Scintillation proximity assay in lead discovery

Khawaja, Xavier; Dunlop, John; Kowal, Dianne

doi:10.1517/17460441.3.11.1267

Cited by 8 publications

(8 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Specifically, Leu binds 10–100-fold more tightly but is transported at least 10-fold more slowly than Ala. 26, 27 Both substrates were labeled with tritium, which is ideal for SPA because its low-energy β-particles have short path lengths in aqueous solution; indeed, light is emitted only if the isotope is within 1–8 μm of a bead. 21 We first immobilized LeuT-nanodiscs onto the surface of copper chelate Yttrium silicate (Cu 2+ -Ysi) SPA beads (PerkinElmer) (Figure 2A). In preliminary experiments, the bead-nanodisc suspension was added to binding buffer such that the final concentrations of [ 3 H]Leu, [ 3 H]Ala, and LeuT were 50, 1000, and 20 nM, respectively.…”

mentioning

confidence: 99%

Radioligand Binding to Nanodisc-Reconstituted Membrane Transporters Assessed by the Scintillation Proximity Assay

Nasr

Singh

2013

Biochemistry

View full text Add to dashboard Cite

The scintillation proximity assay (SPA) is a powerful technique for measuring radioligand binding to membrane transporters and has become an integral part of high-throughput drug discovery screening efforts. Here we adapt the method for use with purified LeuT, a prokaryotic secondary transporter, reconstituted into phospholipid bilayer nanodiscs. This application surmounts potential challenges with background interference from endogenously-expressed proteins; aggregation and loss of binding activity often accompanying detergent solubilization from native cell membranes; and heterogeneity in size and transporter orientation, where at least some ligand binding sites are inaccessible, associated with reconstitution into lipid vesicles.

show abstract

mentioning

confidence: 99%

Radioligand Binding to Nanodisc-Reconstituted Membrane Transporters Assessed by the Scintillation Proximity Assay

Nasr

Singh

2013

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Z ′ values for the AMI-MS assay could have been improved with even a minor increase in enzyme, substrate, and cofactor concentrations. Since the SPA format is generally a very sensitive platform and fairly resistant to nuisance mechanisms, our expectations are that against other HTS platforms, AMI-MS may show lower interference.…”

Section: Discussionmentioning

confidence: 99%

Acoustic Mist Ionization-Mass Spectrometry: A Comparison to Conventional High-Throughput Screening and Compound Profiling Platforms

Belov¹,

Kozole²,

Bean³

et al. 2020

Anal. Chem.

View full text Add to dashboard Cite

Drug discovery usually begins with a high-throughput screen (HTS) of thousands to millions of molecules to identify starting points for medicinal chemistry. Conventional HTS platforms require expensive reagents and typically have complex assay formats. HTS platforms based on radioactivity are expensive, both in terms of reagent cost and disposal. Furthermore, nonspecific interferences common to these technologies result in an extensive attrition of hits during validation experiments. Mass spectrometry (MS) is a highly selective, label-free technology that can quantify multiple analytes in a single experiment. However, most commercial MS platforms typically involve a separation or cleanup prior to analysis and are too slow for large-scale screening campaigns. Recently, an MS platform (AMI-MS) was introduced that uses acoustically generated droplets to deliver analyte molecules directly from microtiter plates into the mass spectrometer at subsecond per well sampling rates. Here, we demonstrate the application of AMI-MS by developing an HTS-compatible assay that measures the inhibition of histone acetyltransferase activity. Real-time kinetic measurements from a single well were used to determine enzyme K m and V max values. We compare the AMI-MS readout with conventional platforms in single-shot screening and multipoint profiling modes. The AMI-MS assay identified 86% of hits previously identified, with a pIC50 ≥ 5.0, in a scintillation proximity assay (SPA) HTS at a lower hit rate and with a significantly reduced cost per well compared to the SPA-based readout. Furthermore, pIC50s, as measured by AMI-MS, showed a good correlation with values generated by RapidFire-MS. AMI-MS has the potential to provide significant improvements to high-throughput bioassays.

show abstract

“…44 Inside the beads is a scintillant that emits light aer stimulation which occurs when radiolabeled compounds attach to the surface. 45 This setup offers more high-throughput capabilities compared to the time-consuming paper chromatography analysis. The substrate for these assays is UDP-[ 3 H]GlcNAc which is a precursor for Lipid II and is more readily available than radioactively labeled Lipid II.…”

Section: Radioactive Labelingmentioning

confidence: 99%