BellBrook Labs looks forward to connecting with attendees at the upcoming SLAS 2024 conference in Boston, Massachusetts. SLAS is a beacon for the life sciences community, showcasing companies from diverse backgrounds that converge onto the same mission: accelerate life sciences research and technologies. BellBrook plans to contribute to this unified mission by showcasing our cutting-edge technologies and innovative assay solutions that will help make drug discovery programs flourish. Our team of HTS assay and services experts is excited to engage with fellow scientists, researchers, and industry partners. We offer a suite of high throughput screening tools compatible with 1000’s of enzyme targets, including HTS enzyme assay kits, assay development, lead discovery services, DNA Damage Response and Innate Immunity resources, and more.
Discover our booth & posters!
SLAS 2024 Conference
Boston Convention & Exhibition Center
Boston, Massachusetts USA
February 3rd – February 7th
Find Us @ Booth #964!
Interrogating RIG-I and MDA5 with the Transcreener ADP2 Assay
RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) are members of the RLR (RIG-I-like receptors) family of immune sensors that bind double-stranded RNA (dsRNA) and trigger an IFN1 response in response to microbial infection and tumorigenesis. RLR agonists are being investigated for enhancing tumor immunity, and related RNA helicases in the DEAD box (DDX) helicase family are being targeted for cancer; therefore robust HTS assays for RIG-I and MDA5 are needed. RNA helicases use cycles of ATP binding and hydrolysis to drive local unwinding of dsRNA, so activity can be measured by detecting ADP formation.
Here, we used the Transcreener ADP2 Assay, which relies on direct, homogenous immunodetection of ADP to develop high throughput assays for MDA5 and RIG-I with FP, FI, and TR-FRET readouts. We produced recombinant RIG-I and MDA5 in baculovirus-infected insect cells, determined their RNA and ATP requirements, and optimized assays for initial velocity detection of RNA-dependent ATPase activity with Z’ values greater than 0.7. We validated the assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming the hits in dose response assays. These assays will facilitate screening and hit-to-lead/SAR for RIG-I and MDA5 agonists and for assessing off target activity of DDX helicase inhibitors.
Leveraging the Molecular Devices SpectraMax® i3x and the Transcreener ADP2 Assay for Discovery of POLQ Helicase Inhibitors
POLQ is a multifunctional enzyme with template-dependent DNA polymerase, ATP-dependent helicase, and dNTP-dependent endonuclease activities that plays important roles in DSB repair. POLQ is synthetic lethal with BRCA-1 and ATM mutations, and both the polymerase and ATPase activities are being targeted with small molecules for anti-cancer drug discovery.
Here, we used the Transcreener ADP2 Assay combined with Molecular Devices SpectraMax® i3x Multi-Mode Microplate reader to detect POLQ ssDNA-dependent ATPase activity, a component of its helicase function. Transcreener assays use highly selective antibodies to detect nucleotides in a homogenous format with far-red fluorescent readouts, including fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Förster energy transfer (TR-FRET). Their single addition mix-&-read format and outstanding reagent stability makes them well-suited for automated workflows. The SpectraMax i3x reader measures absorbance, fluorescence, and luminescence with user-installable detection modules that expand its capabilities to include FP, TR-FRET, dual injection, western blot detection, and more.
We demonstrated robust detection of ssDNA-dependent POLQ ATPase initial velocity activity with FP, TR-FRET, or FI readouts, each yielding a Z’ > 0.7. The FP assay was used for a pilot screen of 1280 bioactives, resulting in a hit rate of 1.8% and an interference rate of 0.1%. Dose-response assays in FP, TR-FRET, and FI detection modes yielded similar IC50 values for one of the hits and the probe inhibitor, suramin. This study clearly demonstrates the utility of the Transcreener ADP2 Assay combined with the Molecular Devices SpectraMax i3x reader for the discovery of POLQ helicase inhibitors.
AptaFluor® SAH: A Homogenous, Universal Assay for Histone, RNA, and DNA Methyltransferases. Case Study for PRMT5, MLL4, METTL3/14, and NSP14
The AptaFluor SAH Methyltransferase Assay is a high-throughput enzymatic assay for SAM-dependent methyltransferases. The assay relies on a naturally occurring SAH-sensing RNA aptamer, or riboswitch, that binds SAH with nanomolar affinity and exquisite selectivity. Upon binding to as low as 1 nM SAH, these split-aptamer sensors generate positive time resolved Förster resonance energy transfer (TR-FRET) signals. As a result, the AptaFluor SAH Methyltransferase Assay overcomes technical gaps of current detection methods that detect specific methylation events or are not sensitive enough for enzymes with low turnovers and/or high SAM affinities.
Here, we demonstrate how the Aptafluor SAH assay will provide a reliable and robust tool for detecting activity of four methyltransferases (MTs) using various acceptor substrates, including peptides, DNA and RNA. Selected enzymes are representative of typical low Km (100 nM to 5 μM) and have attracted interest as therapeutic targets. Among them, PRMT5 and MLL4 are histone methyltransferases and catalyze the methylation of arginine or lysine, respectively. METTL3/14 methylates adenosine at the N6 position (m6A) in mRNA substrates with a DRACH motif while NSP14 catalyzes the methylation of viral RNAs at N7-guanosine in the cap formation process. We show the sensitivity and selectivity of the assay, reagent stability, and tolerance for different types of acceptor substrates. Although the assay is based on an RNA aptamer, it tolerates dsRNAs and certain ssRNAs well. Then, we illustrate that the assay allows robust detection of SAH (Z’>0.7) at nM concentration of enzymes. We use the assay to profile dose response curves for known inhibitors.
The Aptafluor SAH assay is a powerful tool for discovery of methyltransferase antagonists that will accelerate efforts to validate the targets pharmacologically.
Available Guides / Application Notes
There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorder. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.
In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets.
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).
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.
Connect with BellBrook Labs’ team members at our booth to explore potential collaborations, share your research, and gain insights into how our technologies can enhance your projects. Learn more about SLAS 2024: