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Discoveries Featured

Targeting Cancer Through K-Ras

By: Carol A. Rouzer, VICB Communications
Published: May 30, 2012

The discovery of small molecule inhibitors of K-Ras activation could pave the way for a new class of targeted anti-cancer drugs.

The Ras family of proteins comprises a group of small GTPases that plays an important role in cell growth, differentiation, and survival. Ras proteins exist in an inactive, GDP-bound form and an active, GTP-bound form. Conversion of inactive GDP-Ras to active GTP-Ras is mediated by GTP exchange proteins in response to cell signaling events. GTP-Ras then promotes the activity of a wide range of effector kinases and related signaling enzymes. Hydrolysis of GTP regenerates GDP-Ras, bringing an end to the response (Figure 1).

Figure 1. Diagram of RAS pathway signaling.  In this example, a tyrosine kinase growth factor receptor (RTK) binds its ligand (L), and dimerizes.  This activates the kinase, leading to autophosphorylation of the receptor.  The adaptor protein Grb2 then binds to the phosphate groups and recruits the GTP exchange protein Sos.  Sos binds the inactive Ras-GDP, promoting conversion to the active Ras-GTP.  The activated Ras protein, in turn, activates numerous effector proteins, including PI3-K, MEKK1, Raf, RalGEF, and PLC, which leads to a wide range of cellular responses.

Because of its role in cell growth and survival, aberrant Ras signaling can contribute to carcinogenesis. In fact, mutations in RAS genes are among the most common in malignant tumors, being present in up to 30% of human cancers. Consequently, inhibitors of excessive Ras signaling are of great interest as potential cancer chemotherapeutic agents. However, attempts to discover small molecules that bind to Ras proteins and interfere with their function have met with limited success. Now the outlook is improving through the work of Vanderbilt Institute of Chemical Biology member Stephen Fesik and his laboratory, who report the discovery of small molecules that bind to the K-Ras protein and inhibit its activation [Q. Sun et al. (2012) Angew, Chem. Int. Ed., published online May 8, DOI: 10.1002/anie.201201358].

A major challenge facing investigators who seek to discover Ras inhibitors is the fact that Ras proteins function primarily through interactions with other proteins. These interactions occur over large regions of the protein’s surface. Consequently, the “active sites” of Ras proteins lack the small, well-defined binding pockets found on enzymes and hormone receptors that are the usual targets of small molecule inhibitors. This has led to the conclusion that proteins such as Ras are “undruggable”. Refusing to accept this defeatist conclusion, Steve Fesik has invested his career, first at Abbott Laboratories and now at Vanderbilt, in developing methods to discover small molecule modulators of “undruggable” proteins. His approach begins by identifying small “fragment” molecules that interact with the target protein’s surface, and then using structural information to rationally enlarge or link fragments until a high-affinity, biologically effective molecule is obtained.

To identify small molecules that bind to K-Ras, the Fesik lab used a high-throughput NMR-based screen of a library containing 11,000 fragments. They first expressed large quantities of a constitutively active mutant K-Ras (G12D) that was uniformly labeled with 15N. The presence of the 15N label allowed them to use HSQC (heteronuclear single quantum correlation) NMR, which provides a spectrum of signals representing each hydrogen atom that is attached to a nitrogen. Because all amino acids except proline contain at least one N-H bond, the HSQC spectrum provides an information-rich picture of the protein. Of particular importance, the binding of a small molecule causes a shift in the signals of atoms involved in the binding interaction (Figure 2). Thus, changes in the HSQC spectrum provide a sensitive indicator of ligand binding and can also yield information on the specific region of the protein where binding occurs. Using this screen, the investigators identified 140 molecules that bound to G12D K-Ras with affinities in the range of 1.3 to 2 mM. Structural features common to many of the “hit” molecules were the presence of an indole, phenol, or sulfonamide group. These molecules also bound to wild-type K-Ras, the constitutively active G12V K-Ras, and wild-type H-Ras.

Figure 2. 1H-15N-correlation spectra of a uniformly 15N-labeled oncogenic GDP-K-Ras mutant (G12V) in acquired in the absence (black) or presence (red) of a small molecule ligand (inset).  The presence of the ligand causes shifts in the position of signals from regions of the molecule involved in the binding interaction.  Figure kindly provided by the Fesik Laboratory..

To obtain structural information necessary to improve binding affinity, the Fesik lab obtained X-ray crystal structures of the most promising compounds bound to wild-type and/or G12V K-Ras (Figure 3). The structures revealed that the molecules interacted with a hydrophobic pocket located between the α2 helix (amino acids 60 - 74) and the central β-sheet of the protein. This binding pocket was not visible in the ligand-free form of the protein.

Figure 3.  Ribbon and molecular surface representations of the X-ray structures of Ras-GDP bound to four different “hit” ligands.  Reproduced with permission from Sun et al. (2012) Angew. Chem. Int. Ed., published online May 8, DOI: 10.1002/anie.201201358. Copyright 2012 John Wiley & Sons.

Rather, a conformational change involving movement of Tyr-71 and Met-67 leads to opening of the pocket and creation of a secondary binding cleft that is electronegative, due to the presence of Glu-37 and Asp-38 (Figure 4). Further investigations of the K-Ras structure under other experimental conditions suggested that the “open” and “closed” conformations, in which the pocket was present, or absent, respectively, exist in equilibrium when the protein is in solution.

Figure 4. Electrostatic surface representation of the “closed” conformation of GDP-K-Ras (a), and the “open” conformation (b) showing the primary hydrophobic binding pocket and the electronegative (red) secondary binding cleft.  Reproduced with permission from Sun et al. (2012) Angew. Chem. Int. Ed., published online May 8, DOI: 10.1002/anie.201201358. Copyright 2012 John Wiley & Sons.

The structural data revealed an important H-bond interaction between the ligands and the protein, which was confirmed by the low affinity of molecules lacking the necessary -NH or -OH substituents. The electronegativity of the secondary binding pocket suggested that positively charged molecules should exhibit increased binding affinity. This hypothesis was confirmed through the synthesis of analogues containing positively charged amine groups. The best of these analogues, comprising an indole nucleus and an isoleucine side-chain (Figure 5) exhibited a binding affinity for wild-type K-Ras of 190 μM.

Figure 5. Structure of the most active K-Ras activation inhibitor.

The binding of a small molecule to K-Ras is of little value unless it affects the protein’s function. Thus, the Fesik lab tested the effects of their compounds in an assay of K-Ras activation. This assay measures the exchange of fluorescently labeled BODIPY-GDP for unlabeled GTP catalyzed by the exchange factor Sos. The assay showed that all of the small molecules that exhibited an affinity constant of less than 500 μM for K-Ras binding inhibited Sos-mediated nucleotide exchange. The most potent molecule (Figure 5) inhibited K-Ras activation by 78%. HSQC NMR spectra confirmed that the presence of an inhibitor blocks K-Ras-Sos binding. Molecular modeling of the X-ray crystal structure of an inhibitor-K-Ras complex suggests that the bound inhibitor interferes with the interaction between K-Ras and Sos.

These exciting results suggest that small molecule inhibitors of K-Ras function can be discovered and that Ras proteins may be druggable after all. This current series of molecules show great promise as tools to explore K-Ras function and its role in the malignant phenotype. To the end of developing a K-Ras-targeted therapeutic agent, the Fesik Lab is actively working to make further improvements in binding affinity and the “drug-like” properties of these molecules. Their work has also recently been highlighted in Science-Business eXchange [J. Kotz, (2012) SciBx, 5(21), DOI:10.1038/scibx.2012.536].

























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