BellBrook Labs will exhibit and present posters at the upcoming SLAS 2023 conference in San Diego, California. At the SLAS conference, BellBrook will demonstrate applications for its suite of high throughput screening tools, including residence time determination, targeting kinases involved in the innate immune response, navigating hit prioritization after screening using biochemical assays, assay development, lead discovery services, and more. Come find our booth!
SLAS 2023 Conference
San Diego Convention Center
San Diego, California USA
February 25th – March 1st
Chat With Us @ Booth #1757!
Interrogating CD38 and PARG with the Transcreener ADPR FP Assay
The Transcreener® ADPR FP Assay is a biochemical HTS assay for quantitative measurement of ADP-ribose (ADPR) production in enzymatic reactions. The assay uses a coupling enzyme to convert ADPR into AMP, which then detects using a far-red, competitive fluorescence polarization (FP) assay. As examples, the Transcreener ADPR FP assay can detect the activity of human Cluster of Differentiation 38 (CD38) or Poly (ADP-ribose) glycohydrolase (PARG). CD38 modulates cellular NAD homeostasis by degrading NAD to produce ADPR. It has implications for several pathophysiological conditions including infection, aging, and tumorigenesis. PARG produces ADPR from the breakdown of poly-ADP-ribose. It plays an important role in DNA damage repair and has been seen as a potential target for anticancer therapy.
Here, we demonstrate how the Transcreener ADPR FP Assay will provide a reliable and robust tool for the discovery of CD38 and PARG inhibitors. We show the sensitivity and selectivity of the assay, followed by the ability to obtain robust assay signals (>100mP) with less than 10 pM of enzyme and a Z’ value greater than 0.7. The assay was validated in a pilot screen of 1280 pharmacologically active molecules, which identified one inhibitor for CD38. The Transcreener ADPR FP Assay is a powerful tool for discovery of CD38 and PARG antagonists that will accelerate efforts to modulate these targets pharmacologically.
Interrogating TREX1 with the Transcreener dAMP Exonuclease Assay
Three Prime Repair Exonuclease 1 (TREX1) is a crucial component of the innate immune response where it acts as the main exonuclease responsible for degrading cytosolic DNA. Reduction of cytosolic DNA leads to inactivation of the cGAS/STING Pathway, which is essential for intrinsic antitumor immunity. As a result, TREX1 plays a pro-tumorigenic role in cancer and its inactivation is a promising strategy for future monotherapy and combination treatments.
Though TREX1 Inhibitors are being pursued, there are no peer-reviewed reports with quantitative data on specific molecules. To enable screening for small molecule modulators of TREX1, and for selectivity profiling of TREX1 modulators, we developed a robust, HTS-compatible assay method for measuring TREX1 activity. In this method, TREX1 cleaves Interferon Stimulatory DNA (ISDna) to produce dAMP, which is then detected by the Transcreener dAMP Exonuclease Assay, a far-red, competitive fluorescence polarization (FP) assay.
Throughout this poster, we demonstrate how the Transcreener dAMP Exonuclease Assay will provide a reliable and robust tool for the discovery of TREX1 inhibitors. Here, we show the sensitivity and selectivity of the assay, followed by the ability to obtain robust assay signals (>100mP) with less than 250 pM of TREX1 and a Z’ Value of 0.88. The assay was validated in a pilot screen of 3056 pharmacologically active molecules, which identified several inhibitors. The assay allows easy triaging of non-stoichiometric inhibitors and profiling selectivity against similar targets. Overall, the availability of the Transcreener dAMP Exonuclease Assay from BellBrook Labs will boost the discovery of TREX1 antagonists, providing possible molecules that may be utilized for future cancer treatment regimens.
Available Guides / Application Notes
Clinical trials continue to determine whether therapeutic modulation of kinases can suppress the immune system in autoimmune diseases or stimulate for antiviral and cancer immunotherapy. The discovery and characterization of small molecule modulators have aided these efforts. The Transcreener ADP² Kinase Assay meets this need as it relies on direct ADP detection to measure the activity of virtually any kinase. The method has been extensively validated for kinase discovery programs since 2007. This article provides five examples of how Transcreener allowed rapid assay development to enable screening and dose-response measurements.
So you have performed your screen. What’s next? This guide focuses on how biochemical assays characterize and prioritize compounds following a primary screen with an enzyme target, whether using high throughput screening (HTS) or virtual screening (VS). A typical screening funnel is shown below, with the many applications of the biochemical activity assay highlighted.
During drug development initiatives, analysis of drug-target residence times can improve efficacy, increase therapeutic window, and reduce the risk of premature focus on candidate compounds that are likely to have undesirable side effects. This guide provides technical background on concepts and techniques for use of Transcreener® biochemical assays to measure drug-target residence times, along with examples and case studies.