Method for the Rapid Screening of Drug Candidates Using Single‐Protein Tracking in a Living Cell

2020 ◽  
Author(s):  
Dong‐Kyun Kim ◽  
Young Sook Kim ◽  
Chan Sik Kim ◽  
Nam Ki Lee
Keyword(s):  
Living Cell ◽  
Rapid Screening ◽  
Drug Candidates ◽  
Single Protein ◽  
Protein Tracking

scholarly journals Single Protein Tracking of P450-Reductase in an Endoplasmic Reticulum Biomimetic Reveals a NADPH Dependent Interaction with the Membrane

2017 ◽  
Vol 112 (3) ◽  
pp. 508a
Author(s):  
James A. Brozik ◽  
Carlo Barnaba ◽  
Adam O. Barden ◽  
Linda Agyen ◽  
Sean L. Sheridan
Keyword(s):  
Endoplasmic Reticulum ◽  
P450 Reductase ◽  
Single Protein ◽  
Protein Tracking ◽  
Dependent Interaction

scholarly journals Liraglutide improves hepatic steatosis and metabolic dysfunctions in a 3-week dietary mouse model of nonalcoholic steatohepatitis

2019 ◽  
Vol 317 (4) ◽  
pp. G508-G517
Author(s):  
Thibaut Duparc ◽  
François Briand ◽  
Charlotte Trenteseaux ◽  
Jules Merian ◽  
Guillaume Combes ◽  
...  
Keyword(s):  
Drinking Water ◽  
Mouse Model ◽  
Hepatic Steatosis ◽  
Cholic Acid ◽  
Nonalcoholic Steatohepatitis ◽  
Dietary Intervention ◽  
Liver Steatosis ◽  
Rapid Screening ◽  
Drug Candidates ◽  
Stress Markers

Nonalcoholic steatohepatitis (NASH) is an emerging health problem worldwide. However, efficacious pharmacological treatment for NASH is lacking. A major issue for preclinical evaluation of potential therapeutics for NASH is the limited number of appropriate animal models, i.e., models that do not require long-term dietary intervention and adequately mimic disease progression in humans. The present study aimed to evaluate a 3-wk dietary mouse model of NASH and validate it by studying the effects of liraglutide, a compound in advanced clinical development for NASH. C57BL6/J mice were fed a diet high in fat (60%), cholesterol (1.25%), and cholic acid (0.5%), along with 2% hydroxypropyl-β-cyclodextrin in drinking water (HFCC-CDX diet). Histological and biological parameters were measured at 1 and 3 wk. After 1-wk diet induction, liraglutide was administrated daily for 2 wk and then NASH-associated phenotypic aspects were evaluated in comparison with control mice. Prior to treatment with liraglutide, mice fed the HFCC-CDX diet for 1 wk developed liver steatosis and had increased levels of oxidative-stress markers and hepatic and systemic inflammation. For mice not treated with liraglutide, these aspects were even more pronounced after 3 wk of the dietary period, with additional liver insulin resistance and fibrosis. Liraglutide treatment corrected the diet-induced alterations in glucose metabolism and significantly reduced hepatic steatosis and inflammation. This study provides a novel 3-wk dietary model of mice that rapidly develop NASH features, and this model will be suitable for evaluating the therapeutic efficacy of compounds in preclinical drug development for NASH. NEW & NOTEWORTHY We propose a diet high in fat (60%), cholesterol (1.25%), and cholic acid (0.5%) along with 2% hydroxypropyl-β-cyclodextrin in drinking water (HFCC-CDX diet) as a new dietary model of nonalcoholic steatohepatitis. We used the HFCC-CDX model to reproduce the main features of disease development in humans for the purpose of facilitating the rapid screening of drug candidates and prioritizing the more promising candidates for advanced preclinical assessment and subsequent clinical trials.

scholarly journals A Novel High-Throughput Screening Assay for Sickle Cell Disease Drug Discovery

2009 ◽  
Vol 14 (4) ◽  
pp. 330-336 ◽  
Author(s):  
Eszter Pais ◽  
John S. Cambridge ◽  
Cage S. Johnson ◽  
Herbert J. Meiselman ◽  
Timothy C. Fisher ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rapid Screening ◽  
Cell Disease ◽  
Screening Assay ◽  
Drug Candidates ◽  
Test Molecule ◽  
High Throughput Screening Assay

Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This approach has been adapted to wells in a 384-well microplate. Results can be obtained by optical scanning: The size of the red dot is proportional to the antisickling effect of the test molecule. The new assay is simple, inexpensive, reproducible, requires no special reagents, and should be readily adaptable to robotic HTS systems. It has the potential to identify novel drug candidates, allowing the development of new therapeutic options for individuals affected with SCD. ( Journal of Biomolecular Screening. 2009:330-336)

scholarly journals Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells

2021 ◽  
Vol 22 (9) ◽  
pp. 4747
Author(s):  
Jeahee Ryu ◽  
Euiyeon Lee ◽  
Chungwon Kang ◽  
Minhyeong Lee ◽  
Soyoun Kim ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Glucocorticoid Receptor ◽  
Inflammatory Responses ◽  
Rapid Screening ◽  
Peppermint Oil ◽  
Cellular Regulation ◽  
Drug Candidates ◽  
Split Intein ◽  
Proinflammatory Responses ◽  
Anti Inflammatory

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

Using Single-Protein Tracking to Study Cell Migration

2018 ◽  
pp. 291-311 ◽  
Author(s):  
Thomas Orré ◽  
Amine Mehidi ◽  
Sophie Massou ◽  
Olivier Rossier ◽  
Grégory Giannone
Keyword(s):  
Cell Migration ◽  
Single Protein ◽  
Protein Tracking

scholarly journals Fast activation cycles of Rac1 at the lamellipodium tip trigger membrane protrusion

2017 ◽  
Author(s):  
Amine Mehidi ◽  
Olivier Rossier ◽  
Anaël Chazeau ◽  
Fabien Binamé ◽  
Amanda Remorino ◽  
...  
Keyword(s):  
Cell Migration ◽  
Rho Gtpases ◽  
Guanine Nucleotide ◽  
Membrane Protrusion ◽  
Cell Edge ◽  
Rhoa Activation ◽  
Cell Protrusion ◽  
Single Protein ◽  
Protein Tracking ◽  
Fast Control

AbstractThe spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE complex, effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Yet, correlation of Rho GTPases activation with cycles of membrane protrusions, suggested that Rac1 activation is not synchronized with membrane protrusion and occurs behind the lamellipodium. However, RhoA activation is maximal at the cell edge and synchronized with edge progression. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function of Rho GTPases mutants, we demonstrate that Rac1 immobilizations at the lamellipodium tip are correlated with Rac1 activation, on the contrary to RhoA. We show that Rac1 effector WAVE and Rac1 regulator IRSp53 accumulate at the lamellipodium tip by membrane free-diffusion and trapping. Nevertheless, wild-type Rac1, which directly interacts with WAVE and IRSp53, only displays slower diffusion at the lamellipodium tip, suggesting fast local activation/inactivation cycles. Local optogenetic activation of Rac1, triggered by Tiam1 membrane recruitment, proves that Rac1 activation must occur at the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. Furthermore, coupling tracking with optogenetic activation of Rac1 demonstrates that Rac1-WT diffusive properties are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model where Rac1 is rapidly switching between activation and inhibition at the lamellipodium tip, ensuring a local and fast control of Rac1 actions on its targets.SignificanceRac1 and RhoA GTPases are molecular switches controlling the actin cytoskeletal during cell migration. WAVE, Rac1 effector during cell protrusion, is concentrated at the lamellipodium tip. But, recent biosensor imaging studies suggested that Rac1 activation occurs behind the lamellipodium, while RhoA activation is maximal at the cell edge. Using single-molecule imaging and optogentics Rac1 activation we solved this apparent contradiction. We revealed a strong correlation between Rac1 activation and transient immobilizations at the lamellipodium tip, unlike RhoA. Furthermore, we demonstrated that Rac1 must be activated at the lamellipodium tip and not away from it to stimulate protrusion. Thus, fast cycling between activation and inhibition at the proximity of Rac1 targets ensures a local and fast control over Rac1 actions.AbbreviationsArp2/3actin related proteins 2/3Ddiffusion coefficientF-actinactin filamentsFMNL2formin-like protein-2FNfibronectinGAPGTPase-activating proteinGDIGuanine-nucleotide Dissociation InhibitorGEFGuanine-nucleotide Exchange FactorIRSp53insulin receptor tyrosine kinase substrate p53LMlamellipodiumNPFnucleation promoting factorMSDmean squared displacementPALMphotoactivation localization microscopyPSDpost synaptic densityrconfconfinement radiussptsingle protein trackingVASPvasodilator-stimulated phosphoproteinWAVEWASP-family verprolin homologue

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