SLAS 2025 – Join Us!
BellBrook Labs looks forward to connecting with attendees at the upcoming SLAS 2025 conference in San Diego, California. The Society for Laboratory Automation and Screening (SLAS) hosts the SLAS international conference and exhibition to celebrate the latest in technology and discovery as well as laboratory automation and screening. At SLAS, we plan to exhibit our complete HTS assay solutions that are designed to tackle the toughest targets, and will be sharing example data in multiple posters using these tools. Our team of HTS assay and services experts are excited to engage with fellow scientists, researchers, and industry partners. We offer a suite of high throughput screening tools, including HTS enzyme assay kits, assay development, lead discovery services, DNA Damage Response and Innate Immunity resources, and more.
Find our booth & posters!
SLAS 2025 Conference
San Diego Convention Center
San Diego, California USA
January 27th – January 29th
Find Us @ Booth #418!
Poster Presentations
Development of a Screening and Profiling Platform for RNA Helicases Using the Transcreener ADP2 Assay
RNA helicases are a broad class of enzymes that bind and modify RNA in an ATP-dependent manner and play diverse roles in RNA metabolism, regulation of gene expression, and innate immunity. Several RNA helicases, especially in the dead box helicase (DExH/D) family, have been shown to be dysregulated in cancer and other diseases, and there have been limited efforts to develop small molecule inhibitors. To accelerate development of selective helicase inhibitors while avoiding off target effects, especially with helicases that promote tumor immunity, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP2 assay for homogenous detection of RNA-dependent ATPase activity with a far-red fluorescence polarization (FP) readout. Here we describe development of assays for six helicases of interest as potential therapeutic targets (DDX1, DDX3, DDX5, DHX9, DDX17 and DDX5), and two critical off-targets, the Rig-1-like receptor (RLR) helicases, RIG-1 and MDA5, which trigger expression of type I interferons important for tumor immunity. We produced the recombinant enzymes using BaV-infected insect cells, as full-length polypeptides, or in some cases truncated to remove disordered N-terminal domains. We determined RNA substrate and cofactor requirements and kinetic parameters with the purified enzymes and optimized the assays for initial velocity detection of RNA-dependent ATPase activity with a Z’ value greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays. We validated each of the helicase ATPase assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming hits in dose response mode. These assays will enable screening and hit-to-lead/SAR for DExH/D helicases that drive tumorigenesis and/or autoimmunity as well as selectivity profiling to avoid off target effects with RLR helicases that promote tumor immunity.
An HTS Platform for Discovery of Selective VPS4A Inhibitors Using the Transcreener ADP2 Assay
VSP4A and VSP4B (Vacuolar Sorting Protein) are members of the meiotic clade ATPases, which function as oligomers and use ATP hydrolysis to drive protein translocation through their central pore. VPS4B is frequently deleted in several cancers, and VPS4A has been shown to be synthetic lethal with VPS4B in cells and animals, causing cell death accompanied by induction of inflammatory cytokines and anti‐tumor responses. To accelerate development of selective VPS4A inhibitors while avoiding off target effects with related ATPases, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP2 assay for homogenous detection of ATPase activity with a far-red fluorescence polarization (FP) readout. Here we describe development of assays for VPS4A, VPS4B, as well as Spastin, Katanin and Fidgetin, related meiotic clade ATPases that play important roles in microtubule metabolism. Using purified recombinant enzymes, we established reaction conditions, determined kinetic parameters, and optimized the assays for initial velocity detection of ATPase activity with Z’ values greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays. We validated the VPS4A assay for HTS by screening a collection of bioactives (Tocris 2.0) and confirming two hits in dose response mode: BVT948, a PTP inhibitor, and polygodial, an antifungal that has been shown to inhibit a mitochondrial ATPase, both inhibited VPS4A and VPS4B with low micromolar potency. We then profiled BVT948 and polygodial for inhibition of Spastin, Katanin and Fidgetin: both molecules showed modest selectivity (2-5-fold) vs. Katanin and Fidgetin but had similar or greater potency for inhibition of Spastin. These assays will aid efforts to exploit the VPS4A/4B synthetic lethality therapeutically, including screening and SAR-driven hit-to-lead to minimize off-target effects with related ATPases.
Interrogating WRN Helicase and Exonuclease Activities with the Transcreener HTS Assay Platform
Werner syndrome helicase (WRN), a double strand break repair enzyme with ATP-dependent DNA-helicase and 3’- >5’ exonuclease activities, is under intense focus as a therapeutic target because of its synthetic lethality in tumors with microsatellite instability. To accelerate discovery of WRN inhibitors, we developed and validated homogenous, fluorescence-based high throughput assays for WRN helicase and exonuclease enzymatic activities using the Transcreener HTS Assay platform, which relies on highly selective antibodies to detect nucleotides in a homogenous (mix-and-read) format with far red fluorescent readouts. We produced the WRN helicase domain (aa 500-946, N-terminal 6xHis) in BaV-infected insect cells and optimized detection of its dsDNA-dependent ATPase activity using the Transcreener ADP2 Assay in FP, TR-FRET, and FI detection modes. Optimal reaction conditions included 50 μM ATP (approximate Km) and saturating concentration (100 nM) of a 3’ flapped duplex DNA oligomer; these conditions yielded a good signal with 250 pM WRN, a kcat of 300 min-1, and a Z’ of 0.85. An IC50 of 35 nM was determined for the probe inhibitor NSC-617145, and a 1,280 compound pilot screen (Tocris 2.0 Micro) identified multiple hits that were confirmed in dose response experiments. We produced the WRN 3’-5’ exonuclease domain (aa 1-133-6xHis) in E. coli and used the Transcreener dAMP Exonuclease Assay to allow direct, FP-based detection of dAMP monomers released from a 52 bp dsDNA oligomer with a 5’ overhang. With saturating DNA present (1 μM), the assay produced a good signal with 1 nM enzyme; the Z’ value was 0.89. There are no published inhibitors for the WRN exonuclease, but suramin inhibited with an IC50 of approximately 100 nM. These assays allow sensitive, robust, quantitative detection of WRN ATPase and exonuclease activities using an extensively validated HTS platform; they should be valuable tools for screening and optimizing small molecules targeting specific WRN functions.
Development of a Homogenous poly(ADP-ribose) Assay for Screening and Profiling PARP Inhibitors
Poly(ADP-ribose) polymerases (PARPs) initiate DNA damage repair (DDR) by binding to double strand breaks and creating a scaffold of poly(ADP-ribose) (pADPr) chains for assembly of other DDR proteins. The use of PARP inhibitors for ovarian tumors with BRCA-1 mutations was the first clinical exploitation of synthetic lethality, and PARP inhibitors are now used for breast, prostate, pancreatic, and ovarian cancers. New types of PARP inhibitors are being sought to broaden the scope of indications. To facilitate this effort, we developed a high throughput screening (HTS) assay for PARPs that enables quantitative detection of pADPr formation in a homogenous format. The assay relies on enzymatic conversion of pADPr to AMP, which is then detected using the Transcreener AMP/GMP assay with a fluorescence polarization (FP) or time-resolved Förster resonance energy transfer (TR-FRET) readout. To minimize interference from screening compounds, we first optimized the concentration of coupling enzymes to ensure that the pADPr will be completely converted to AMP even if the conversion reaction is inhibited by as much as 80%. Using this optimized system, the assay generated a robust signal (Z’ > 0.8) using 0.8 nM PARP1 with sub-saturating NAD (100 μM) and saturating DNA (0.25 mg/mL) under initial velocity conditions. The assay was validated for HTS using a 1,280 compound bioactive library, resulting in 18 hits, 5 of which proved to be interference. We also screened a 3,200 compound diversity library, resulting in 44 hits, 3 of which were identified as interference. Four known PARP1 inhibitors, Olaparib, PJ 34 HCl, Veliparib, and Rucaparib Camsylate, were detected in the bioactive screen and tested in dose response mode with both FP and TR-FRET readout; the measured IC50 values (0.45 nM, 29 nM, 1.4 nM and 0.4 nM, respectively) were consistent with published values. By enabling sensitive, quantitative detection of pADPr formation in a simple, mix-and-read format, the Transcreener pADPr assay should facilitate efforts to develop more effective PARP inhibitors.
Available Guides / Application Notes
A Guide to the DNA Damage Response and Innate Immunity
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.
Targeting Kinases Involved in the Innate Immune Response
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.
A Guide to Navigating Hit Prioritization After Screening Using Biochemical Assays
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 Guide to Measuring Drug-Target Residence Times with Biochemical Assays
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 2025:
