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Chemical Approaches to Deciphering and Controlling Signal Transduction Pathways

Research Summary

Kevan Shokat has developed chemical methods to decipher the role of individual kinases and GTPases in cellular signaling networks. His goals are to understand each kinase's role in the body and to learn which kinases should be targeted to treat diseases such as cancer and immune dysfunction.

Research in my laboratory is focused on using the tools of synthetic organic chemistry, biochemistry, structural biology, and genetics to gain insight into how signaling networks transmit information in normal and disease settings.  Our guiding principle is to use chemistry to answer questions that cannot be addressed by the use of biochemistry or genetics alone—in this way we seek to provide tools which fill in the gaps left behind by more traditional approaches. 

A major thrust of the lab is to develop chemical tools enabling the identification of the direct substrates of individual protein kinases.  There are at least two challenges to identifying the direct substrates of kinases: (1) all kinases accept ATP as the phosphodonor, making distinction between individual kinases based on the cofactor ATP impossible, and (2) substrates of kinases are typically present in low abundance, making their purification and isolation from cells a challenge.  We solved the first problem by developing a modified form of ATP which is only accepted by a mutated form of the kinase of interest.  We have solved the second problem more recently by using thiophosphate as a tag for direct substrates through development of both antibody and chemical capture methods for isolation of the thiophosphate modified substrates in cells. Using these methods we and others have identified hundreds of direct kinase substrates.

Movie 1: Starting with the full domain structure of c-Src, the catalytic domain (blue) is shown, with the residues in the ATP-binding pocket (highlighted in yellow) surrounding ATP (shown as stick models). The remaining protein-protein interaction domains are shown in orange and purple. As the movie zooms into the active site, a cutaway through the surface of the protein shows the substrate ATP-binding pocket and surface of the c-Src kinase. Superimposed upon ATP is PP1, an inhibitor of the wild-type kinase. This molecule, a general inhibitor of many tyrosine kinases, fits into the active site analogously to how ATP binds.

As ATP disappears from view, the surface of the gatekeeper residue (T338 in c-Src) is highlighted in red to draw attention to the close contact with the phenyl ring of PP1. Next, an analog of PP1, 1-NAPP1, appears and makes direct contact (pokes through) the surface of the protein produced by the gatekeeper T338 residue. The PP1 (wild-type inhibitor) is removed, and the movie zooms in closer to the steric clash of 1-NAPP1 with the gatekeeper residue (red). The mutation of the gatekeeper residue to glycine (T338G) is introduced next, which allows 1-NAPP1 unimpeded access to the mutant kinase active site. The surface produced by Gly338 is much deeper and smaller than the Thr338 surface of the wild-type protein, providing the basis for exclusive selectivity for mutant protein kinases with a glycine at the gatekeeper position.

Finally the movie "rocks" to allow a three-dimensional impression of the features of the active site. The final frame of the movie represents how 1-NAPP1 encounters the conserved large gatekeeper residue in all wild-type kinases, providing exclusive specificity for engineered kinases containing an enlarged ATP-binding site.

Daniel Rauh, UCSF, HHMI postdoctoral fellow

In an attempt to develop a drug to treat Parkinson's disease we have designed a small molecule which helps protect neurons from dying due to various types of stress. Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. We have shown that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1G309D and wild-type PINK1.  Discovery of neo-substrates for kinases could provide a novel modality for regulating kinase activity.

Inhibiting oncogenic K-Ras

In the area of cancer, we have recently made a breakthrough by discovering a way to block the function of the GTPase, K-Ras.  Somatic mutations in the K-Ras gene are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to directly target this oncogene have faced difficulties due to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. We developed small molecules that irreversibly bind to a common oncogenic mutant, K-RasG12C. These compounds rely on the mutant cysteine for binding and therefore do not affect the wild type protein (WT). Using crystallography, we identified a new pocket beneath the effector binding switch-II region of Ras that was not apparent in previous structures of the molecule. Our results provide structure-based validation of a novel allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

Scientist Profile

Investigator
University of California, San Francisco
Cancer Biology, Chemical Biology