Cracking the Undruggable Code: Top 10 Key Takeaways

Posted on by Sara Christenson

For decades, the concept of “undruggable” targets has presented one of the most significant challenges in drug discovery. At our recent virtual event, Illuminating New Frontiers: Cracking the Undruggable Code, leading researchers and industry experts gathered to showcase cutting-edge technologies and fresh perspectives that are expanding the boundaries of therapeutic development. Over three engaging days, participants explored groundbreaking advances in targeting RAS signaling, leveraging protein degradation and induced proximity strategies, and exploring RNA as a therapeutic target.

Target engagement of RAF dimer inhibitor LXH254 at RAF kinases, in complex with KRAS (blue). RAF inhibitor LXH254 engages BRAF or CRAF protomers (orange), but spares ARAF (red). Unoccupied ARAF is competent trigger downstream mitogenic signaling (lightning bolts). Red cells in the background are fluorescently labeled RAS proteins, expressed in live cells.
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Live-Cell Imaging: It’s Time to See What Else Your Luminescence Assays Can Tell You

Posted on by Kelly Grooms
luminescent cells behind a molecular structure

Luminescent live-cell assays are powerful tools for cellular biology research. They offer both qualitative and quantitative insights into processes such as gene expression, cell viability, metabolic activity, protein and small molecule interactions, and targeted protein degradation. But what if you could go beyond the numbers and actually see what is happening in your cells? With luminescent imaging, you have the opportunity to uncover more dynamic data by visualizing what happens with your cells in real time.

Why Luminescent Imaging?

Bioluminescent reporters such as NanoLuc® Luciferase reporters are well-suited for use in bioluminescent imaging studies. The extreme brightness means that exposure times can be reduced, compared to the time required for other luminescent reporter proteins. Its small size also makes it less likely to perturb the normal biology or functionality.

Another benefit of bioluminescence for imaging is the inherent stability and sustainability of the bioluminescent signal, which does not require external excitation like fluorescent tags.  This allows direct visualization of protein dynamics in living cells without the need for repeated sample excitation. The lack of external excitation also reduces the risk of phototoxicity and photobleaching, common issues that can adversely affect cell viability and signal integrity over time.

Applications Across Cellular Research

Luminescent imaging complements traditional luminescence assays by adding spatial and temporal dimensions. With luminescent live-cell imaging, researchers can visualize NanoLuc® Luciferase assays to gain a deeper understanding of the real-time cellular processes occurring in each experiment. Applications include:

  • Determining which cells provide signal
  • Analyzing mixed cell populations
  • Identifying rare events
  • Monitoring protein:protein interactions
  • Identifying protein localization and translocation
  • Tracking protein degradation and stability over time
  • Visualizing ligand:protein interactions (target engagement)

Luminescent Imaging in Action

Targeted Protein Degradation

Selectively targeting proteins for removal from the cell—instead of inhibiting protein activity—is a newer approach with therapeutic potential. In this method, the protein is targeted for degradation using the cell’s natural ubiquitin proteasome system (UPS). The degradation process is initiated by compounds such as molecular glues and proteolysis targeting chimeras (PROTACs) linking the target protein to an E3 ligase. Once this linkage occurs, the cell’s UPS does the rest.

Luminescent substrates with increased signal stability, such as the Nano-Glo® Extended Live Cell Substrate, enables researchers to image targeted protein degradation in their cells in real time. In the example shown below, Nano-Glo® Vivazine™ Live Cell Substrate was used to image degradation of the GSPT1 protein by the CC-885 degrader over 5 hours.

gif showing luminescent signal disappearing with protein degradation

Targeted protein degradation over time. HEK293 cells expressing endogenous HiBiT-tagged GSPT1 and stably expressing LgBiT were treated with CC-885 degrader or DMSO control treatment. Assayed with Nano-Glo® Vivazine™ Live Cell Substrate and imaged over 5 hours using GloMax® Galaxy Bioluminescence Imager.

Combining Luminescent and Fluorescent Imaging to Detect Protein:Small Molecule Interactions

Using bioluminescence resonance energy transfer (BRET)-based assays such as NanoBRET® assays allows you to detect protein:protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. These assays can be used to monitor changes in protein interactions over time, making them a useful tool for small-molecule screening.

The schematic below illustrates how the NanoBRET® NanoGlo® Detection Systems can be used to visualize target engagement. The cells on the left are expressing a NanoLuc® fusion protein, resulting in a luminescent signal. Adding a fluorescent small tracer (center) results in energy transfer and a fluorescent signal (right). Using an imaging platform that has luminescence and fluorescence imaging capabilities will let you see this energy transfer in action.

schematic showing cells detected by luminescent and fluorescent imaging
Detecting protein:small molecule interactions with NanoBRET® NanoGlo® Detection Systems.  HCT116 cells expressing a PRMT5–NanoLuc® fusion were supplemented with a fluorescent small molecule tracer (center panel). Before tracer addition, luminescent signal indicates energy is present on the donor protein (left; 3-minute exposures for 15 minutes). Binding of fluorescent tracer results in energy transfer and fluorescent signal (right; 3-minute exposures for 60 minutes). Images were captured on the GloMax® Galaxy Bioluminescence Imager.

Bringing the Power of Luminescent Imaging to Your Lab

glomax galaxy imager and computer screen

Having the right tools is critical to unlocking the full potential of bioluminescence imaging. The GloMax® Galaxy Bioluminescence Imager is uniquely positioned to offer researchers the power of imaging in an accessible, benchtop instrument. The Galaxy is a fully equipped microscope that can visualize output from NanoLuc® Technologies and offers luminescence, fluorescence and brightfield imaging capabilities. By offering a user-friendly platform for live-cell luminescent imaging, the GloMax® Galaxy empowers researchers to enrich their understanding of functional and dynamic cellular events across a cell population.

Conclusion

Luminescent imaging can enrich what we learn from live-cell assays and offers an unprecedented view into the dynamics of cellular processes. From monitoring drug responses to visualizing protein interactions, this technology delivers insights that go beyond the capabilities of traditional assays.

Whether you’re studying cancer biology, drug development or cellular signaling, luminescent imaging can help you uncover what’s hidden in your data and see your research in a whole new light.

Additional Resources

GloMax® Galaxy Luminescent Imager, NanoBRET® Nano-Glo® Detection Systems and Nano-Glo® Vivazine live Cell Substrate are for Research Use Only. Not for Use in Diagnostic Procedures.

Academic Access to Cutting-Edge Tools Fuels Macular Degeneration Discovery

Posted on by Jordan Villanueva

Age-related macular degeneration (AMD) is a common eye disease that can result in progressive loss of vision. While AMD typically affects older adults, a specific rare type of AMD called Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) can appear as early as the teenage years. Although ML/DHRD is rare, its study may provide insights into broader mechanisms of retinal degeneration, which could benefit millions affected by AMD.  

While the genetic cause of ML/DHRD is known, there have been no small molecule inhibitors identified that reduce the production of the disease-causing protein. However, researchers from the University of Texas Southwestern Medical Center and the University of Minnesota recently published a paper that describes a small-molecule inhibitor that addresses the primary pathology of ML/DHRD. In the paper, titled “GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology,” the team used CRISPR-engineered cell lines acquired through the Promega Academic Access Program to study production of the disease-causing protein in response to treatment with inhibitors. Their results point to future strategies for developing therapeutics at the currently incurable disease.

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Ice Age Secrets: The Discovery of a Juvenile Sabretooth Cat Mummy 

Posted on by Shannon Sindermann

In the permafrost of Siberia, a remarkable discovery has been made—a mummified juvenile sabretooth cat, Homotherium latidens, frozen in time for over 35,000 years. This discovery, made along the Badyarikha River in the Indigirka River Basin of Yakutia, Russia, offers an exciting glimpse into a species that has no modern analog (a living equivalent of something extinct) (1). For paleontologists and evolutionary biologists, it provides an unprecedented look at an ancient predator that roamed the Earth during the Ice Age. So, how is this cub mummy truly fascinating scientists?  

A Rare Find  

Homotherium Sabretooth mummy
The frozen mummy of Homotherium latidens: (A) external appearance; (B) skeleton, CT-scan, dorsal view (1).

The permafrost of Siberia is a treasure trove of Ice Age fossils, but the discovery of a mummified Homotherium cub stands out for its rarity and significance. While bones can tell us a lot about the history of an extinct species, mummies—where the animal’s soft tissues, such as fur, skin and sometimes internal organs, are preserved—offer far more detailed information. ‘Mummies’ refer to animals (or humans) that have been preserved with their soft tissues intact, often through natural or intentional processes like drying or embalming. This preservation allows scientists to gain insights into the organism’s diet, health, development and adaptations—details that bones alone can’t reveal! 

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Thanksgiving Cooking: Lessons in Chemistry

Posted on by Shannon Sindermann
Thanksgiving dinner

With Thanksgiving just around the corner, kitchens across the country will soon be alive with the sights, smells and sounds of cooking. But what if we dove deeper into those recipe books and looked beyond the instructions? You might find you’re more than just a cook; you’re quite the chemist!  

Inspired by the popular show Lessons in Chemistry, an adaptation of Bonnie Garmus’s bestselling novel, cooking is presented as applied science, encouraging viewers to think critically about their cooking and the chemical reactions that create the flavors and textures they love. In this blog, we’ll explore the chemistry behind Thanksgiving cooking, revealing how different techniques bring out the best in each dish. 

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Exploring the World Through the Promega and Eppendorf Family Exchange Program

Posted on by Riley Bell

The Eppendorf Family Exchange program offers a unique opportunity for Promega families to participate in an international exchange initiative designed to bring our global community closer together. Through this program, children of Eppendorf and Promega employees immerse themselves in new cultures, gain language skills, and forge lifelong friendships.

The program began in 2019 during the 40th anniversary of Promega when we received the generous gift of an exchange program from a friend in the industry: Eppendorf. Every year, ten children ages 14-18 have the opportunity to participate in this enriching exchange, experiencing daily life from a new cultural perspective in a different country for two to four weeks. In return, Promega families will host a child from an Eppendorf family.

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Soft Skills for the Science Lab: Develop Yourself with Promega

Posted on by Promega
Soft-skills such as presentation skills, time management and project management are essential for success in the science lab.

Preparing samples, conducting test series with cell cultures, or writing laboratory reports. Laboratory tasks cover a broad range of activities. Technical assistants support researchers in performing and evaluating experiments or carrying out laboratory tests in the medical field. A lab without them? Hard to imagine. However, it is not just scientific and technical understanding that is important. “Certain soft skills are necessary to be successful in your job. This also applies to the scientific field,” says Anette Leue, Head of Digital Marketing & Communications at Promega GmbH. “The focus is often on technical skills, while personal development is neglected. This inspired us to come up with our ‘Develop Yourself with Promega’ program.”

What is Develop Yourself with Promega?

“Develop Yourself with Promega” is a training series for laboratory personnel, focusing on personal development. It covers topics such as “How do I present my results in an interesting and structured way?” or “What do I need to make my lab more sustainable?” The aim is to expand professional competencies through soft-skill training. “At the beginning, we conducted a survey with our partner, the Life Science Learning Lab (in German Glaesernes Labor) in Berlin, among technical assistants to find out which topics are important to them,” Leue continues. These insights became the starting point for the first four trainings:

  • Green your lab: How can my lab become more sustainable?
  • Presentation training: A few steps to a good presentation
  • Project management in everyday lab work
  • Time management for technical assistants
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Unlocking the Secrets of ADP-Ribosylation with Arg-C Ultra Protease, a Key Enzyme for Studying Ester-Linked Protein Modifications 

Posted on by Michele Arduengo

Post-translational modifications of proteins are critical for proper protein function. Modifications such as phosphorylation/dephosphorylation can act as switches that activate or inactivate proteins in signaling cascades. The addition of specific sugars to membrane proteins on cells are critical for recognition, interaction with the extracellular matrix and other activities. While we know volumes about some types of protein modifications, ADP-ribosylation on aspartate and glutamate residues has been more difficult to study because of the chemical instability of these ester-linked modifications. 

Matić Lab (Eduardo José Longarini and Ivan Matić) recently published a study that explored mono-ADP-ribosylation (ADPr) on aspartate and glutamate residues by the protein PARP1 and its potential reversal by PARG. PARP1 and PARG signaling are central to DNA repair and apoptosis pathways, making them potentially powerful therapeutic targets in cancer or neurodegenerative diseases in which DNA repair processes are often disrupted. 

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Don’t Flush Your Kitty Litter! Toxoplasmosis Is a Growing Threat to Sea Otters and Other Marine Mammals

Posted on by Kelly Grooms
Sea otter in water with an overlay of Toxoplasma gondii oocysts.

Southern sea otters (Enhydra lutris nereis), endangered marine mammals along California’s coastlines, are facing an unexpected threat. The menace comes not from pollution, habitat loss or natural predators, but from a microscopic enemy—Toxoplasma gondii (T. gondii). This protozoan parasite, typically associated with domestic cats, has found its way into marine ecosystems with sometimes deadly consequences for sea otters. Recently, scientists identified transmission of virulent, atypical strains of T. gondii from terrestrial felids to sea otters along the southern California coast, with lethal consequences (1).

Understanding T. gondii and Its Hosts

T. gondii is a versatile parasite that can infect nearly all warm-blooded animals, including humans and marine mammals. However, the T. gondii lifecycle depends upon felids (e.g., domestic cats and their wild relatives) who serve as definitive hosts. It is in their intestines that the parasite completes its sexual reproductive stage. The resulting oocysts are excreted in the animals’ feces. T. gondii oocysts exhibit remarkable resilience, surviving in soil, freshwater and seawater for extended periods. They are even resistant to standard wastewater treatment processes, which means oocysts in cat waste disposed of by flushing will pass through the treatment plant and be discharged into the environment. ​(2,3).

Oocysts can also be washed from soil contaminated with cat waste and carried via storm drains and rivers into the ocean, dispersing them into coastal waters. Once there, the oocysts settle on kelp or in sediments where they can be picked up by marine invertebrates like snails, mussels and clams. Marine mammals such as sea otters become infected when they consume these contaminated invertebrates. Otters can also ingest oocysts during grooming sessions​ (1,3).

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Novel Promega Enzyme Tackles Biggest Challenge in DNA Forensics

Posted on by Jordan Villanueva

On September 25, Promega Research Scientist David Mokry addressed a full audience at the International Symposium on Human Identification. The event brings together people from the forensic DNA industry – criminalists, analysts, lab directors and more – eager to learn about advancements in the field. Over the next 20 minutes, David unveiled a novel enzyme designed to tackle a challenge that has plagued DNA forensics for decades.

Known as “Reduced Stutter Polymerase,” the new enzyme virtually eliminates confounding stutter artifacts in forensic DNA analysis. When incorporated into STR analysis kits, it will dramatically simplify mixed sample deconvolution and help forensic analysts generate accurate profiles of multiple contributors. This technology is the result of years of collaboration between the Genetic Identity R&D Group and the Advanced Technology Group at Promega.

Here’s how they did it, and why it’s so important.

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