targets for a wide range of modern drugs. Detergent-free technologies such as styrene-maleic acid lipid particles (SMALP), diisobutylene-maleic acid lipid particles (DIBMALP), and native cell membrane nanoparticles (NCMN) systems have recently emerged as revolutionary alternatives to the traditional detergent-based approaches for membrane protein research. NCMN systems aim to create a membrane-active polymer library suitable for high-resolution structure determination. Herein, we report our design, synthesis, characterization and comparative application analyses of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x and NCMNP21b-x. Although each NCMN polymer can solubilize various model membrane proteins and conserve native lipids into NCMN particles, only the NCMNP21b-x series reveals lipid-protein particles with good buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. Consequently, the NCMNP21b-x polymers that bring out high-quality NCMN particles are particularly attractive for membrane protein structural biology.

scholarly journals An automated platform for structural analysis of membrane proteins through serial crystallography

2021 ◽  
Author(s):  
Robert D Healey ◽  
Shibom Basu ◽  
Anne-Sophie Humm ◽  
Cedric Leyrat ◽  
Xiaojing Cong ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
High Throughput ◽  
Drug Targets ◽  
Protein Crystallization ◽  
Integral Membrane Protein ◽  
Structural Level ◽  
Ligand Discovery ◽  
Serial Crystallography ◽  
Automated Platform

Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze at a structural level. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins due to lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperature and in a single support. We apply this approach to two human integral membrane proteins, which allowed us to capture different conformational states of intramembrane enzyme-product complexes and analyze the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting and serial crystallography enabling screening of small molecule libraries with membrane protein crystals grown in meso. This approach brings badly needed automation for this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.

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