When targeting a specific protein with a small molecule drug in order to treat a disease, scientists often use a molecule that mimics the natural substrate of the enzyme and targets the active site. However, this approach has met with limited success in the case of the oncogenic GTPase K-Ras. GTPases are regulatory proteins that act like binary switches for cellular pathways. In its “on” state, K-Ras is bound to GTP and activates signaling cascades responsible for cell growth, survival, and differentiation. When GTP gets hydrolyzed to GDP, K-Ras is turned “off.” Mutations that prolong the lifetime of GTP when bound to K-Ras, such as the G12C (glycine at position 12 is changed to cysteine) mutant, are highly oncogenic and lead to cancer. The high affinity of K-Ras for GTP and GDP makes drug targeting of the K-Ras active site difficult, but researchers Ostrem, Peters, et al. have discovered an alternate site on K-Ras that can be targeted for cancer therapies.
The researchers set out to find a small molecule that could specifically bind to the oncogenic G12C mutant protein while avoiding the wild type K-Ras by screening a disulfide library, which would be expected to react with the thiol group of the cysteine. Intact protein mass spectrometry revealed which compounds bound to the G12C mutant without targeting the wild type. The two strongest binders were unaffected by the presence of excess GDP, indicating that they do not compete with GDP for binding. X-ray crystallography showed that one of the strong binders was binding in a previously allosteric pocket of K-Ras.
In order to further characterize the novel allosteric site, the researchers examined libraries containing electrophiles, acrylamides, and vinyl sulphonamides for G12C K-Ras binding. Co-crystals of potent binders with K-Ras revealed that the switch-I and switch-II domains of the protein are disrupted, which also disturbs magnesium ion binding. Previously studied mutations in the residues that coordinate the magnesium ion result in a preference for GDP over GTP, thus the researchers tested the compounds for this activity as well. Indeed, exchange assays reveal a shift in K-Ras’s preference from GTP to GDP when the potent electrophiles are bound. Additionally, the compounds can block nucleotide exchange by exchange factors, though EDTA still effectively catalyzes the exchange of GDP for GTP.
It was also noted that the potent compounds occupied a position normally reserved for G60 when K-Ras is active. Known mutants of G60 have impaired binding to partner effector proteins such as Raf. Studies in cell lines show that compound binding impairs the association of K-Ras with Raf. Lastly, in order to show the effectiveness of the identified compounds as chemotherapeutic drugs, the researchers treat various cancer cell lines, some of which contain the G12C mutation. As expected, the cells with the mutation demonstrated significantly decreased viability in the presence of the compounds.
Overall, this is an elegant approach to small molecule drug development that fortuitously revealed a novel regulatory site of K-Ras. Drugs that target this site can be designed specifically for oncogenic mutations, and do not have to overcome the significant barrier of trying to out compete GDP and GTP for binding. The extensive crystal structure and enzymatic characterizations lay the groundwork for further drug development on K-Ras and may open up a whole new class of chemotherapeutic drugs.