Thanks to all who stopped by the booth at SLAS 2018! We appreciate the great conversation and look forward to continued discussion. Its great to hear the success stories of current Transcreener users, and to meet a new group of individuals who are searching for an HTS assay for their challenging target. For those of you interested in kinases or a current hot target CD39, please find the following two posters presented at the conference. If methyltransferases are your current area, check out our new AptaFluor SAH Methyltransferase Assay, a hot topic of conversation at the booth.
Kinases are one of the most highly validated target families, yet only a small fraction of the kinome has been exploited therapeutically. ADP detection enables a generic assay method that has been broadly adopted for HTS efforts targeting kinases. The Transcreener ADP² Kinase assay uses homogenous detection of ADP with a choice of FP, FI, or TR-FRET readouts. Although other methods of ADP detection are available, Transcreener is the only with direct detection of ADP. Alternative assays use complex coupling mechanisms making them prone to assay interference that generate false positives and thus require time consuming counter-screens to triage. The assay provides sensitive detection of kinase initial velocity over a broad range of ATP concentrations, critical for screening such a diverse class of targets. Compatibility with 1536 well-miniaturized formats along with outstanding overnight and reagent signal stability provide flexibility in liquid handling while producing data that yields dependable results. And while Transcreener’s flexibility and simplicity make it an excellent fit for HTS, the assay also has distinct advantages in SAR and MOA studies. Sensitivity affords use of the assay at low enzyme concentrations and helps determine accurate IC50 measurements for potent inhibitors. Transcreener can also be used kinetic mode, which simplifies early stage assay development and enables high throughput assessment of drug-target residence time. Here we demonstrate the Transcreener ADP² Kinase Assay as a powerful HTS approach for discovering, evaluating, selecting, and improving small molecule kinase inhibitors.
Ectonucleotidases are plasma membrane-bound enzymes with externally oriented active sites that metabolize nucleotides to nucleosides and are crucial for maintaining immune homeostasis. The ectonucleoside triphosphate diphosphohydrolase-1, also known as CD39, ENTPD1, or NTPDase1 hydrolyzes ATP and ADP to AMP. AMP can further be processed to adenosine leading to a significant impact on many disease states. Recent studies have shown a key role for adenosine in immunosuppression in the tumor microenvironment, and ectonucleotidases are emerging as promising immuno-oncology targets. As the only HTS method capable of direct detection of nucleotides, the Transcreener platform is uniquely suited for measuring ectonucleotidase activity with the high sensitivity and low levels of interference required for a successful HTS campaign. The homogenous assays use a far-red fluorescence polarization (FP) or TR-FRET readout and they can be broadly applicable to ectonucleotidases. We developed a simple biochemical assay for measuring CD39 activity using the Transcreener AMP² Assay. The assay provides robust detection of AMP production (Z’ > 0.6) with sub-nanomolar amounts of CD39. Initial pilot screens have demonstrated robust assay performance (Z’ = 0.6 – 0.7) and IC50s determined for tool compounds of CD39 were consistent with published values. The availability of HTS-compatible enzyme assay methods will accelerate the discovery of inhibitors for CD39 and related ectonucleotidases that play a role in tumor immunity and other diseases impacted by adenosine signaling.