ras

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.

Continue reading “RAS-Targeted Drug Discovery: From Challenge to Opportunity”

High-Throughput Drug Screening Using 3D Cell Cultures

Posted on by Johanna Lee

For a long time, the drug industry has relied on flat 2D cell cultures grown on a plate to screen for potential drugs. However, 2D models do not accurately reflect the native environment of cells in vivo. 3D cell cultures, on the other hand, better represent the numerous cell-cell and cell-matrix interactions and hypoxic conditions that have a profound effect on the behavior of cells. In a 2018 study published in Oncogene, Kota et al. developed a high-throughput 3D spheroid-based screening assay to identify drug candidates that target RAS proteins.

RAS proteins are GTPases that transmit extracellular signals into cellular signaling pathways, which could activate cell proliferation, differentiation and survival mechanisms. Oncogene mutation in the three human RAS genes (HRAS, NRAS and KRAS) are found in 30% of all cancers, making RAS proteins the most common oncogene. In fact, mutations in KRAS are found in >90% of pancreatic cancers. Despite the prevalence of RAS mutations, targeting RAS proteins with drugs is extremely challenging due to the complex nature of the protein.

The authors in this study wanted to test a new approach using a 3D spheroid-based screening assay to find drugs that target RAS proteins. They first harvested 2D monolayer cultures of pancreatic epithelial tumor cells that express either wild-type KRAS or mutant oncogenic KRAS, and tested their ability to form 3D spheroids. They confirmed spheroid growth using the CellTiter-Glo® 3D Cell Viability Assay with linearity of detection in the range of 1,000–10,000 cells seeded.

The 3D spheroids were then treated with a library of 1,280 known drugs. From the high-throughput screen, they identified one compound with the greatest selective inhibition against oncogenic KRAS. The compound is called Proscillaridin A, a cardiac glycoside that is known for treating congestive heart failure and cardiac arrhythmia. In 3D spheroids, Proscillardin A inhibited oncogenic KRAS at a >90% inhibition rate, with <10% inhibition of wild-type KRAS. In 2D cultures, however, there was no selective inhibition of oncogenic KRAS (inhibition rates for both oncogenic and wild-type KRAS were about 50%). This means that Proscillaridin A would not have been identified as a candidate if the screen was done using only 2D cultures.

Next, the authors wanted to determine how Proscillaridin A impacts tumor cell viability. Could it induce apoptosis in tumor cells? To test this, they used the RealTime-Glo™ Annexin V Apoptosis Assay. This bioluminescent assay is able to detect apoptosis in real time, based on the exposure of phosphatidylserine on the outer leaflet of the cell membrane when apoptosis occurs. Using this assay, they found that Proscillaridin A induced apoptosis at earlier time points and higher rates in 3D spheroids expressing oncogenic KRAS compared with wild-type KRAS. In 2D cultures, there was no difference in the rate of apoptosis.

This study shows that high-throughput screening in 3D spheroids can identify potential drugs that would not have been discovered in a 2D format. This provides hope for finding drugs against difficult target proteins such as RAS.

Reference: Kota S., et al. (2018) A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype. Oncogene. Epub ahead of print.

A Better GTPase Assay for Drug Development

Posted on by Sara Klink

Cover of October Issue of Assay and Drug Development Technologies featuring GTPase-Glo™ Assay.The path to drug development is strewn with obstacles: Identifying targets; configuring assays to help identify targets or drugs; uncovering the right compound to affect the selected target without off-target effects and screening multiple compounds to eliminate or identify potential drugs. Without the right tools, compounds or target, identifying potential disease therapies becomes nearly impossible.

When it comes to a drug target for cancer, the Ras protein family is at the top of the list because the proteins are expressed ubiquitiously and found mutated in many types of cancer. Because Ras proteins are involved in transducing signals from the surface of cells, many of the resulting mutations produce an activated Ras, inducing uncontrolled expression of the genes that Ras controls. Ras proteins are small GTPases (20–25kDa) that comprise a larger superfamily of proteins divided into five subfamilies: Ras, Rho, Rab, Arf, and Ran. These proteins control diverse cellular activities, including cellular differentiation, proliferation, cell division, nuclear import and export, and vesicle transport. GTPases are guanosine-nucleotide-binding proteins with affinity for GDP or GTP and are able to hydrolyze GTP. When bound to GTP, GTPases are active (turned on) and interact with downstream proteins in the signaling cascade. When GTPases are bound to GDP, the proteins are inactivated (turned off) and no longer transduce signals. Continue reading “A Better GTPase Assay for Drug Development”