Cell Based Assays

Drug Target Confirmed? Tivantinib’s Lesson on the Importance of Cellular Target Engagement

Posted on by Promega

This guest blog post is written by Tian Yang, Associate Product Manager at Promega.

There are often challenges with translating results from a test tube into a living system, demanding more physiologically relevant assays. In drug discovery, demonstrating a compound’s ability to modulate its target protein in live cells is a critical step in the hit-to-lead workflow. A variety of cell-based assays can be used to assess a compound’s activity in live cells. Take kinase inhibitors as an example, these assays can range from substrate phosphorylation assays that more directly report on the activity of target kinases, to genetic reporter assays or cell viability assays that assess the downstream effects of target modulation.

In the case of Tivantinib, several pieces of data from its development were used to establish its role as an inhibitor of MET kinase. MET Kinase is a prominent target for anti-cancer therapeutics due to frequent MET dysregulation in a wide range of tumors. For example, over-activation of MET drives cancer proliferation and metastasis. In the initial report on Tivantinib, in addition to biochemical activity assays performed with purified MET, the activity of Tivantinib in cells was verified by several methods, including: 1) inhibition of phosphorylation of MET and downstream signaling pathways, 2) cytotoxicity in cancer cell lines expressing MET, and 3) antitumor activity in xenograft mouse models (1). Additionally, a co-crystal structure of the MET-Tivantinib complex was solved, seemingly confirming that Tivantinib is a bona fide MET inhibitor capable of engaging MET in live cells (2). Based on these observations and other pre-clinical data, Tivantinib appeared to be a promising drug candidate and was taken through phase 3 clinical trials targeting cancers with MET overexpression. However, Tivantinib ultimately was not approved as a new therapeutic, failing to show efficacy in these phase 3 clinical trials (3,4).  

Within three years of the initial publication on Tivantinib, two separate articles challenged the mechanism of action in Tivantinib-induced cytotoxicity of tumor cells (5,6). Authors for both articles showed that Tivantinib can kill both MET-addicted and nonaddicted cells with similar potency. Both articles also concluded that perturbation of microtubule dynamics, instead of MET inhibition, is likely responsible for the cytotoxicity observed with Tivantinib. Considering the failed clinical trials and uncertainties regarding the mechanism of action, one may wonder if the original pre-clinical work adequately determined if Tivantinib effectively binds and inhibits MET in cells? If Tivantinib’s cellular engagement to MET was assessed directly rather than by MET phosphorylation analysis, would a different pre-clinical recommendation have been made?

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Non-Pharmacological Approaches to ADHD: Exploring Inflammation and Omega-3s

Posted on by Simon Moe

Attention-Deficit/Hyperactivity Disorder (ADHD) is a complex neurodevelopmental disorder that affects millions worldwide. Current therapeutic treatment relies on pharmaceutical approaches, but emerging research suggests that dietary supplements, such as omega-3 fatty acids, may offer complementary therapeutic options. A recent study published in the Journal of Psychiatric Research explores the relationship between inflammation and dietary supplements to determine how they might influence ADHD pathology. This work was conducted in Dr. Edna Grünblatt’s lab at the University of Zurich and was supported through Promega’s Academic Access Program. I had the chance to interview Dr. Natalie Walter, the lead author, to learn more about how her work offers potential opportunities for non-pharmacological interventions.

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Bioluminescence vs. Fluorescence: Choosing the Right Assay for Your Experiment 

Posted on by Anna Bennett

From enzyme activity to gene expression, light-based assays have become foundational tools in life science research. Among these, fluorescence and bioluminescence are two of the most widely-used approaches for detecting and quantifying biological events. Both rely on the emission of light, but the mechanisms generating that light—and the practical implications for experimental design—are quite different. 

Choosing between a fluorescence or bioluminescence assay isn’t as simple as picking between two reagents off the shelf. Each has strengths and limitations depending on the application, instrumentation, and biological system. In this blog, we’ll walk through how each method works, where they shine (and where they don’t), and what to consider when deciding which approach is right for your experiment. 

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Glo-ing Above and Beyond: Simplifying Science with MyGlo Reagent Reader

Posted on by Simon Moe

When it comes to laboratory tools, few things resonate more than the experiences of researchers who rely on them daily. At the University of Cincinnati the MyGlo Reagent Reader has quickly become an indispensable lab companion, due to its compact design, affordability, and intuitive interface with tailored apps for Promega assays. But what truly sets the MyGlo Reagent Reader apart is how it empowers scientists to focus on their research.

Take Ipsita Kundu, a third-year PhD student at the University of Cincinnati working in Dr. Tim Phoenix’s lab. The Phoenix lab, dedicated to studying innovative brain tumor therapies, faced challenges with their outdated luminescence reader. They needed an affordable, reliable solution to streamline Ipsita’s experiments without compromising accuracy or efficiency.

The MyGlo Reagent Reader was the answer. This blog highlights how this integrated solution is redefining laboratory workflows, enabling researchers to maximize productivity and maintain focus on groundbreaking discoveries. Let’s delve into Ipsita’s story and explore how MyGlo transformed her research.

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The Greatness of Glycogen: A Central Storage Molecule in Energy Metabolism

Posted on by Simon Moe

Introduction

Glycogen is a fundamental molecule in energy metabolism, serving as the critical storage form of glucose that supports cellular health and energy homeostasis. As a polysaccharide, glycogen is essential for maintaining stable energy levels, particularly during periods of fasting and physical exertion. This article will examine glycogen’s synthesis, storage, and utilization, along with its broader significance in human health and disease. Understanding glycogen’s role can provide valuable insight into energy regulation and metabolic health.

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The Benefits of BCAAs: Branched-Chain Amino Acids in Health and Disease

Posted on by Simon Moe

Introduction

Branched-chain amino acids (BCAAs) are essential nutrients that play a significant role in muscle metabolism and overall health. Comprised of leucine, isoleucine, and valine, BCAAs cannot be synthesized by the body and must be obtained through diet. Recent research has highlighted how the metabolic pathways are influenced by BCAAs, such as their ability to activate mTOR signaling, which is vital for muscle protein synthesis (Choi, 2024). Beyond muscle growth, BCAAs may support cognitive function and metabolic health, with ongoing research exploring their broader benefits in disease management. This article explores the diverse roles of BCAAs and their impact on health and diseases

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The Brilliance of BHB: A Key Ketone Body in Metabolic Health

Posted on by Simon Moe

Introduction

β-Hydroxybutyrate (BHB), the most abundant ketone body, is a crucial molecule that sustains energy production during periods of glucose deprivation. Whether you are fasting, adhering to a ketogenic diet, or simply interested in metabolic flexibility, BHB offers key insights into how our bodies adapt to alternative energy sources. This article will delve into how BHB is produced, the diverse roles it plays, and its implications for health and disease.

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Visualize Protein:Protein Interactions with Bioluminescence Imaging

Posted on by Simon Moe

If you’re familiar with bioluminescence, you’ve probably used it in plate-based experiments to track various biological processes. You understand it provides distinct advantages over traditional fluorescence assays, particularly when it comes to sensitivity. However, there’s always that one nagging question: how representative is the signal on a cell-to-cell level?

Traditional approaches to decipher cell-to-cell signal rely on complex, time-intensive measures that only approximated the findings acquired through bioluminescence. That’s where the GloMax® Galaxy Bioluminescence Imager comes in. Built to visualize the NanoLuc®-based assays we engineered, the Galaxy goes beyond simple numeric outputs to reveal what’s happening in individual cells.

NanoLuc® luciferase is up to 150X brighter than firefly or Renilla luciferase, giving it the sensitivity to detect subtle protein interactions in complex biological systems. This bright luminescent enzyme enables a much broader linear range than fluorescence, improving detection of small changes in protein activity, such as proteins interacting. Microplate readers measuring NanoLuc® assays rely on signal generated from many cells. This results in an approximation of what is occurring biologically. Truly validating those luminescent readings within a cell population has been challenging—until now. The GloMax® Galaxy allows you to perform bioluminescence imaging, moving beyond the numbers, offering the power to visualize protein interactions directly.

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Immunometabolism: The Dynamic Interplay of Cytokines and Metabolites

Posted on by Kendra Hanslik

Immunometabolism is the study of how metabolic processes influence immune cell functions and how immune responses, in turn, shape cellular metabolism. This field examines the roles of cytokines and metabolites, which act as signaling molecules and energy sources, respectively. Cytokines can trigger or modulate metabolic pathways in immune cells, affecting their activation, growth, and response capabilities. Similarly, metabolites provide the necessary energy and building blocks that enable immune cells to proliferate, function optimally, and sustain their activity during immune responses. This dynamic interplay is crucial for maintaining health and combating disease. Together, cytokines and metabolites orchestrate a complex network that links metabolic health with immune competence on a systemic and cellular level. This blog discusses how cytokines and metabolites not only influence but also drive immune cell functions, revealing new avenues for therapeutic interventions across a range of diseases.

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Rethinking Cell Proliferation Assays

Posted on by Simon Moe

In the field of cancer research, accurately measuring cell proliferation is crucial for assessing the efficacy of therapeutic agents. This is particularly difficult with CDK 4/6 inhibitors, which arrest cells in the G1 phase without stopping their growth. This continued growth can skew results from proliferation assays which rely on factors that naturally scale with cell growth. These include mitochondrial activity (ATP levels), total cell protein, or mRNA as measured through the PRISM molecular barcoding strategy. Even though these cells are not dividing, the increase in these measurements can misleadingly suggest active proliferation.

There is growing awareness among researchers of these challenges.  A recent study highlights these limitations by demonstrating the discrepancies that arise when using metabolic assays to assess cell proliferation after treatment with drugs that induce cell cycle arrest. This blog post delves into the study’s implications and demonstrates how one of Promega’s latest developments is poised to address these challenges.

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