Small GTPase Modulation: GDP Assays to Accelerate Discovery

Cancer is complex, which makes determining the culprit as well as individual treatment extremely challenging. A variety of cancer types exist with a multitude of contributors. As the second leading cause of death in the US, cancer has been a target scientists have been battling for decades. When DNA is damaged or mutations occur, cells can become cancerous, leading to unwanted maladies. Many genes can be affected and contribute to cancer, from tumor suppressor genes to oncogenes. Ras genes, particularly, are commonly found oncogenes in cancer.

The Role of a Small GTPase

Small GTPase enzymes such as those in the Ras family play a significant role in cellular functions that, when aberrant, can have deadly consequences. These enzymes are known as molecular switches since they move to and from an active GTP-bound form and an inactive GDP-bound form. In the active GTP-bound state, GTPases can bind to other proteins to regulate cellular functions involved in cell growth, survival, movement, and gene expression.²  To further complicate this signaling, several factors regulate the cycling between active and inactive small GTPase.

Guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and, for the Rho and Rab sub-families, guanine nucleotide dissociation inhibitors (GDIs) act in slightly different ways, further compounding the complexity of studying the cascade. ¹

Mutations in small GTPases have been linked to various types of cancer. Specifically, Ras GTPase mutations are found in approximately 20-30% of cancer. ³ With the multitude of proteins/pathways involved in regulating the small GTPases themselves, many opportunities exist for potential therapies. Gray et al. found several drug discovery challenges when they reviewed recent small molecule and biologics strategies to target small GTPases but noted the improvements underway.² Additionally, as we continue to understand the structural data of these small GTPases, we increase our chances of discovering molecules that target GTPase regulators.

Small GTPase Activity Assay

The universal GDP detection obtained using the Transcreener GDP GTPase Assay is perfect for measuring any small GTPase activity; therefore, an asset in discovering drugs that can inhibit and potentially be used for therapeutic purposes. The benefits and procedures for the activity assay can be found here.  Briefly, the nature of small GTPases helps transfer a molecule from a donor substrate to an acceptor substrate in an enzymatic reaction, forming GDP as a product that can be measured by using a specific antibody labeled with a fluorophore. This assay can test hundreds of samples at once, making it ideal for drug discovery.

While it has been challenging to find anti-cancer therapies that target small GTPases, particularly those within the Ras family, advances in technology and the increasing knowledge of signaling pathways and protein structural data make future attempts more likely to succeed.

References

1. Evelyn, C. R., Biesiada, J., Duan, X., Tang, H., Shang, X., Papoian, R., … Zheng, Y. (2015). Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a ras-activating enzyme. Journal of Biological Chemistry, 290(20), 12879–12898. https://doi.org/10.1074/jbc.M114.634493
2. Gray, J. L., von Delft, F., & Brennan, P. E. (2020). Targeting the Small GTPase Superfamily through Their Regulatory Proteins. Angewandte Chemie – International Edition, 59(16), 6342–6366. https://doi.org/10.1002/anie.201900585
3. Yuan Lin (Research Fellow) & Yi Zheng (Director of Experimental Hematology and Cancer Biology, Katherine Stewart Waters Endowed Chair, Co-Director of Cancer and Blood Diseases Institute, Professor of UC Department of Pediatrics) (2015) Approaches of targeting Rho GTPases in cancer drug discovery, Expert Opinion on Drug Discovery, 10:9, 991-1010, https://www.tandfonline.com/doi/abs/10.1517/17460441.2015.1058775