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Development of a Rapid Fluorescence-Based High-Throughput ...- a detergent-based assay for the detection of promiscuous inhibitors blood ,Feb 08, 2018·The assay was shown to be linear with respect to time up to 50 min in the fluorescence-based assay system and also in the UHPLC assay ... to equally favor detection of inhibitors of all classes, ... Effect of Detergent on “Promiscuous” Inhibitors. J. Med. Chem.A detergent-based assay for the detection of promiscuous ...Jan 01, 2006·Here we describe a protocol for identifying aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific mechanisms. As a convenient proxy for promiscuous, aggregate-based inhibition, we monitor inhibition of beta-lactamase in the absence and presence of detergent.



Cell viability assays: Alternatives to the MTT assay

May 01, 2017·ATPases then deplete any remaining cytoplasmic ATP, meaning that any ATP leftover will come from metabolically active cells. The detection reagent used in this assay contains detergent to lyse cells, ATPase inhibitors, a substrate (luciferin) and firefly luciferase, which catalyzes the production of light photons from luciferin and ATP.

Gallotannins are non‐specific inhibitors of α‐amylase ...

Sep 05, 2020·For classifying a compound as a promiscuous aggregation-based inhibitor, the following results should be obtained: (a) decreased inhibition in the presence of a detergent (Feng & Shoichet, 2006; Ryan, Gray, Lowe, & Chung, 2003), (b) inhibition of at least three unrelated enzymes, (c) decreased inhibition with a multiple-fold increase in enzyme ...

Structure of Mpro from SARS-CoV-2 and discovery of its ...

Apr 09, 2020·Using a previously described detergent-based assay 25, we found that TDZD-8 is an aggregate-based inhibitor that might nonspecifically inhibit M pro (Extended Data Fig. 5); it …

The Effect of Promiscuous Aggregation on in Vitro Drug ...

Oct 25, 2019·In a high-throughput screen of a large compound library, 95% of the molecules found to act as promiscuous inhibitors were classified as aggregate-based inhibitors . The phenomenon of aggregation-based inhibition has been widely reported to lead to false positives during enzyme-based high-throughput screens that are used for drug discovery ( 6 ...

DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive ...

Schematic representation of enzyme detection using DIANA. (A) A covalent conjugate comprising an oligonucleotide (reporter DNA) and small-molecule competitive inhibitor of the target enzyme is used as a detection probe.The probe binds to the active site of the enzyme, which has been captured on the solid support by binding to the immobilized antibody.

Assessing Compound Self-aggregation in Drug Discovery and ...

1.2 Detection methods of compound aggregation Several computational methods and biophysical tests are currently used to detect small molecule aggregates in aqueous solution. One of the most common methods is enzyme-based inhibition assays such as lactate dehydrogenase, β-lactamase and chymotrypsin (Chan et al., 2009) that are

Efficient Elimination of Nonstoichiometric Enzyme ...

May 21, 2009·In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits.

Discovery of Pyrrolidine-2,3-diones as Novel Inhibitors of ...

To discover novel, non-β-lactam inhibitors against PBP3 of Pseudomonas aeruginosa, we optimised a fluorescence assay based on a well-known thioester artificial substrate and performed a target screening using a focused protease-targeted library of 2455 compounds, which led to the identification of pyrrolidine-2,3-dione as a potential scaffold ...

Filtering promiscuous compounds in early drug discovery ...

Jun 01, 2016·A detergent-based assay was described, using a nonionic detergent, for the detection of aggregate-based inhibitors. Compounds that showed attenuated inhibition in the presence of detergent are potential aggregators. This assay can distinguish aggregate-type effect from small molecules that mediate its effects via specific mechanisms.

High-Throughput Identification of Promiscuous Inhibitors ...

The MSTI assay enables the detection of thiol-reactive compounds in a multiwell plate format in contrast to many reported fluorescent sulfur-based probes that have been developed for the detection of thiol-disulfide equilibria in biological samples. 15 –17 To our knowledge, the MSTI assay enables researchers to detect thiol-reactive compounds ...

HDAC2 Fluorogenic Assay Kit - BPS Bioscience

The Fluorogenic HDAC2 Assay Kit is a complete assay system designed to measure histone deacetylase 2 (HDAC2) activity for screening and profiling applications. It comes in a convenient 96-well format, with all the reagents necessary for 100 fluorescent HDAC2 activity measurements. In addition, the kit includes purified HDAC2 enzyme and a potent ...

Identifying Promiscuous Inhibitors with Wyatt's DynaPro

Jul 11, 2017·Learn how use of Wyatt's DynaPro Plate Reader is being used by Brian Shoichet to detect aggregation in drug discovery

A Detergent-Based Assay for the Detection of Promiscuous ...

At micromolar concentrations, many small molecules self-associate into colloidal aggregates that non-specifically inhibit enzymes and other proteins. Here we describe a protocol for identifying aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific ...

The Ecstasy and Agony of Assay Interference Compounds ...

A detergent-based assay for the detection of promiscuous inhibitors Nat. Protoc. 2006, 1, 550 – 553 DOI: 10.1038/nprot.2006.77 [ Crossref ], [ PubMed ], [ CAS ], Google Scholar 37

High-Throughput Screening for Novel Inhibitors of ...

Sep 25, 2012·Feng BY, Shoichet BK (2006) A detergent-based assay for the detection of promiscuous inhibitors. Nat Protoc 1: 550–553. View Article Google Scholar 36. Shoichet BK (2006) Screening in a spirit haunted world. Drug Discov Today 11: 607–615. View Article Google Scholar 37.

The food colorant erythrosine is a promiscuous protein ...

Mar 15, 2011·To investigate the possibility of aggregation effects, we repeated the CD40–CD154 binding assays in the presence of a non-ionic detergent (Triton X-100, 0.05% and 0.5%) as recommended for the detection of promiscuous inhibitors , but observed no significant effect on IC 50 s for 1 (data not shown).

Fragment-based drug design (FBDD) | Journal of Organic and ...

Mar 23, 2017·Fragment-based drug design (FBDD) is one of the modern techniques used for developing new drugs, and an alternative to the widely used high throughput screening. The main methodological approaches of FBDD, as well as the methods of optimization for the identified “fragments“ when transferring them to the drug-like molecules have been described.

Identification of an antibody‐based immunoassay for ...

Dec 06, 2017·Using the TEAR1 assay, we have validated the direct binding of specific RIPK1 inhibitors in cells, blood and tissues following treatment with benzoxazepinone (BOAz) RIPK1 inhibitors. The TEAR1 assay is a valuable tool for facilitating the clinical development of the lead RIPK1 clinical candidate compound, GSK2982772, as a first-in-class RIPK1 ...

Growing PAINS in academic drug discovery | Future ...

Jun 06, 2011·A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J. Med. Chem. 45(8),1712–1722 (2002)rossref, Medline, CAS, Google Scholar; 4 Roche O, Schneider P, Zuegge J et al. Development of a virtual screening method for identification of ‘frequent hitters’ in compound libraries. J. Med.

Shoichet Lab : Aggregator Screening

The results of a high-throughput assay for the detection of promiscuous aggregates was used to screen 70,563 molecules from the pubchem library. Aggregates are detected by screening for detegergent-dependant inhibition of AmpC beta-lactamase, an enzyme sensitive to aggregate-based inhibition. An eprint of the paper is available here. The ...

Facile, Fluorogenic Assay for Protein Histidine ...

Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved ...

Improving the Human Hazard Characterization of Chemicals ...

Assay for promiscuous and specific inhibitors of cruzain (with and without detergent) NTP b: 1476, 1478: DNA polymerase III: Assay for inhibitors of DNA polymerase III holoenzyme system: NTP b: 603: Glucocerebrosidase: Assay for inhibitors that could potentially act as molecular chaperones on mutant forms: NTP b: 360: HADH560

Novel Broad Spectrum Inhibitors Targeting the Flavivirus ...

Jun 22, 2015·High-throughput assays for promiscuous inhibitors. Nat Chem Biol. 2005;1(3):146–8. Epub 2006/01/13. pmid:16408018. View Article PubMed/NCBI Google Scholar 41. Feng BY, Shoichet BK. A detergent-based assay for the detection of promiscuous inhibitors. Nat …

DR GYÖNGYI GYÉMÁNT (Orcid ID : 0000-0003-2374-3408 ...

detergent [12-13]. For classifying a compound as a promiscuous, aggregation-based inhibitor, the following results should be obtained: (1) decreased inhibition in the presence of a detergent [14-15], (2) inhibition of at least three unrelated enzymes, (3) decreased inhibition with a multiple-fold increase in enzyme concentration,

Complex I Enzyme Activity Microplate Assay Kit ...

Complex I Enzyme Activity Assay Kit (ab109721) is a kit designed for the analysis of mitochondrial OXPHOS Complex I enzyme activity from human, rat, mouse and bovine cell and tissue extracts. Capture antibodies specific for Complex I are pre-coated in the microplate wells. Samples are added to the microplate wells which have been precoated with ...

Interference correction in biochemical assays

Mar 07, 2010·I mentioned 'Giannetti et al, Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors, J. Med. Chem. 2008, 51, 574-580' in the Feb 11 post on SPR. I may do a more detailed review of that paper because SPR has a lot of advantages for fragment screening.