scholarly journals Estimating Protein−Ligand Binding Affinity Using High-Throughput Screening by NMR

2008 ◽  
Vol 10 (6) ◽  
pp. 948-958 ◽  
Author(s):  
Matthew D. Shortridge ◽  
David S. Hage ◽  
Gerard S. Harbison ◽  
Robert Powers
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

1999 ◽  
Vol 4 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Ilona Kariv ◽  
Michelle E. Stevens ◽  
Davette L. Behrens ◽  
Kevin R. Oldenburg
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transmembrane Domain ◽  
Receptor Function ◽  
Camp Production ◽  
Ligand Interaction ◽  
G Protein Coupled ◽  
Native Ligand

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

scholarly journals Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

1999 ◽  
Vol 4 (4) ◽  
pp. 193-204 ◽  
Author(s):  
Sheri Miraglia ◽  
Elana E. Swartzman ◽  
Julia Mellentin-Michelotti ◽  
Lolita Evangelista ◽  
Christopher Smith ◽  
...  
Keyword(s):  
Ligand Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Laser Scanner ◽  
Peptide Ligand ◽  
Binding Assays ◽  
Using Data ◽  
Immunological Assays ◽  
The Individual ◽  
Homogeneous Assays

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm2 digitized image of a 100-μm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twister™ (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

scholarly journals High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

2001 ◽  
Vol 6 (6) ◽  
pp. 429-440 ◽  
Author(s):  
Michael W. Pantoliano ◽  
Eugene C. Petrella ◽  
Joseph D. Kwasnoski ◽  
Victor S. Lobanov ◽  
James Myslik ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Ligand Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
General Strategy ◽  
General Applicability ◽  
High Throughput Drug Screening ◽  
Compound Libraries ◽  
Thermal Shift

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

scholarly journals An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

2017 ◽  
Vol 22 (8) ◽  
pp. 950-961 ◽  
Author(s):  
Chih Hung Lo ◽  
Nagamani Vunnam ◽  
Andrew K. Lewis ◽  
Ting-Lan Chiu ◽  
Benjamin E. Brummel ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Ligand Binding ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Tumor Necrosis ◽  
Critical Role ◽  
Receptor Activation ◽  
Computational Docking ◽  
Necrosis Factor

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor–receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

scholarly journals High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography

IUCrJ ◽  
2019 ◽  
Vol 6 (6) ◽  
pp. 1074-1085 ◽  
Author(s):  
Tadeo Moreno-Chicano ◽  
Ali Ebrahim ◽  
Danny Axford ◽  
Martin V. Appleby ◽  
John H. Beale ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Radiation Damage ◽  
High Throughput ◽  
High Throughput Screening ◽  
Room Temperature ◽  
Data Sets ◽  
Binding Studies ◽  
X Ray ◽  
Temperature Damage ◽  
Serial Femtosecond Crystallography

High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.

scholarly journals An efficient strategy to enhance binding affinity and specificity of a known isozyme inhibitor

2016 ◽  
Vol 14 (28) ◽  
pp. 6833-6839 ◽  
Author(s):  
Joo-Eun Jee ◽  
Jaehong Lim ◽  
Yong Siang Ong ◽  
Jessica Oon ◽  
Liqian Gao ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Binding Affinity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Peptide Libraries ◽  
Binding Profile ◽  
Efficient Strategy

The binding profile of a known inhibitor, benzenesulfonamide, against a family of carbonic anhydrase isozymes was efficiently enhanced via high-throughput screening of customized combinatorial one-bead-one-compound peptide libraries.

scholarly journals High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding

2010 ◽  
Vol 394 (1) ◽  
pp. 194-199 ◽  
Author(s):  
Mohd Hafeez Faridi ◽  
Dony Maiguel ◽  
Brock T. Brown ◽  
Eigo Suyama ◽  
Constantinos J. Barth ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening

High-Throughput Screening Assays for Estrogen Receptor by Using Coumestrol, a Natural Fluorescence Compound

2013 ◽  
Vol 19 (2) ◽  
pp. 253-258 ◽  
Author(s):  
Caihua Wang ◽  
Changhao Li ◽  
Haibing Zhou ◽  
Jian Huang
Keyword(s):  
Estrogen Receptor ◽  
Binding Affinity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Binding Assay ◽  
Convex Curve ◽  
Nonspecific Adsorption ◽  
Plot Analysis ◽  
Detection And Identification ◽  
Reported Data

Estrogen receptor (ER) is a ligand-inducible transcriptional factor involving in cell growth, differentiation, and diseases, so detection and identification of compounds having estrogenic effects are of great importance in the drug discovery industry. We have developed and validated a rapid, simple, and homogeneous method that can detect estrogenic compounds. This human ERα/β binding assay uses fluorescence polarization (FP) by applying an autofluorescent phytoestrogen, coumestrol (CS). A nonspecific adsorption assay shows that no obvious nonspecific adsorption is detected between CS and ERs. In the Scatchard plot analysis, the convex curve exhibits a positive cooperative binding, indicating that the binding of CS induces a conformational change of the ER to form a dimer and increases the affinity for the additional CS. In the Hill plot analysis, CS shows moderate binding affinity with both ERα and ERβ, and the measured Kd of CS is 32.66 µM and 36.14 µM, respectively, indicating that CS is applicable to the ER binding assay for determination of potent ligands of moderate binding affinity. Four typical ligands are selected to verify the ER binding assays, and the results are consistent with the reported data. All of above make the FP method based on CS suitable for high-throughput screening.

scholarly journals Supercomputer-aided Drug Repositioning at Scale: Virtual Screening for SARS-CoV-2 Protease Inhibitor

2020 ◽  
Author(s):  
Sangjae Seo ◽  
Jung Woo Park ◽  
Dosik An ◽  
Junwon Yoon ◽  
Hyojung Paik ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Inhibitor ◽  
Binding Affinity ◽  
High Throughput ◽  
Active Site ◽  
High Throughput Screening ◽  
Drug Repositioning ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Drug Candidates

Coronavirus diseases (COVID-19) outbreak has been labelled a pandemic. For the prioritization of treatments to cope with COVID-19, it is important to conduct rapid high-throughput screening of chemical compounds to repurposing the approved drugs, such as repositioning of chloroquine (Malaria drug) for COVID-19. In this study, exploiting supercomputer resource, we conducted high-throughput virtual screening for potential repositioning candidates of the protease inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the three dimensional structure of main protease (Mpro) of SARS-CoV-2, we evaluated binding affinity between Mpro and drug candidates listed in SWEETLEAD library and ChEMBL database. Docking scores of 19,168 drug molecules at the active site of Mpro were calculated using Autodock Vina package. Among the calculated result, we selected 43 drug candidates and ran molecular dynamics (MD) simulation to further investigate protein-drug interaction. Among compounds that bind to the active site of SARS-CoV-2, we finally selected the 8 drugs showing the highest binding affinity; asunaprevir, atazanavir, dasabuvir, doravirine, fosamprenavir, ritonavir, voxilaprevir and amprenavir, which are the antiviral drugs of hepatitis C virus or human immunodeficiency virus. We expect that the present study provides comprehensive insights into the development of antiviral medication, especially for the treatment of COVID-19.<div><br></div><div>* Attached excel file contains a full list of results of docking calculations</div>

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