SPR-Based Direct Binding Assays in Drug Discovery

2011 ◽  
pp. 269-282 ◽  
Author(s):  
Walter Huber
Keyword(s):  
Drug Discovery ◽  
Binding Assays ◽  
Direct Binding

Interaction of Mycoplasma gallisepticum with sialyl glycoproteins

1980 ◽  
Vol 30 (2) ◽  
pp. 353-361
Author(s):  
L R Glasgow ◽  
R L Hill
Keyword(s):  
Sialic Acid ◽  
Mycoplasma Gallisepticum ◽  
Binding Assays ◽  
Molecular Weights ◽  
Acetylneuraminic Acid ◽  
Direct Binding ◽  
Membrane Bound ◽  
Strict Specificity ◽  
Human Glycophorin ◽  
Acid Structure

The binding of several glycoproteins to freshly grown and harvested cells of Mycoplasma gallisepticum was examined. Only human glycophorin, the major sialoglycoprotein of the erythrocyte membrane, bound tightly as judged by direct binding assays with 125I-labeled glycoproteins. Neuraminidase-treated glycophorin did not bind, suggesting that binding is mediated through sialic acid groups. Although other sialoglycoproteins did not appear to bind M. gallisepticum by direct binding assays, some inhibited the binding of glycophorin. The best inhibitors had a mucin-like structure, with high molecular weights and high sialic acid contents. N-acetylneuraminic acid appeared to be the favored sialic acid structure for binding, but there was no strict specificity for its anomeric linkage. Neuraminidase activity could not be detected on the surface of M. gallisepticum, suggesting that this enzyme is not involved in the mechanism of adherence of sialoglycoproteins. Binding of sialoglycoproteins was time dependent, however, and markedly diminished with increasing ionic strength, but was largely unaffected between pH 4 and 9.

Abstract 15297: Discovery and Preclinical Optimization of Selective ABCA1 Inducers as Multifunctional Therapeutic Candidates

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Cutler Lewandowski ◽  
Md. Wasim Khan ◽  
Manel Ben Aissa ◽  
Brian Layden ◽  
Gregory Thatcher
Keyword(s):  
Liver Steatosis ◽  
Acid Synthesis ◽  
Luciferase Assay ◽  
Pharmacokinetic Parameters ◽  
Lead Compound ◽  
Binding Assays ◽  
Drug Candidates ◽  
Direct Binding ◽  
Isoform Selectivity

Introduction: Reduced expression of cholesterol transporter ABCA1 is critical in pathogenesis of type 2 diabetes (T2D) and related conditions, such as cardiovascular disease (CVD) and Alzheimer’s disease (AD). Thus, increasing ABCA1 represents a novel therapeutic strategy for these conditions. However, prior drug development efforts have achieved limited success at increasing ABCA1 (controlled by liver X receptor [LXR] β) while avoiding unwanted liver triglyceride production (through LXRα via transcription factor SREBP1c). Hypothesis: We hypothesized that phenotypic screening for selective ABCA1 inducers followed by medicinal chemistry optimization would bypass the isoform selectivity issues encountered in traditional target-based drug discovery and enable development of lead therapeutic candidates with preclinical efficacy and safety. Methods/Results: We screened 20k compounds for ABCA1 and SREBP1c-linked luciferase activity, followed by qPCR to validate and prioritize selective ABCA1-inducing hits. We synthesized ~70 structural analogs of the best hit, achieving substantial EC 50 (4.5 μM to 270 nM) and E max (3.5-fold to 6.0-fold vs. vehicle) improvements in ABCA1 luciferase assay while maintaining selectivity against SREBP1c. Direct binding assays confirmed selectivity for LXRβ vs. LXRα, corroborating cell-based data. Lead compounds enhanced cellular cholesterol efflux, reduced inflammation in vitro , and attenuated high-fat diet (HFD) induced weight gain, insulin resistance, and inflammation in mice. Metabolomics analysis revealed that our lead compound corrected HFD-induced perturbations in liver glucose and fatty acid synthesis. Finally, side effects associated with published LXR agonists - liver steatosis and neutropenia - were not observed with our compound. Conclusions: We established a platform to develop selective ABCA1 inducers as drug candidates. Via this platform, we identified a safe and efficacious lead compound for T2D. Our study also represents the first report of an LXR agonist characterized by metabolomics - a powerful tool to complement biochemical readouts. Continued optimization to improve pharmacokinetic parameters, plus evaluation in CVD and AD models, is ongoing.

scholarly journals Fibrinogen Hershey IV: A novel dysfibrinogen with a γV411I mutation in the integrin αIIbβ3 binding site

2008 ◽  
Vol 99 (06) ◽  
pp. 1008-1012 ◽  
Author(s):  
Veronica Flood ◽  
Hamid Al-Mondhiry ◽  
Chantelle Rein ◽  
Kristine Alexander ◽  
Rehana Lovely ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Biological Effects ◽  
Terminal Segment ◽  
Wild Type ◽  
Binding Assays ◽  
Normal Platelet ◽  
Platelet Receptor ◽  
Direct Binding ◽  
Aggregation Inhibition ◽  
Carboxyl Terminal

SummaryThe carboxyl terminal segment of the fibrinogen γ chain from γ408–4ll plays a crucial role in platelet aggregation via interactions with the platelet receptor αIIbβ3. We describe here the first naturally-occurring fibrinogen point mutation affecting this region and demonstrate its effects on platelet interactions. DNA sequencing was used to sequence the proband DNA, and platelet aggregation and direct binding assays were used to quantitate the biological effects of fibrinogen Hershey IV. The Hershey IV proband was found to be heterozygous for two mutations, γV411I and γR275C. Little difference in aggregation was seen when fibrinogen Hershey IV was compared to normal fibrinogen. However, less aggregation inhibition was observed using a competing synthetic dodecapeptide containing the V411I mutation as compared to the wild-type dodecapeptide. Purified fibrinogen Hershey IV also bound to purified platelet αIIbβ3 with a lower affinity than wild-type fibrinogen. These findings show that the γV411I mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available wild-type fibrinogen appears to be sufficient to support normal platelet aggregation.

Novel interaction of selenium-binding protein with glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase of Arabidopsis thaliana

2006 ◽  
Vol 33 (9) ◽  
pp. 847 ◽  
Author(s):  
Adamantia Agalou ◽  
Herman P. Spaink ◽  
Andreas Roussis
Keyword(s):  
Arabidopsis Thaliana ◽  
Metabolic Regulation ◽  
Binding Protein ◽  
Binding Assays ◽  
Fructose Bisphosphate ◽  
Metabolic Role ◽  
Fructose Bisphosphate Aldolase ◽  
Direct Binding ◽  
High Degree

The metabolic role and regulation of selenium, particularly in plants, is poorly understood. One of the proteins probably involved in the metabolic regulation of this element is the selenium-binding protein (SBP) with homologues present across prokaryotic and eukaryotic species. The high degree of conservation of SBP in different organisms suggests that this protein may play a role in fundamental biological processes. In order to gain insight into the biochemical function of SBP in plants we used the yeast two-hybrid system to identify proteins that potentially interact with an Arabidopsis thaliana (L.) Heynh. homologue. Among the putative binding partners of SBP, a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a fructose-bisphosphate aldolase (FBA) were found as reliable positive candidates. The interaction of these proteins with SBP was confirmed by in vitro binding assays. Previous findings in Escherichia coli, demonstrated the direct binding of selenium to both GAPDH and aldolase. Therefore our results reveal the interaction, at least in pairs, of three proteins that are possibly linked to selenium and suggest the existence of a protein network consisting of at least SBP, GAPDH and FBA, triggered by or regulating selenium metabolism in plant cells.

scholarly journals Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues

1989 ◽  
Vol 258 (1) ◽  
pp. 109-113 ◽  
Author(s):  
R W Loveless ◽  
T Feizi ◽  
R A Childs ◽  
T Mizuochi ◽  
M S Stoll ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Human Milk ◽  
Bovine Serum ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Human Milk Oligosaccharides ◽  
Binding Assays ◽  
Carbohydrate Recognition ◽  
Milk Oligosaccharides ◽  
Direct Binding

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.

scholarly journals Combining Label‐free and Flourometric Functional Assays with Biophysical Binding Assays in Drug Discovery: A Comparison of Positives from an HTS Campaign

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
Matthew Wayne Olson ◽  
Jukka Kervinen ◽  
Carsten Schubert ◽  
Jennifer Kirkpatrick ◽  
James Kranz ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Label Free ◽  
Binding Assays ◽  
Functional Assays

pH-responsive stabilization of glutamate dehydrogenase mRNA in LLC-PK1-F+cells

2003 ◽  
Vol 285 (2) ◽  
pp. F258-F265 ◽  
Author(s):  
Jill M. Schroeder ◽  
Wenlin Liu ◽  
Norman P. Curthoys
Keyword(s):  
Glutamate Dehydrogenase ◽  
Direct Repeat ◽  
Ph Responsive ◽  
Binding Assays ◽  
Chronic Metabolic Acidosis ◽  
Ph Response ◽  
High Affinity ◽  
Base Sequences ◽  
F Cells ◽  
Direct Binding

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3′-untranslated region (3′-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds ζ-crystallin/NADPH:quinone reductase (ζ-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that ζ-crystallin also binds to the full-length 3′-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind ζ-crystallin with different affinities. Insertion of the 3′-UTR of GDH cDNA into a β-globin expression vector (pβG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a βG construct that contained only the single GDH4 ARE and a destabilizing element from phospho enolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.

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