drug discovery

Genome-Wide CRISPR Screening: Putting Death on Hold

Posted on by Ken Doyle

We share this planet with approximately 8.7 million species of plants and animals. Within such a diverse environment, it’s only natural that many complex relationships have developed among different species. Some relationships are mutually beneficial, some are parasitic—and some are lethal.

Genome wide - crisper screening to help with toxic compounds to humans

Natural toxins and venoms are biologically active compounds produced by normal metabolic processes in an organism but are harmful to other organisms. Typically, toxins are encountered passively or ingested by the affected organisms, and have a specific mode of action and binding site within a cell. In contrast, venoms are introduced directly into the victim through a specialized delivery mechanism, and they may consist of a mixture of compounds that affect a range of cell types and tissues (1). Both types of poisons are produced for predation, defense, or to offer a competitive advantage (1).

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PARP and DDR Pathways: Targeting the DNA Damage Response for Cancer Treatment

Posted on by Ken Doyle

Our cells, and the DNA they contain, are under constant attack from external factors such as ionizing radiation, ultraviolet light and environmental toxins. Internal cellular processes can also generate metabolites, such as reactive oxygen species, that damage DNA. In most cases, DNA damage results in permanent changes to DNA molecules, including DNA mismatches, single-strand breaks (SSBs), double-strand breaks (DSBs), crosslinking, or chemical alteration of bases or sugars. If left unchecked, DNA damage can cause genome instability, mutations and aberrant transcription, and oncogenic transformation.

PARP DDR pathway for drug discovery

Fortunately, our cells have also evolved multiple pathways to repair damaged DNA, collectively known as the DNA damage response (DDR). The type of repair mechanism depends on the nature of the damage, and whether the damage occurs in mitochondrial or nuclear DNA. These mechanisms have been reviewed extensively (1,2). Recently, considerable attention has focused on pathways for repairing SSBs and DSBs, mediated by the ADP-ribosylating enzyme known as poly (ADP-ribose) polymerase 1, or PARP-1.

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MISpheroID: A Knowledgebase to Improve Reproducibility in Spheroid Research

Posted on by Johanna Lee
Spheroid research is now a common component of cell biology and drug discovery science

Advantages of Spheroids

In the past decade, there has been a sharp rise in studies using spheroids as cell models for basic research and drug discovery. Spheroids are self-organized aggregation of cells that form a spherical mass, and they have become widely popular because they are much more physiologically relevant compared to flat 2D cell cultures.

In spheroids, the inner cells have less access to nutrients and oxygen compared to the outer layer, forming a natural gradient. As a result, metabolite concentration and cellular state such as proliferation and differentiation, can be very different at the periphery compared to the inner core. This phenomenon, known as “heterogeneity”, makes 3D tumor spheroids much more representative of actual tumors in the human body.

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Save Precious Time with Same-Well Multiplexing

Posted on by AnnaKay Kruger
Scientist performing a multi-well assay. Same-well multiplexing enables you to look at one event from several perspectives.

A graduate student believes he has mastered the art of “the assay”. No need to run duplicates, he knows exactly which one will get him the answers he needs right away.  

To challenge this, his PI proposes an exercise. He asks of the graduate student, “What happens when you treat cells with doxorubicin?”

The graduate student raises his cells, treats them accordingly, and decides to run a cell viability assay to determine their fate. He returns to the PI with the final verdict: his cells are dead.

The PI takes a look at the data and asks the graduate student to repeat the experiment with an additional assay for cytotoxicity―but the cytotoxicity assay shows that the cell membranes are intact, which only puzzles the graduate student. The PI asks him to run a third assay for apoptosis, and when the student does so, it becomes clear that the cells are dying.

The PI uses this opportunity to make his point: “Now do you see why I ask for more than one assay?”

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Permeability Possibilities with the New NanoClick Assay

Posted on by Natalie Larsen

Peptide Predicament

For decades now, peptides have been a molecule of interest for drug discovery research. Peptides offer a unique opportunity for therapeutic intervention that closely mimics natural pathways, as many physiological functions utilize peptides as intrinsic signaling molecules. Macrocyclic peptides, in particular, have recently proven to be promising candidates for targeting intracellular protein–protein interactions (PPIs), an attractive but hard-to-reach therapeutic target for conventional small molecule and biological drugs.  

As with any opportunity, there are also challenges that accompany the peptide therapeutic development. Peptide ligands typically have poor membrane permeability, so thus far the majority of peptide therapeutics predominantly target extracellular proteins and receptors. There are also multiple mechanisms for cellular uptake of peptides, including both energy-dependent routes like endocytosis, and energy-independent, like passive diffusion or membrane translocation. Multiple mechanisms of cellular uptake paired with poor permeability makes engineering enough membrane permeability into peptides in order to advance them through drug discovery pipelines extremely difficult.  

There are other factors to consider in developing peptide therapeutics, such as solubility, protein/lipid binding and stability, which can also have an affect on the overall cytosolic concentration and, ultimately impact the ability of the peptide to effectively engage its desired intracellular targets.

With so many challenging factors, the ability to have a predictive, high-throughput assay to assess cell permeability, independent of the mechanism(s) of entry, would be a critical and invaluable tool to support peptide drug discovery research.

In a recent study published in ACS Chemical Biology, researchers sought to develop such a tool, and demonstrated a new application for Promega NanoBRET™ technology: the NanoClick assay.

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Overcoming Challenges to Detect Apoptosis in 3D Cell Structures

Posted on by Promega

This blog is written by guest author, Maggie Bach, Sr. Product Manager, Promega Corporation.

Researchers are increasingly relying on cells grown in three-dimensional (3D) structures to help answer their research questions. Monolayer, or 2D cell culture, was the go-to cell culture method for the past century. Now, the need to better represent in vivo conditions is driving the adoption of 3D cell culture models. Cells grown in 3D structures better mimic tissue-like structures, better exhibit differentiated cellular functions, and better predict in vivo responses to drug treatment.

Switching to 3D cell culture models comes with challenges. Methods to interrogate these models need to be adaptable and reliable for the many types of 3D models. Some of the most popular 3D models include spheroids grown in ultra-low attachment plates, and cells grown in an extracellular matrix, such as Matrigel® from Corning. Even more complex models include medium flow over the cells in microfluidic or organ-on-a-chip devices. Will an assay originally developed for cells grown in monolayer perform consistently with various 3D models? How is measuring a cellular marker different when cells are grown in 3D models compared to monolayer growth?

Close up of cells in 3D culture apparatus. 3D Cell Structures Provide Challenges for Measuring Markers of Cellular Activitiy
3D Cell Structures Provide Challenges for Measuring Markers of Cellular Activitiy
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RAS-Targeted Drug Discovery: From Challenge to Opportunity

Posted on by Ken Doyle
cancer cell, ras-targeted drug discovery

In 1963, Jennifer Harvey was studying Moloney murine leukemia virus (MMLV) at the cancer research department of the London Hospital Research Laboratories. After routine transfers of plasma from MMLV-infected rats to mice, she made an unusual discovery. In addition to the expected leukemia, the mice that received the plasma developed solid tumors (soft-tissue sarcomas), primarily in the spleen (1). A few years later, Werner Kirsten at the University of Chicago observed similar results working with mouse erythroblastosis virus (MEV) (2).

Subsequent research, with the advent of genome sequencing, showed that a cellular rat gene had been incorporated into the viral genome in both cases (3). These genomic sequences contained a mutation later shown to be responsible for the development of sarcomas, and the word “oncogene” became a common part of the vocabulary in cancer publications during the early 1980s (4). Harvey’s discovery led to the naming of the corresponding rat sarcoma oncogene as HRAS, while Kirsten’s related oncogene was named KRAS. Several laboratories, working independently, cloned the human homolog of the viral HRAS gene in 1982 (3). The human KRAS gene was cloned shortly thereafter, as well as a third RAS gene, named NRAS (3). Additional studies showed that a single point mutation in each of these genes led to oncogenic activation, and they have been popular targets for anticancer drug discovery efforts ever since.

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RNA-Protein Interactions: A New Frontier for Drug Discovery

Posted on by Jordan Villanueva

Almost 90% of the human genome is transcribed into RNA, but only 3% is ultimately translated into a protein. Some non-translated RNA is thought to be useless, while some play a significant yet often mysterious role in cancer and other diseases. Despite its abundance and biological significance, RNA is rarely the target of therapeutics.

“We say it’s undruggable, but I would say that ‘not-yet-drugged’ is a better way to put it,” says Amanda Garner, Associate Professor of Medicinal Chemistry at the University of Michigan. “We know that RNA biology is important, but we don’t yet know how to target it.”

Amanda’s lab develops systems to study RNA biology. She employs a variety of approaches to analyze the functions of different RNAs and study their interactions with proteins. Her lab recently published a paper describing a novel method for studying RNA-protein interactions (RPI) in live cells. Amanda says that with the right tools, RPI could become a critical target for drug discovery.

“It’s amazing that current drugs ever work, because they’re all based on really old approaches,” Amanda says. “This isn’t going to be like developing a small molecule kinase inhibitor. It’s a whole new world.”

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Measuring Changing Metabolism in Cancer Cells

Posted on by Isobel Maciver

Because of the central role of energy metabolism in health and disease, and its effect on other cellular processes, assays to monitor changes in cellular metabolic state have wide application in both basic research and drug discovery. In the webinar “Tools for Cell Metabolism: Bioluminescent NAD(P)/NAD(P)H-Glo™ Assays” Jolanta Vidurigiene, a Senior Research Scientist at Promega, introduces three metabolism assays for measuring oxidized and reduced forms of NAD and NADP.

In this webinar, Jolanta provides background information on why it is important to be able to accurately measure metabolites such as NAD/NADH and NADP/NADPH. She outlines the roles of each, and highlights some of the challenges involved in developing assays that can accurately measure these metabolites. She discusses key considerations for successful NAD(P)/NAD(P)H assays and provides examples of how to use these assays to measure either total (both oxidized and reduced) forms of NAD and NADP, or to measure oxidized and reduced forms individually in a single assay plate.

NAD(P)H-Glo™ Assay Mechanism
NAD(P)H-Glo™ Assay Mechanism

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Paving New Ways for Drug Discovery & Development: Targeted Protein Degradation

Posted on by Riley Bell

The Dana-Farber Targeted Protein Degradation Webinar Series discusses new discoveries and modalities in protein degradation.

In this webinar, Senior Research Scientist, Dr. Danette Daniels, focuses primarily on proteolysis-targeting chimeras, or PROTACs. A variety of topics are covered including the design, potency, and efficacy of PROTACs in targeted protein degradation. Watch the video below to learn more about how PROTACs are shifting perspectives through fascinating research and discoveries in targeted protein degradation.

Learn more about targeted protein degradation and PROTACS here.