scholarly journals A molecular toolbox for ADP-ribosyl binding proteins

2021 ◽  
Author(s):  
Sven T Sowa ◽  
Albert Galera-Prat ◽  
Mirko M Maksimainen ◽  
Heli I Alanen ◽  
Sarah Wazir ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Viral Infections ◽  
Binding Assay ◽  
Fluorescent Proteins ◽  
Protein Fusion ◽  
E Coli ◽  
Wide Range ◽  
Gi Protein ◽  
Protein Alpha Subunit

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or in viral infections, which makes them attractive targets for the development of inhibitors. Our goal was to develop a robust and accessible assay technology that is suitable for high-throughput screening and applicable to a wide range of proteins acting as either hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. As a foundation of our work, we developed a C-terminal protein fusion tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups as well as chemical ADP-ribosyl analogs. By fusion of the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we were able to generate robust FRET signals and the interaction with 22 previously described ADP-ribosyl-binders was confirmed. To demonstrate the applicability of this binding assay for high-throughput screening, we utilized it to screen for inhibitors of the SARS-CoV-2 nsp3 macrodomain and identified the drug suramin as a moderate yet unspecific inhibitor of this protein. To complement the binding technology, we prepared high-affinity ADP-ribosyl binders fused to a nanoluciferase, which enabled simple blot-based detection of mono- and poly-ADP-ribosylated proteins. These tools can be expressed recombinantly in E. coli using commonly available agents and will help to investigate ADP-ribosylation systems and aid in drug discovery.

scholarly journals High Throughput Screening of FDA-Approved Drug Library Reveals the Compounds that Promote IRF3-Mediated Pro-Apoptotic Pathway Inhibit Virus Replication

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 442 ◽  
Author(s):  
Anna Glanz ◽  
Karan Chawla ◽  
Stephanie Fabry ◽  
Gayatri Subramanian ◽  
Julie Garcia ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transcriptional Activity ◽  
Viral Infections ◽  
Antiviral Effect ◽  
Apoptotic Pathway ◽  
Antiviral Genes ◽  
Wide Range ◽  
Infected Cells

Interferon (IFN) regulatory factor 3 (IRF3) is the key transcription factor for the induction of IFN and antiviral genes. The absence of antiviral genes in IRF3 deficiency leads to susceptibility to a wide range of viral infections. Previously, we uncovered a function for nontranscriptional IRF3 (nt-IRF3), RLR (RIG-I-like receptor)-induced IRF3-mediated pathway of apoptosis (RIPA), which triggers apoptotic killing of virus-infected cells. Using knock-in mice expressing a transcriptionally inactive, but RIPA-active, IRF3 mutant, we demonstrated the relative contribution of RIPA to host antiviral defense. Given that RIPA is a cellular antiviral pathway, we hypothesized that small molecules that promote RIPA in virus-infected cells would act as antiviral agents. To test this, we conducted a high throughput screen of a library of FDA-approved drugs to identify novel RIPA activators. Our screen identified doxorubicin as a potent RIPA-activating agent. In support of our hypothesis, doxorubicin inhibited the replication of vesicular stomatitis virus, a model rhabdovirus, and its antiviral activity depended on its ability to activate IRF3 in RIPA. Surprisingly, doxorubicin inhibited the transcriptional activity of IRF3. The antiviral activity of doxorubicin was expanded to flavivirus and herpesvirus that also activate IRF3. Mechanistically, doxorubicin promoted RIPA by activating the extracellular signal-regulated kinase (ERK) signaling pathway. Finally, we validated these results using another RIPA-activating compound, pyrvinium pamoate, which showed a similar antiviral effect without affecting the transcriptional activity of IRF3. Therefore, we demonstrate that the RIPA branch of IRF3 can be targeted therapeutically to prevent virus infection.

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 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.

scholarly journals Directed Evolution of Aptamer Discovery Technologies

2021 ◽  
Author(s):  
Diana Wu ◽  
Chelsea Gordon ◽  
John Shin ◽  
Michael Eisenstein ◽  
Hyongsok Tom Soh
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Click Reaction ◽  
Clinical Diagnostics ◽  
High Quality ◽  
Affinity Reagents ◽  
Wide Range ◽  
Target Binding ◽  
Modified Nucleobases ◽  
Parallel Fashion

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

Rapid generation of Hybrid Biochemical/Mechanical Cues in Heterogeneous Droplets for High-Throughput Screening of Cellular Response

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Xing Zhao ◽  
Gaozhi Ou ◽  
Mengcheng Lei ◽  
Yang Zhang ◽  
Lina Li ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cellular Response ◽  
Wide Range ◽  
Rapid Generation ◽  
P Cells

Cells in native microenvironment are subjected to varying combinations of biochemical cues and mechanical cues in a wide range. Despite many signaling pathways have been found to be responsive for...

SoftBV – a software tool for screening the materials genome of inorganic fast ion conductors

2019 ◽  
Vol 75 (1) ◽  
pp. 18-33 ◽  
Author(s):  
Haomin Chen ◽  
Lee Loong Wong ◽  
Stefan Adams
Keyword(s):  
Force Field ◽  
High Throughput ◽  
High Throughput Screening ◽  
Density Functional ◽  
Inorganic Compounds ◽  
Computational Cost ◽  
Lewis Base ◽  
Computational Screening ◽  
Wide Range ◽  
Dft Simulations

The identification of materials for advanced energy-storage systems is still mostly based on experimental trial and error. Increasingly, computational tools are sought to accelerate materials discovery by computational predictions. Here are introduced a set of computationally inexpensive software tools that exploit the bond-valence-based empirical force field previously developed by the authors to enable high-throughput computational screening of experimental or simulated crystal-structure models of battery materials predicting a variety of properties of technological relevance, including a structure plausibility check, surface energies, an inventory of equilibrium and interstitial sites, the topology of ion-migration paths in between those sites, the respective migration barriers and the site-specific attempt frequencies. All of these can be predicted from CIF files of structure models at a minute fraction of the computational cost of density functional theory (DFT) simulations, and with the added advantage that all the relevant pathway segments are analysed instead of arbitrarily predetermined paths. The capabilities and limitations of the approach are evaluated for a wide range of ion-conducting solids. An integrated simple kinetic Monte Carlo simulation provides rough (but less reliable) predictions of the absolute conductivity at a given temperature. The automated adaptation of the force field to the composition and charge distribution in the simulated material allows for a high transferability of the force field within a wide range of Lewis acid–Lewis base-type ionic inorganic compounds as necessary for high-throughput screening. While the transferability and precision will not reach the same levels as in DFT simulations, the fact that the computational cost is several orders of magnitude lower allows the application of the approach not only to pre-screen databases of simple structure prototypes but also to structure models of complex disordered or amorphous phases, and provides a path to expand the analysis to charge transfer across interfaces that would be difficult to cover by ab initio methods.

scholarly journals A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

2010 ◽  
Vol 15 (9) ◽  
pp. 1088-1098 ◽  
Author(s):  
Adam Shapiro ◽  
Haris Jahic ◽  
Swati Prasad ◽  
David Ehmann ◽  
Jason Thresher ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
New Drugs ◽  
E Coli ◽  
Cellular Processes ◽  
The Difference ◽  
Cellular Replication

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

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 Screening of Metal–organic Frameworks for Macroscale Heteroepitaxial Alignment

2018 ◽  
Author(s):  
Andrew Tarzia ◽  
Masahide Takahashi ◽  
Paolo Falcaro ◽  
Aaron Thornton ◽  
Christian Doonan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Defect Formation ◽  
Metal Organic Frameworks ◽  
Rectangular Lattice ◽  
Copper Hydroxide ◽  
Computational Screening ◽  
Screening Algorithm ◽  
Wide Range ◽  
Metal Organic

The ability to align porous metal–organic frameworks (MOFs) on substrate surfaces on a macroscopic scale is a vital step towards integrating MOFs into functional devices. But macroscale surface alignment of MOF crystals has only been demonstrated in a few cases. To accelerate the materials discovery process, we have developed a high-throughput computational screening algorithm to identify MOFs that are likely to undergo macroscale aligned heterepitaxial growth on a substrate. Screening of thousands of MOF structures by this process can be achieved in a few days on a desktop workstation. The algorithm filters MOFs based on surface chemical compatibility, lattice matching with the substrate, and interfacial bonding. Our method uses a simple new computationally efficient measure of the interfacial energy that considers both bond and defect formation at the interface. Furthermore, we show that this novel descriptor is a better predictor of aligned heteroepitaxial growth than other established interface descriptors, by testing our screening algorithm on a sample set of copper MOFs that have been grown heteroepitaxially on a copper hydroxide surface. Application of the screening process to several MOF databases reveals that the top candidates for aligned growth on copper hydroxide comprise mostly MOFs with rectangular lattice symmetry in the plane of the substrate. This result indicates a substrate-directing effect that could be exploited in targeted synthetic strategies. We also identify that MOFs likely to form aligned heterostructures have broad distributions of in-plane pore sizes and anisotropies. Accordingly, this suggests that aligned MOF thin films with a wide range of properties may be experimentally accessible.

scholarly journals High-Throughput Screening of Metal–organic Frameworks for Macroscale Heteroepitaxial Alignment

2018 ◽  
Author(s):  
Andrew Tarzia ◽  
Masahide Takahashi ◽  
Paolo Falcaro ◽  
Aaron Thornton ◽  
Christian Doonan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Defect Formation ◽  
Metal Organic Frameworks ◽  
Rectangular Lattice ◽  
Copper Hydroxide ◽  
Computational Screening ◽  
Screening Algorithm ◽  
Wide Range ◽  
Metal Organic

The ability to align porous metal–organic frameworks (MOFs) on substrate surfaces on a macroscopic scale is a vital step towards integrating MOFs into functional devices. But macroscale surface alignment of MOF crystals has only been demonstrated in a few cases. To accelerate the materials discovery process, we have developed a high-throughput computational screening algorithm to identify MOFs that are likely to undergo macroscale aligned heterepitaxial growth on a substrate. Screening of thousands of MOF structures by this process can be achieved in a few days on a desktop workstation. The algorithm filters MOFs based on surface chemical compatibility, lattice matching with the substrate, and interfacial bonding. Our method uses a simple new computationally efficient measure of the interfacial energy that considers both bond and defect formation at the interface. Furthermore, we show that this novel descriptor is a better predictor of aligned heteroepitaxial growth than other established interface descriptors, by testing our screening algorithm on a sample set of copper MOFs that have been grown heteroepitaxially on a copper hydroxide surface. Application of the screening process to several MOF databases reveals that the top candidates for aligned growth on copper hydroxide comprise mostly MOFs with rectangular lattice symmetry in the plane of the substrate. This result indicates a substrate-directing effect that could be exploited in targeted synthetic strategies. We also identify that MOFs likely to form aligned heterostructures have broad distributions of in-plane pore sizes and anisotropies. Accordingly, this suggests that aligned MOF thin films with a wide range of properties may be experimentally accessible.

Sign in / Sign up

Export Citation Format

Share Document