GTPase targeting approach paves the way to drug discovery

Scientists at the University of California, San Francisco (UCSF) have discovered how to specifically target a class of molecular switches called GTPases. These are involved in a variety of diseases – from Parkinson's disease to cancer – and were long considered untreatable with drugs.

GTPases have largely evaded modern drug discovery, with the exception of one cancer-causing GTPase, K-Ras. On a hunch, the team led by Kevan Shokat, PhD, a UCSF professor in the Division of Cellular and Molecular Pharmacology, tested a dozen drugs that target K-Ras against a handful of GTPases they had mutated to make them more susceptible to the drugs. Their approach revealed new drug-binding sites that could not have been predicted using computational drug discovery tools.

The results open up the possibility of developing new treatment strategies for the various diseases caused by GTPase dysfunction. “We have known about GTPases for decades, but we lacked a way to reliably treat them with drugs,” said Shokat. “This really puts all of these GTPases on the drug discovery list, so it is possible to target them if they are associated with diseases.”

Shokat is lead author of the researchers’ published paper in cellentitled “Targeting Ras-, Rho-, and Rab-family GTPases.” In their article, the researchers concluded: “We systematically examined members of the Ras, Rho, and Rab family of GTPases and found that many GTPases contain targetable Switch II pockets. Notable differences in composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously untreatable family.”

Our cells rely on networks of GTPases to oversee everything from the movement of molecules to cell growth and division. “Together with their regulators and effectors, GTPases act as molecular switches that control many fundamental cellular processes,” the authors write. “The majority of these proteins belong to the Ras superfamily of small GTPases, which includes the GTPases Ras, Rho, Rab, Arf, and Ran.”

When something goes wrong with these switches, disease can result. “Ras GTPases are involved in proliferation and migration, and their aberrant regulation is associated with cancer and developmental diseases, so-called RASopathies,” the team continued. And although small molecules that specifically target individual members of the GTPase superfamily could be valuable tools to dissect signaling function and enable treatment of diseases involving GTPases, “…there are very few examples of such molecules, and unlike ATP-binding proteins, GTPases are still widely considered 'undruggable' targets.”

In 2013, Shokat and colleagues discovered a “pocket” – a cryptic allosteric pocket (switch II [SII] pocket) – where drugs could bind to K-Ras, a GTPase responsible for up to 30% of all cancers. Since then, nearly a dozen drugs have been developed to target mutations in K-Ras, but the other GTPases have remained untouched.

In their newly published study, Shokat's team, led by Johannes Morstein, PhD, a postdoctoral fellow at UCSF and first author, inserted one of the cancer-causing K-Ras mutations, G12C, into a representative group of GTPases. They suspected that G12C, which attaches a chemical “hook” to a protein, might help them figure out which of the ten K-Ras G12C drugs could bind to other GTPases that bear little resemblance to K-Ras itself. “To investigate the ability of K-Ras (G12C) inhibitors to target other GTPases, we introduce the corresponding cysteine ​​mutations into the GTPases of interest,” they explained.

Their laboratory experiments yielded gold: With the help of G12C, some of the K-Ras drugs bound to the otherwise structureless GTPases. After removing G12C, these drugs still bound to the GTPase.

The chemical-genetic approach took advantage of the flexibility of GTPases and allowed the drugs to poke open the SII pocket in the protein where they could lodge. Previous attempts to computationally predict where drugs might bind had failed to reach the pocket. “Here we show that a targetable cryptic SII pocket exists in many GTPases beyond K-Ras,” the team noted.

“Because these GTPases switch between 'on' and 'off' states, the pocket is usually not visible, especially to the standard software used for drug discovery,” Shokat said. “Instead, the drug binds to an intermediate state that freezes and inactivates the GTPases.”

The authors further stated: “…The ability to covalently target genetically engineered cysteine-containing mutants of other GTPases holds great potential for chemical genetic approaches to selectively study GTPases in a proteome.”

The researchers are sharing their methods openly in the hope that others will use them to target the GTPase of their interest, whether it's a Rab GTPase linked to Alzheimer's disease or a Rac GTPase involved in breast cancer. Among the hundreds of GTPases, there is great potential to make progress for patients. “For those interested in GTPases not explicitly studied here, we have included a flow chart for sequence analysis and assignment of candidate inhibitors based on sequence alignment of each small GTPase family member to K-Ras,” they wrote.

“With these enzymes, it was critical for us to test our ideas experimentally in the lab first to actually see what works,” Morstein said. “We hope this can really accelerate drug discovery.”