Promega (United States)

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Protein-protein interactions (PPIs) are integral to cellular signaling networks and are frequently disrupted in cancer, neurodegeneration, inflammation, and metabolic disorders. Targeting dysregulated PPIs presents a promising strategy for the development of therapeutic compounds. However, traditional drug discovery platforms often rely on plasmid-driven overexpression models that fail to replicate the complexity and dynamics of PPI in native cellular contexts. This study aims to evaluate the use of NanoLuc Binary Technology (NanoBiT) and NanoLuc Bioluminescence Resonance Energy Transfer (NanoBRET) for quantifying interactions of endogenously regulated proteins in live cells. To achieve this, CRISPR-mediated genome engineering was used to integrate NanoBiT and NanoBRET fusion tags at the loci for EGFR/GRB2 and KRas/CRAF in DLD-1 and HCT 116 cell lines. Assays using the engineered cell lines were then conducted in monolayer cultures using endpoint and kinetic measurements, as well as luminescence imaging. The approach was further expanded to investigate PPI in cancer-associated isogenic cell lines and 3D spheroid models that better preserve additional aspects of cellular organization. Collectively, these findings establish a robust and modular workflow for generating endogenously regulated PPI reporter cell lines to improve the relevance and predictive power of live-cell assays. By capturing interaction dynamics in a more representative background, this approach offers a potentially valuable tool for elucidating signaling mechanisms and characterizing therapeutic compounds targeting PPIs.

Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m7GpppX cap present at the 5′ terminus of coding mRNAs, with an m7G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technology’s potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.

O laboratório escolar de Física pode desempenhar um papel fundamental no desenvolvimento tanto de aprendizagens conceituais, quanto de habilidades, estratégias, procedimentos, hábitos e disposições. No Brasil, a Base Nacional Comum Curricular (BNCC) e a matriz de referência do Enem (MR) são documentos oficiais que sinalizam a importância dessas aprendizagens. Neste trabalho, analisamos as habilidades da MR com potencial de serem desenvolvidas em uma atividade prática de cunho investigativo sobre forças de atrito. Buscamos indícios de desenvolvimento dessas habilidades pelos estudantes na análise do desempenho em questões do próprio Enem, aplicadas pelo professor da disciplina, que abordam pelo menos uma das habilidades trabalhadas pela atividade sobre atrito. O estudo foi desenvolvido em uma escola pública federal localizada na capital de um dos estados do sudeste brasileiro. A análise dos dados envolveu aspectos qualitativos e quantitativos (teste de Wilcoxon). Identificamos o potencial de desenvolvimento de três habilidades da MR na atividade analisada. O desempenho dos estudantes em questões do Enem, aplicadas antes e depois da atividade, sugerem um efeito médio da intervenção sobre o mesmo. Apesar disso, as estatísticas do testes de Wilcoxon sinalizam falta de significância estatística na melhoria de desempenho dos estudantes.

Gene therapy with AAV vectors is a promising approach for treating numerous genetic disorders but is often hindered by preexisting antibodies that neutralize the vectors. Given that females may exhibit stronger immune responses than males, this study hypothesizes that females may have higher preexisting antibody titers against AAV. Serum samples from two U.S. cohorts were analyzed for antibody titers, antibody subtypes, and transduction inhibition activity against AAV serotypes AAV1, AAV2, AAV5, AAV8, and AAV9. We found that among seropositive samples, females had higher preexisting antibody levels and neutralizing activities against AAV9 and other serotypes. Immunoglobulin subclass analysis showed IgG1 dominance in both sexes, but females had higher IgA levels, whereas males had higher levels of IgG2. We further evaluated the cellular level of this differential immune response to AAV by stimulation of male and female human PBMCs. We observed dose-dependent increase in cytokines and chemokines in female PBMCs which suggests a differential inflammatory response. Altogether, our findings suggest that the enhanced immune response in females could lead to neutralization and faster clearance of AAV vectors with potential to impact the efficacy of gene therapy.

Abstract N6-methyladenosine (m6A) is a modified nucleoside derived from adenosine, distinguished by the presence of a methyl group at the N6 position. This structural modification enables m6A to play unique roles in cellular signaling, gene expression regulation, and metabolic processes, positioning it as a crucial player in cellular function and disease pathology. Its involvement ushered the beginning of the field of in epitranscriptomics, particularly N6-methyladenosine (m6A)-related pathways, which modulate mRNA stability, translation, and splicing have lent tremendous significance to this form of RNA modification. There are about 7000 mRNAs with m6A modifications and they are highly conserved. Non-coding RNA is mainly regulated by m6A methyltransferase and demethylase, which plays an important biological role in the occurrence and development of tumors. Recent studies suggest that m6A and its derivatives could serve as potential biomarkers for disease diagnosis and as therapeutic targets. Recognizing how important the early detection of m6A is, we have developed an assay that monitors the concentration of m6A so it can be used for screening of m6A-RNA from patients with cancer such as liver cancer. The assay relies on new concept of novel nanoluciferase (nanoluc) complementation where a small peptide (SmBiT) linked to m6A through a linker (m6A tracer) can be recognized by an anti m6A antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in a bioluminescence upon the addition of nanoluc substrate. The presence of m6A-RNA that is generated in the reaction with RNA substrate, S-adenosylmethionine (SAM) and METTL 3/14 complex can be detected with this assay. Using MTTL3/14 complex we show that SAM is an excellent substrate for this enzyme with a Km of 240nM. The assay does not recognize Adenosine, AMP, ADP, or ATP in the reaction. We will show data in support of this technology to monitor the presence of m6A in methylated RNA. The assay is homogenous, HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contributions for investigating modulators of the MTTL3/14 complex or FTO demethylating enzymes and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, Dareen Mikheil, Kevin Hsiao, WeiWei An, Matt Larsen. High throughput bioluminescent homogenous assay to monitor N6-methyladenoisne (m6A) [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 5491.

Abstract We present a robust, homogeneous, and luminescent microplate-based immunoassay utilizing Lumit® technology for fast and quantitative detection of human Ki-67, a marker of cell proliferation. This assay enables efficient screening of antiproliferative agents, demonstrating exceptional performance in assessing antiproliferative activity across multiple mechanisms of action. We evaluated the Lumit® hKi-67 immunoassay in HCT116 colorectal cancer cells treated with increasing concentrations of four agents known to inhibit proliferation via different mechanisms: palbociclib (CDK4/6 inhibitor), samuraciclib (CDK7 inhibitor), nutlin-3A (MDM2/p53 interaction inhibitor), and thymidine (dNTP pool perturbation). With all four test agents, the Lumit® hKi-67 immunoassay detected a marked, dose-dependent 63-82% reduction in Ki-67 after only 24 hours, and an even greater 79-98% reduction at 48 hours, demonstrating a fast and reliable measure of antiproliferative effects. A same-well multiplex with a cell-impermeant fluorogenic DNA dye demonstrated no cell death provoked by these compounds at the doses and times tested. In contrast to the large decrease in the proliferation marker Ki-67 elicited within 24 h of treatment by these antiproliferative agents, the response windows achieved with assays of viable cell number were significantly smaller at the early time point. Only 25-40% less viable-cell DNA was observed after 24 h compound treatment vs. untreated controls, with 48 h required to produce response windows of 57-89%. Similar observations were obtained with two other viability assays, one detecting ATP levels and the other live-cell protease activity. However, ATP levels were reduced more than expected by 24 h treatment with thymidine, likely due to perturbation of metabolic activity. Equally concerning, at 48 h, the live-cell protease assay and, especially, detection of ATP levels dramatically underreported the decrease in viable cell number (relative to time-matched untreated controls) in response to palbociclib and nutlin-3A. This latter effect is likely due to the substantial increase in cell size induced by palbociclib and nutlin-3A even as they inhibit cell proliferation. The strong antiproliferative effects induced by palbociclib and nutlin-3A and detected with the hKi-67 immunoassay, however, were further validated by two additional assays: Ki-67 immunofluorescence and BrdU ELISA. Notably, the Lumit® assay provides faster, more robust data compared to these more labor-intensive alternatives. This study highlights the Lumit® hKi-67 immunoassay as an attractive, high-throughput alternative for screening antiproliferative agents, offering a simpler, faster, and more consistent approach than traditional methods. It is poised to be an invaluable tool in drug discovery and preclinical screening. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. Homogeneous bioluminescent immunoassay for hKi-67 provides a simple and robust screening tool for antiproliferative agents [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 5337.

Abstract Bioluminescence prosubstrates are chemically protected bioluminescence substrates which, upon introduction to cells, can gradually and slowly release free substrate for generating luminescent signals in cellular assays. Prosubstrates can generate steady signals that last for several hours or days, enabling live-cell, kineticassays. Noticeable examples of bioluminescence prosubstrates include Nano-Glo Endurazine and Vivazine Live Cell Substrates. Although these prosubstrates have been serving the scientific community for years, they still have drawbacks, including reduced brightness, sensitivity, and signal duration. Herein, we describe a new generation of live cell bioluminescence prosubstrates that can overcome the drawbacks of current prosubstrates. Citation Format: Chao Gao, Brock Binkowski, Hui Wang, Wenhui Zhou. Longer and brighter live cell bioluminescence prosubstrates [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 3167.

The PRMT5 methyltransferase plays a global role in cell physiology and is an established therapeutic target in cancer. In 10% of human cancers, deletion of the MTAP gene results in accumulation of MTA, exposing a synthetic lethality and opportunity for precision medicine by selective targeting of PRMT5 in this context. Reported small molecule PRMT5 inhibitors engage either cosubstrate (SAM) or peptide-substrate pockets through diverse mechanisms. A subset of chemotypes demonstrate uncompetitive engagement with SAM or its inhibitory metabolic precursor, MTA. Here we describe the development of a cell-permeable BRET probe CBH-002 that acts as a dynamic biosensor of the intracellular SAM/MTA pool. Using this probe, we evaluate a range of diverse PRMT5 inhibitors to mechanistically characterize and quantify uncompetitive target engagement and ternary complex formation at PRMT5-SAM and -MTA complexes in live cells, enabling direct insights into drug mechanism-of-action and metabolite-dependent response to inhibitors.

The cyclic GMP-AMP synthase-stimulator of the interferon gene (cGAS-STING) signaling pathway is considered an essential pattern recognition and effector pathway in the natural immune system and is mainly responsible for recognizing DNA molecules present in the cytoplasm and activating downstream signaling pathways to generate type I interferons (IFN-I) and other inflammatory factors. STING, a crucial junction protein in the innate immune system, exerts an essential role in host resistance to external pathogen invasion. The DNA introduced by pathogens or tumors is recognized by the cytoplasmic nucleic acid receptor cGAS, and a second messenger, cGAMP, is generated using intracellular guanosine triphosphate (GTP) and adenosine triphosphate (ATP). Furthermore, cellular and extracellular cGAMP concentrations are also controlled by ENPP1, an enzyme that breaks down cGAMP to AMP and GMP. Therefore, the role of the cGAS-STING signaling pathway has generated great interest in inflammatory and cancer research. To advance our understanding of innate immune system and in particular the STING pathway, we have developed a homogeneous, bioluminescent cGAMP detection assay that is very sensitive and highly selective against other nucleotides, cyclic nucleotides, and dicyclic nucleotides. The assay can be also used to monitor the activity of cGAS and ENPP1 to enable the development of inhibitors of both enzymes which might be used for therapeutic applications.