Bellbrook Labs (United States)

Abstract Disclosure: K.N. Islam: None. R.K. Islam: None. A.H. Penn: None. Diabetes is a major cause of morbidity and mortality. Elevation of oxidative stress is associated with diabetic patients because of increased levels of blood sugar concentration. Hydrogen Sulfide (H2S) is a gasotransmitter and antioxidant with many health benefits for many disease conditions. The pancreatic β cells are insulin-secreting. The present study investigated the effects of H2S on reducing sugar-induced oxidative stress in pancreatic β cells (HIT) from a Chinese hamster. Here, we determined the effects of reducing sugar on the induction of oxidative stress, as it was measured by the formation of thiobarbituric acid reactive substances (TBARS) and protein carbonyl (CO) formation in serum-starved β cells. We observed marked oxidative stress in the serum-starved β cells after treatment with reducing sugar. We tested the effects of H2S on reducing sugar-induced oxidative stress in serum-starved β-cells, and we found that H2S markedly inhibited sugar-induced oxidative stress in serum-starved β cells. Treatment with H2S caused significant induction of cell viability and a marked reduction of cellular cytotoxicity in β cells under serum starvation incubated without or with sugar. H2S treatment of starved β cells antagonized expression levels of inflammatory cytokines, TNF-α and inhibited the activation of the transcription factor, NF-κB signaling pathway. In addition, H2S treatment increased antioxidant activity and activated the Nrf2 signaling pathway in stressed β cells. Our findings suggest that H2S/its donors may be used as therapy to cure diabetes by inhibiting oxidative stress, thereby decreasing blood sugar levels in diabetic patients. Presentation: Sunday, July 13, 2025

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. Citation Format: Anibal Ramos Martinez, Camdon Yemm, Ha Pham, Robert Lowery. Development of HTS enzymatic assays for VPS4A and related ATPases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5671.

Abstract 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 saturated sheared salmon sperm 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. Citation Format: Mahbbat Ali, Ha Pham, Anibal Ramos-Martinez, Robert Lowery. Development of a homogenous poly(ADP-ribose) assay for screening and profiling PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4184.

Abstract 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. Citation Format: Ha Pham, Mahbbat Ali, Anibal Ramos-Martinez, Robert Lowery. Homogenous detection of O-Acetyl-ADP-ribose as a generic HTS assay method for SIRTs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4237.

College Board is in the final stages of rolling out changes to the AP® Computer Science A course and exam for 2025--2026. This is routine, in the sense that such revisions occur periodically to maintain the course alignment, but in this instance some of the changes are fairly substantial. Early announcements of the general plans have reliably generated much discussion and many questions, both in formal presentations and on social media.

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) – the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z’ > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

&lt;p&gt;&lt;b&gt;ABSTRACT &lt;/b&gt;&lt;/p&gt; &lt;p&gt;Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to an output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM – 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small molecule targets.&lt;/p&gt;

We report a flexible and modular design for biosensors based on exploiting semiconductor quantum dots (QDs) and their excellent Forster resonance energy transfer (FRET) acceptor properties along with the long-lived fluorescent lifetimes of lanthanide donors. We demonstrate the format’s wide application by developing a broad adenosine diphosphate (ADP) sensor with quantitative and high-throughput capabilities as a kinase/ATPase assay method. The sensor is based on a Terbium (Tb)-labeled antibody (Ab) that selectively recognizes ADP versus ATP. The Tb-labeled Ab (Ab-Tb) acts as a FRET donor to a QD, which has an ADP modified His6-peptide conjugated to its surface via metal-affinity coordination. This strategy of using self-assembly, modified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays of interest. We utilize time-resolved FRET (TR-FRET) to measure the amounts of Ab-Tb bound to the QD by looking at the emission ratio of the QD and Tb in a time-gated manner, mi...

The emergence of new capabilities such as virtualization and elastic (private or public) cloud computing infrastructures has made it possible to deploy multiple applications, on demand, on the same cloud infrastructure. A major challenge to achieve this possibility, however, is that modern applications are typically distributed, structured systems that include not only computational and storage entities, but also policy entities (e.g., load balancers, firewalls, intrusion prevention boxes). Deploying applications on a cloud infrastructure without the policy entities may introduce substantial policy violations and/or security holes. In this paper, we present PACE: the first systematic framework for Policy-Aware Application Cloud Embedding. We precisely define the policy-aware, cloud application embedding problem, study its complexity and introduce simple, efficient, online primal-dual algorithms to embed applications in cloud data centers. We conduct evaluations using data from a real, large campus network and a realistic data center topology to evaluate the feasibility and performance of PACE. We show that deployment in a cloud without considering in-network policies may lead to a large number of policy violations (e.g., using tree routing as a way to enforce in-network policies may observe up to 91% policy violations). We also show that our embedding algorithms are very efficient by comparing with a good online fractional embedding algorithm.

"Regulator of G-protein Signaling" (RGS) proteins constitute a class of intracellular signaling regulators that accelerate GTP hydrolysis by heterotrimeric Gα subunits. In recent years, RGS proteins have emerged as potential drug targets for modulation by small molecules. Described in this unit are high-throughput screening procedures for identifying modulators of RGS protein-mediated GTPase acceleration (GAP activity), for assessment of RGS domain/Gα interactions (most avid in vitro when Gα is bound by aluminum tetrafluoride), and for validation of candidate GAP-modulatory molecules with the single-turnover GTP hydrolysis assay.