scholarly journals Identification of a Small GTPase Inhibitor Using a High-Throughput Flow Cytometry Bead-Based Multiplex Assay

2009 ◽  
Vol 15 (1) ◽  
pp. 10-20 ◽  
Author(s):  
Zurab Surviladze ◽  
Anna Waller ◽  
Yang Wu ◽  
Elsa Romero ◽  
Bruce S. Edwards ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Small Molecule ◽  
High Throughput ◽  
False Negative ◽  
Small Gtpases ◽  
Multiplex Assay ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Gtp Binding

Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small-molecule inhibitors. The authors describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different glutathione-S-transferase (GST)—tagged small GTPases were bound to glutathione beads, each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent—guanosine triphosphate (GTP) binding was used as the readout. This novel multiplex assay allowed the authors to screen a library of almost 200,000 compounds and identify more than 1200 positive compounds, which were further verified by dose-response analyses, using 6- to 8-plex assays. After the elimination of false-positive and false-negative compounds, several small-molecule families with opposing effects on GTP binding activity were identified. The authors detail the characterization of MLS000532223, a general inhibitor that prevents GTP binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live-cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high-throughput screening via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.

scholarly journals Identification and characterization of a new isoform of small GTPase RhoE

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Yuan Dai ◽  
Weijia Luo ◽  
Xiaojing Yue ◽  
Wencai Ma ◽  
Jing Wang ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Biological Functions ◽  
Coding Region ◽  
Alternative Translation ◽  
Rho Family ◽  
Binding Capability ◽  
Identification And Characterization

Abstract The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases.

Identification of Small-Molecule Inducers of FOXP3 in Human T Cells Using High-Throughput Flow Cytometry

2017 ◽  
pp. 243-252 ◽  
Author(s):  
Rob Jepras ◽  
Poonam Shah ◽  
Metul Patel ◽  
Steve Ludbrook ◽  
Gregory Wands ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Small Molecule ◽  
High Throughput ◽  
Human T Cells

scholarly journals Identification and Characterization of a Small-Molecule Rabies Virus Entry Inhibitor

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Venice Du Pont ◽  
Christoph Wirblich ◽  
Jeong-Joong Yoon ◽  
Robert M. Cox ◽  
Matthias J. Schnell ◽  
...  
Keyword(s):  
G Protein ◽  
Small Molecule ◽  
High Throughput ◽  
Viral Entry ◽  
Cost Effective ◽  
Molecular Probe ◽  
Entry Inhibitor ◽  
Protein Fusion ◽  
Life Saving

ABSTRACT Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry. IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.

scholarly journals Isolation and Characterization of Effector-Loop Mutants ofCDC42in Yeast

2001 ◽  
Vol 12 (5) ◽  
pp. 1239-1255 ◽  
Author(s):  
Amy S. Gladfelter ◽  
John J. Moskow ◽  
Trevin R. Zyla ◽  
Daniel J. Lew
Keyword(s):  
Cell Polarity ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Cytoskeletal Organization ◽  
Cell Behaviors ◽  
Isolation And Characterization ◽  
Starting Point ◽  
Effector Pathways

The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of “effector-loop” mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants ofSaccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

A high-throughput flow cytometry-on-a-CMOS platform for single-cell dielectric spectroscopy at microwave frequencies

Lab on a Chip ◽  
2018 ◽  
Vol 18 (14) ◽  
pp. 2065-2076 ◽  
Author(s):  
Jun-Chau Chien ◽  
Ali Ameri ◽  
Erh-Chia Yeh ◽  
Alison N. Killilea ◽  
Mekhail Anwar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dielectric Properties ◽  
Single Cell ◽  
High Throughput ◽  
Dielectric Spectroscopy ◽  
Free Flow ◽  
Label Free ◽  
Microwave Frequencies

This work presents a microfluidics-integrated label-free flow cytometry-on-a-CMOS platform for the characterization of the cytoplasm dielectric properties at microwave frequencies.

scholarly journals An acoustic platform for single-cell, high-throughput measurements of the viscoelastic properties of cells

2020 ◽  
Author(s):  
Valentin Romanov ◽  
Giulia Silvani ◽  
Huiyu Zhu ◽  
Charles D Cox ◽  
Boris Martinac
Keyword(s):  
Mechanical Properties ◽  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Adherent Cells ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Membrane Cytoskeleton ◽  
Change Temperature

ABSTRACTCellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 °C and 37 °C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

scholarly journals Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo

2002 ◽  
Vol 367 (3) ◽  
pp. 617-628 ◽  
Author(s):  
Maria L. HENRIKSSON ◽  
Charlotta SUNDIN ◽  
Anna L. JANSSON ◽  
Åke FORSBERG ◽  
Ruth H. PALMER ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Small Gtpase ◽  
Eukaryotic Cells ◽  
Gtpase Activating Protein ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Intracellular Targeting ◽  
Gtp Binding ◽  
Adp Ribosyltransferase

Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.

scholarly journals High-Throughput Fluorescence Assay for Small-Molecule Inhibitors of Autophagins/Atg4

2011 ◽  
Vol 16 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Chih-Wen Shu ◽  
Charitha Madiraju ◽  
Dayong Zhai ◽  
Kate Welsh ◽  
Paul Diaz ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Caspase 3 ◽  
Bioactive Molecules ◽  
Dependent Manner ◽  
Chemical Libraries ◽  
Evolutionarily Conserved ◽  
Identification And Characterization

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA2) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z′ factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules ( n = 1280 for Lopac™ and 2000 for Spectrum™ library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA2 and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA2 reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.

scholarly journals Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1296
Author(s):  
Jonathan Burnie ◽  
Vera A. Tang ◽  
Joshua A. Welsh ◽  
Arvin T. Persaud ◽  
Laxshaginee Thaya ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Single Cells ◽  
Surface Current ◽  
High Sensitivity ◽  
Viral Envelope ◽  
High Throughput Analysis ◽  
Associated Proteins ◽  
Hiv 1

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

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