Join Us at AACR 2025!
BellBrook Labs looks forward to connecting with attendees at the upcoming American Association for Cancer Research® (AACR) Annual Meeting 2025 in Chicago, Illinois. AACR gathers a community of scientists, clinicians, health care providers, and more to showcase and advance the latest cutting-edge technologies for cancer research. 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!
April 27th-30th, 2025
McCormick Place Convention Center
Chicago, Illinois
Find Us @ Booth #110!
Poster Presentations
Development of a homogenous poly(ADP-ribose) assay for screening and profiling PARP inhibitors
Session Start: 4/29/2025 9:00:00 AM
Session End: 4/29/2025 12:00:00 PM
Poster Board Number: 1
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.
Profiling Ras inhibitors for effects on GTP hydrolysis using the Transcreener GDP assay
Session Start: 4/30/2025 9:00:00 AM
Session End: 4/30/2025 12:00:00 PM
Poster Board Number: 19
Ras family GTPases are the most frequently mutated proteins in human cancers, most notably missense mutations at G12 and G13 that prolong the activated, GTP-bound state of the protein. Inhibitors that shift the equilibrium toward GDP-bound Ras, such as sotorasib, are proving effective clinically, and efforts to expand upon this strategy are widespread. Current strategies for biochemical screening and analysis of Ras inhibitors rely on the use of fluorescent GTP and GDP analogs to detect effects on GDP release and GTP binding. Though robust and widely used, these methods do not provide information on GTP hydrolysis, a critical part of the GTPase signal transduction process. We have developed methods for detecting GTP hydrolysis by small GTPases using the Transcreener GDP2 assay, a homogenous, competitive immunoassay for GDP with far-red fluorescence readout. Because GDP release is generally rate limiting, stimulation by GEFs can also be measured by monitoring GDP formation. We have optimized these assays for initial velocity detection of GTPase activity for wild type (WT) and mutant Ras proteins with robustness suitable for high throughput screening and tested a number of compounds reported to inhibit GTP and/or GDP binding. Consistent with expectations, the Ras G12C-specific covalent inhibitor, Sotorasib, potently inhibited KRas G12C (IC50 = 9.6 nM) and did not inhibit the wild-type enzyme. MRTX1133, which binds noncovalently to GDP-bound KRas G12D, inhibited the G12D mutant with an IC50 of 8.4 nM, and was less potent with the G12V and WT enzymes; IC50 = 48 nM and 69 nM, respectively. BI-2865, a pan KRas inhibitor, showed much weaker inhibition of all KRas variants tested, with IC50 values of 427 nM, 228 nM and 236 nM for WT, G12D and G12V KRas. We note that these GTPase assays require nanomolar concentrations of protein, and therefore cannot be used to accurately determine the potency of covalent inhibitors, such as sotorasib, that bind with picomolar affinity. However, for non-covalent inhibitors, such as MRTX1113 and BI-2865, measuring GTPase activity is a robust approach for selectivity profiling and may yield mechanistic insights different from those gleaned from guanine nucleotide binding/release assays or biophysical methods, e.g. SPR.
Homogenous detection of O-Acetyl-ADP-ribose as a generic HTS assay method for SIRTs
Session Start: 4/29/2025 9:00:00 AM
Session End: 4/29/2025 12:00:00 PM
Poster Board Number: 23
Sirtuins (SIRTs) are deacetylases that catalyze removal of acetyl groups from histones and other proteins; they are important components of the epigenetic regulatory landscape and have been targeted for diverse indications, including cancer. SIRTs deacetylate in an NAD-dependent reaction that yields O-acetyl-ADP-ribose (OAADPr) and nicotinamide as products along with the deacetylated protein. Screening and hit-to-lead efforts targeting SIRTs has been hampered by a lack of robust HTS assays methods for detection of SIRT enzymatic activity, a situation that is exacerbated by the diversity of deacetylated protein products and the inherent instability of the OAADPr product. To address this unmet need, we developed a generic activity assay for SIRTs based on enzymatic conversion of OAADPr to AMP in real time, 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. Using recombinant full length Sirt1 and Sirt2, we determined optimal NAD, acetylated peptide substrate, and enzyme concentration for initial velocity detection of deacetylase activity. For both enzymes, a robust signal was observed using concentrations in the nanomolar range, and there was good concordance in activity measured using FP and TR-FRET readouts. The assays were validated for HTS using a 1,280 collection of bioactive compounds. Selected hits were further profiled in dose-response assays. The assay Z’ value was above 0.7, supporting its robustness for screening and hit-to-lead/SAR for Sirt1 and Sirt2 antagonists. To our knowledge, the Transcreener OAADPr assay is the first SIRT activity assay that relies on detection of OAADPr rather than the deacetylated protein product. By enabling homogenous detection of SIRT activity with native acetylated substrates, it should simplify screening and profiling of inhibitors and facilitate drug discovery efforts targeting SIRTs.
Development of HTS enzymatic assays for VPS4A and related ATPases
Session Start: 4/29/2025 2:00:00 PM
Session End: 4/29/2025 5:00:00 PM
Poster Board Number: 16
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 a Z’ value 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.
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 AACR 2025:
