Scientists at the University of California, San Francisco (UCSF), have discovered how to target a class of molecular switches called GTPases that are involved in a myriad of diseases—from Parkinson’s disease to cancer—and have long been thought to be undruggable.
GTPases have remained largely out of reach of modern drug discovery, with the exception of one cancer-causing GTPase, K-Ras. On a hunch, the team, headed by Kevan Shokat, PhD, UCSF professor in the department 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 receptive to the drugs. Their approach revealed new drug binding sites that could not have been predicted by computational drug discovery tools.
The findings open up an opportunity to develop new treatment strategies for the diverse diseases that arise from GTPase dysfunction. “We’ve known about the GTPases for decades but have lacked any way to reliably drug them,” said Shokat “This really puts all those GTPases on the map for drug discovery, so it’s possible to target them when they’re associated with disease.”
Shokat is senior author of the researchers’ published paper in Cell, titled, “Targeting Ras-, Rho-, and Rab-family GTPases.” In their paper, the investigators concluded, “We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family.”
Our cells depend on networks of GTPases that oversee everything from the movement of molecules to cell growth and division. “Together with their regulators and effectors GTPases function as molecular switches that govern many fundamental cellular processes,” the authors wrote. “The majority of these proteins belong to the Ras superfamily of small GTPases, which comprise Ras, Rho, Rab, Arf, and Ran GTPases.”
When something goes wrong among these switches, disease can follow. “Ras GTPases are involved in proliferation and migration, and their aberrant regulation is implicated in cancers and developmental diseases, termed RASopathies,” the team continued. And while small molecules that selectively target individual members of the GTPase superfamily could be valuable tools to dissect signaling function and enable the treatment of diseases in which GTPases are implicated, “… examples of such molecules are very limited 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 that’s responsible for up to 30% of all cancer cases. Since then nearly a dozen drugs have been developed targeting mutations to K-Ras, but the other GTPases have remained untouchable.
In their newly reported study, Shokat’s team, led by UCSF postdoctoral scholar and first author Johannes Morstein, PhD, engineered one of the cancer-causing K-Ras mutations, G12C, into a representative group of GTPases. They guessed that G12C, which places a chemical “hook” onto a protein, could help them fish out which among ten K-Ras G12C drugs might bind to other GTPases, which have only subtle similarity to K-Ras itself. “To study the ability of K-Ras(G12C) inhibitors to target other GTPases, we introduce the equivalent cysteine mutations to GTPases of interest,” they explained.
Their laboratory experiments turned up gold: with the help of G12C, some of the K-Ras drugs bound to the otherwise featureless GTPases. When G12C was removed, those drugs still bound to the GTPase.
The chemical genetics approach exploited the flexibility of the GTPases, enabling the drugs to nudge open the SII pocket in the protein where it could lodge itself. The pocket had evaded previous efforts to predict, computationally, where drugs might bind. “Here, we demonstrate that a targetable cryptic SII pocket exists in many GTPases beyond K-Ras,” the team noted.
“Since these GTPases switch between ‘on’ and ‘off’ states, the pocket is not usually visible, certainly not to the standard software used for drug discovery,” Shokat said. “Instead, the drug binds to an intermediate state, freezing the GTPases and inactivating them.”
The authors further stated, “… The ability to covalently target engineered cysteine-containing mutants of other GTPases bears great potential for chemical genetics approaches to studying GTPases selectively in a proteome.”
The researchers are sharing their methods openly in the hopes that others will use them to drug their GTPase of interest, whether it’s a Rab GTPase, which is implicated in Alzheimer’s, or a Rac GTPase, which plays a role in breast cancer. Among the hundreds of GTPases, there’s rich potential to make progress for patients. “For those interested in GTPases not studied explicitly here, we include a flow chart for sequence analysis and matching of candidate inhibitors based on sequence alignment of any small GTPase family member to K-Ras,” they wrote.
“In the case of these enzymes, it was critical for us to first test our ideas experimentally in the laboratory, to actually see what worked,” Morstein said. “We’re hopeful it can really accelerate drug discovery.”
