scholarly journals Structural and Biological Characterizations of Novel High-Affinity Fluorescent Probes with Overlapped and Distinctive Binding Regions on CXCR4

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2928
Author(s):  
Siyu Zhu ◽  
Qian Meng ◽  
Robert T. Schooley ◽  
Jing An ◽  
Yan Xu ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Competitive Binding ◽  
Calcium Signal ◽  
Binding Modes ◽  
Binding Assays ◽  
High Affinity ◽  
Binding Regions ◽  
Hiv 1 ◽  
Competitive Binding Assays

CXC-type chemokine receptor 4 (CXCR4) is well known as a co-receptor for cellular entry and infection of human immunodeficiency virus type 1 (HIV-1). As an important member of the G protein-coupled receptor (GPCR) family, CXCR4 also mediates a variety of cellular processes and functions, such as cell chemotaxis, proliferation, and calcium signal transductions. Identification and characterization of molecular ligands or probes of CXCR4 have been an intensive area of investigations as such ligands or probes are of significant clinical values for the studies and treatments of HIV-1 infection and other human diseases mediated by the receptor. The crystal structures of CXCR4 in complex with different ligands have revealed two distinctive binding regions or subpockets. Thus, understanding the interactions of diverse ligands with these distinctive CXCR4 binding regions has become vital for elucidating the relationship between binding modes and biological mechanisms of ligand actions. Peptidic CVX15 is the only ligand that has been validated to bind one of these distinctive binding regions (or so called the major subpocket) of CXCR4. Therefore, in this study, we developed an efficient probe system including two high-affinity peptidic fluorescent probes, designated as FITC-CVX15 and FITC-DV1, with the aim of targeting distinctive CXCR4 subpockets. We conducted rational design and chemical characterization of the two CXCR4-specific probes and examined their application in biological experiments including competitive binding assays, flow cytometry analysis, and confocal imaging. Especially these two probes were applied in parallel CXCR4 competitive binding assays to detect and analyze potential binding modes of diverse CXCR4 ligands, together with molecular docking and simulations. Our results have indicated that these peptidic fluorescent probe systems provide novel ligand detecting tools, as well as present a new approach for analyzing distinctive binding modes of diverse CXCR4 ligands.

scholarly journals A Novel CXCR4-Selective High-Affinity Fluorescent Probe and Its Application in Competitive Binding Assays

Biochemistry ◽  
2014 ◽  
Vol 53 (30) ◽  
pp. 4881-4883 ◽  
Author(s):  
Yilei Yang ◽  
Qinghao Zhang ◽  
Mei Gao ◽  
Xiaohong Yang ◽  
Ziwei Huang ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Competitive Binding ◽  
Binding Assays ◽  
High Affinity ◽  
Competitive Binding Assays

scholarly journals Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
David Wensel ◽  
Yongnian Sun ◽  
Zhufang Li ◽  
Sharon Zhang ◽  
Caryn Picarillo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
High Affinity ◽  
Glycosylation Sites ◽  
Spectrum Activity ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Broad Spectrum Activity

ABSTRACT A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

Model for competitive binding assays. Shape and location of the inhibition curves

1974 ◽  
Vol 46 (8) ◽  
pp. 1132-1135 ◽  
Author(s):  
Donald J. Laurence ◽  
Graeme. Wilkinson
Keyword(s):  
Competitive Binding ◽  
Binding Assays ◽  
Competitive Binding Assays

scholarly journals Characterization of MK6240, a tau PET tracer, in autopsy brain tissue from Alzheimer’s disease cases

2020 ◽  
Author(s):  
Mona-Lisa Malarte ◽  
Agneta Nordberg ◽  
Laetitia Lemoine
Keyword(s):  
Brain Tissue ◽  
Binding Sites ◽  
First Generation ◽  
Competitive Binding ◽  
Binding Assays ◽  
Control Brain ◽  
Saturation Binding ◽  
Pet Tracer ◽  
Tau Pet ◽  
Competitive Binding Assays

Abstract Purpose MK6240 is a second-generation tau PET tracer designed to detect the neurofibrillary tangles in the brains of patients with Alzheimer’s disease (AD). The aim of the study was to characterize 3H-MK6240 in AD and control brain tissue and to compare its binding properties with those of first-generation tau PET tracers. Methods Saturation binding assays with 3H-MK6240 were carried out in the temporal and parietal cortices of AD brains to determine the maximum number of binding sites (Bmax) and the dissociation constants (Kd) at these sites. Competitive binding assays were carried out between 3H-MK6240 and unlabelled MK6240, AV-1451 (aka T807, flortaucipir) and THK5117, and between 3H-THK5351 and unlabelled MK6240. Regional binding studies with 3H-MK6240 were carried out in homogenates from six AD and seven control brains and, using autoradiography, on large frozen sections from two AD brains and one control brain. Results The saturation binding assays gave Bmax and Kd values of 59.2 fmol/mg and 0.32 nM in the temporal cortex and 154.7 fmol/mg and 0.15 nM in the parietal cortex. The competitive binding assays revealed two binding sites with affinities in the picomolar and nanomolar range shared by 3H-MK6240 and all the tested unlabelled compounds. There were no binding sites in common between 3H-THK5351 and unlabelled MK6240. Regional binding of 3H-MK6240 was significantly higher in AD brain tissue than in controls. Binding in brain tissue from AD patients with early-onset AD was significantly higher than in brain tissue from patients with late-onset AD. Binding of 3H-MK6240 was not observed in off-target regions. Autoradiography showed high regional cortical binding in the two AD brains and very low binding in the control brain. Conclusions 3H-MK6240 has a high binding affinity for tau deposits in AD brain tissue but also has different binding characteristics from those of the first-generation tau tracers. This confirms the complexity of tau tracer binding on tau deposits with different binding affinities for different binding sites.

scholarly journals Development of a radioligand immunoassay for 1,25-dihydroxycholecalciferol receptors utilizing monoclonal antibody

1984 ◽  
Vol 221 (1) ◽  
pp. 129-136 ◽  
Author(s):  
S Dokoh ◽  
M R Haussler ◽  
J W Pike
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
D3 Receptor ◽  
Sedimentation Analysis ◽  
Binding Assays ◽  
Standard Curve ◽  
High Affinity ◽  
Cell Extracts ◽  
Mouse Myeloma

A radioligand immunoassay for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] receptors was developed utilizing a specific, high-affinity (Kd = 1.8×10(-11 M) monoclonal antibody (9A7 gamma) obtained from suspension cultures of rat spleen X mouse myeloma hybrid SP2/0-9A7. A standard curve was established, based on the competition between 1,25(OH)2[3H]D3-receptor (18 fmol/tube) and increasing concentrations of radioinert 1,25(OH)2D3-receptor (0-240 fmol/tube) for the binding site on 9A7 gamma. Samples, prepared in identical buffer, contained 0-100 fmol of receptor/tube. After an equilibrium incubation of 1,25(OH)2[3H]D3-receptor with either standard or sample (16 h at 4 degrees C), antibody-bound receptor was immunoprecipitated with rabbit anti-(rat immunoglobulin) prelinked to Staphylococcus aureus and quantified. The assay is statistically sensitive to 2 fmol of receptor/tube, with intra- and inter-assay variations of 7 and 12% respectively. Occupied, unoccupied and denatured receptor were observed to compete equally in the assay. This quantitative technique has been successfully applied to the characterization of receptors after fractionation by sedimentation analysis and DNA-cellulose chromatography. Finally, the measurement of total receptor by this assay, in conjunction with 1,25(OH)2D3 binding assays, has revealed that rachitic, normal and 1,25(OH)2D3-injected chicks have respectively 13, 20, and 56% of receptor in the occupied form. From these results we consider that this radioligand immunoassay will be a useful tool in further research focusing on quantifying 1,25(OH)2D3 receptors in tissue and cell extracts.

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