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.

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

Hormesis in high-throughput screening of antibacterial compounds in E coli

2010 ◽  
Vol 29 (8) ◽  
pp. 667-677 ◽  
Author(s):  
Edward J Calabrese ◽  
George R Hoffmann ◽  
Edward J Stanek ◽  
Marc A Nascarella
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Threshold Model ◽  
Concentration Effects ◽  
Data Set ◽  
E Coli ◽  
Model Predictions ◽  
High Throughput Study ◽  
Repeat Measure

This article assesses the response below a toxicological threshold for 1888 antibacterial agents in Escherichia coli, using 11 concentrations with twofold concentration spacing in a high-throughput study. The data set had important strengths such as low variability in the control (2%—3% SD), a repeat measure of all wells, and a built-in replication. Bacterial growth at concentrations below the toxic threshold is significantly greater than that in the controls, consistent with a hormetic concentration response. These findings, along with analyses of published literature and complementary evaluations of concentration-response model predictions of low-concentration effects in yeast, indicate a lack of support for the broadly and historically accepted threshold model for responses to concentrations below the toxic threshold.

scholarly journals High-throughput direct screening of restriction endonuclease using microfluidic fluorescence-activated drop sorter based on SOS response in E. coli

2022 ◽  
Author(s):  
Yizhe Zhang ◽  
Jeremy J Agresti ◽  
Yu Zheng ◽  
David A Weitz
Keyword(s):  
Restriction Endonuclease ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Screening Method ◽  
Sos Response ◽  
Industrial Applications ◽  
Ease Of Use ◽  
Isolation And Characterization ◽  
New Strategy

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route for expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient fashion. Briefly, we construct a host bacterial cell to link the RE genotype to the phenotype of β-galactosidase expression based on the bacterial SOS response, and use a high-throughput microfluidic platform to isolate, detect and sort the REs. We employ this strategy to screen for the XbaI gene from constructed libraries of varied sizes. In single round of sorting, a 30-fold target enrichment was obtained within 1 h. The direct screening approach we propose shows potential for efficient search of desirable REs in natural samples compared to the conventional RE-screening method, and is amenable to being adapted to high-throughput screening of other genotoxic targets.

scholarly journals 1,4-Naphthoquinone Analogues: Potent Antibacterial Agents and Mode of Action Evaluation

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1437 ◽  
Author(s):  
Palanisamy Ravichandiran ◽  
Sunirmal Sheet ◽  
Dhanraj Premnath ◽  
Ae Rhan Kim ◽  
Dong Jin Yoo
Keyword(s):  
Antibacterial Activity ◽  
Mode Of Action ◽  
Bacterial Infections ◽  
Antibacterial Agents ◽  
Annexin V ◽  
Ros Generation ◽  
Redox Potentials ◽  
Bacterial Strains ◽  
E Coli ◽  
New Class

1,4-Naphthoquinones have antibacterial activity and are a promising new class of compound that can be used to treat bacterial infections. The goal was to improve effective antibacterial agents; therefore, we synthesized a new class of naphthoquinone hybrids, which contain phenylamino-phenylthio moieties as significant counterparts. Compound 4 was modified as a substituted aryl amide moiety, which enhanced the antibacterial activity of earlier compounds 3 and 4. In this study, five bacterial strains Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) were used to evaluate the antibacterial potency of synthesized naphthoquinones using the minimal inhibitory concentration (MIC) method. Most of the studied naphthoquinones demonstrated major antibacterial activity with a MIC of 15.6 µg/mL–500 µg/mL. Selected compounds (5a, 5f and 5x) were studied for the mode of action, using intracellular ROS generation, determination of apoptosis by the Annexin V-FITC/PI assay, a bactericidal kinetic study and in silico molecular modelling. Additionally, the redox potentials of the specified compounds were confirmed by cyclic voltammetry (CV).

A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

2021 ◽  
Author(s):  
Kuang-Yu Chen ◽  
Tim Krischuns ◽  
Laura Ortega Varga ◽  
Emna Harigua-Souiai ◽  
Sylvain Paisant ◽  
...  
Keyword(s):  
High Throughput ◽  
Broad Spectrum ◽  
High Throughput Screening ◽  
Large Scale ◽  
Luciferase Assay ◽  
Screening Methods ◽  
Sensitive Cell ◽  
Viral Protease ◽  
Compound Libraries ◽  
Highly Sensitive

Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 micromolar range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.

scholarly journals Discovery of a Novel and Potent Class of FabI-Directed Antibacterial Agents

2002 ◽  
Vol 46 (10) ◽  
pp. 3118-3124 ◽  
Author(s):  
David J. Payne ◽  
William H. Miller ◽  
Valerie Berry ◽  
John Brosky ◽  
Walter J. Burgess ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Enoyl Acp Reductase

ABSTRACT Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI) catalyzes the final step in each elongation cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. High-throughput screening of the Staphylococcus aureus FabI enzyme identified a novel, weak inhibitor with no detectable antibacterial activity against S. aureus. Iterative medicinal chemistry and X-ray crystal structure-based design led to the identification of compound 4 [(E)-N-methyl-N-(2-methyl-1H-indol-3-ylmethyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide], which is 350-fold more potent than the original lead compound obtained by high-throughput screening in the FabI inhibition assay. Compound 4 has exquisite antistaphylococci activity, achieving MICs at which 90% of isolates are inhibited more than 500 times lower than those of nine currently available antibiotics against a panel of multidrug-resistant strains of S. aureus and Staphylococcus epidermidis. Furthermore, compound 4 exhibits excellent in vivo efficacy in an S. aureus infection model in rats. Biochemical and genetic approaches have confirmed that the mode of antibacterial action of compound 4 and related compounds is via inhibition of FabI. Compound 4 also exhibits weak FabK inhibitory activity, which may explain its antibacterial activity against Streptococcus pneumoniae and Enterococcus faecalis, which depend on FabK and both FabK and FabI, respectively, for their enoyl-ACP reductase function. These results show that compound 4 is representative of a new, totally synthetic series of antibacterial agents that has the potential to provide novel alternatives for the treatment of S. aureus infections that are resistant to our present armory of antibiotics.

Accelerated Discovery of High-Refractive-Index Polyimides via First-Principles Molecular Modeling, Virtual High-Throughput Screening, and Data Mining

2019 ◽  
Author(s):  
Mohammad Atif Faiz Afzal ◽  
Mojtaba Haghighatlari ◽  
Sai Prasad Ganesh ◽  
Chong Cheng ◽  
Johannes Hachmann
Keyword(s):  
Data Mining ◽  
Refractive Index ◽  
High Throughput ◽  
First Principles ◽  
High Throughput Screening ◽  
Large Scale ◽  
Computational Study ◽  
High Refractive Index ◽  
Structural Features ◽  
Learning Program

<div>We present a high-throughput computational study to identify novel polyimides (PIs) with exceptional refractive index (RI) values for use as optic or optoelectronic materials. Our study utilizes an RI prediction protocol based on a combination of first-principles and data modeling developed in previous work, which we employ on a large-scale PI candidate library generated with the ChemLG code. We deploy the virtual screening software ChemHTPS to automate the assessment of this extensive pool of PI structures in order to determine the performance potential of each candidate. This rapid and efficient approach yields a number of highly promising leads compounds. Using the data mining and machine learning program package ChemML, we analyze the top candidates with respect to prevalent structural features and feature combinations that distinguish them from less promising ones. In particular, we explore the utility of various strategies that introduce highly polarizable moieties into the PI backbone to increase its RI yield. The derived insights provide a foundation for rational and targeted design that goes beyond traditional trial-and-error searches.</div>

Recent Advances in 1,3,5-Triazine Derivatives as Antibacterial Agents

2020 ◽  
Vol 17 (8) ◽  
pp. 991-1041
Author(s):  
Divya Utreja ◽  
Jagdish Kaur ◽  
Komalpreet Kaur ◽  
Palak Jain
Keyword(s):  
Antibacterial Activity ◽  
Heterocyclic Compounds ◽  
Antibacterial Agents ◽  
Rapid Development ◽  
Pharmacological Action ◽  
Biological Properties ◽  
Vast Array ◽  
Bacterial Diseases ◽  
New Class ◽  
Triazine Derivatives

Triazine, one of the nitrogen containing heterocyclic compounds has attracted the considerable interest of researchers due to the vast array of biological properties such as anti-viral, antitumor, anti-convulsant, analgesic, antioxidant, anti-depressant, herbicidal, insecticidal, fungicidal, antibacterial and anti-inflammatory activities offered by it. Various antibacterial agents have been synthesized by researchers to curb bacterial diseases but due to rapid development in drug resistance, tolerance and side effects, there had always been a need for the synthesis of a new class of antibacterial agents that would exhibit improved pharmacological action. Therefore, this review mainly focuses on the various methods for the synthesis of triazine derivatives and their antibacterial activity.

scholarly journals Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takumi Kayukawa ◽  
Kenjiro Furuta ◽  
Keisuke Nagamine ◽  
Tetsuro Shinoda ◽  
Kiyoaki Yonesu ◽  
...  
Keyword(s):  
Juvenile Hormone ◽  
Cell Line ◽  
Signaling Pathway ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Ex Vivo ◽  
Agricultural Field ◽  
Chemical Library ◽  
Field Development

Abstract Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

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.

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