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A New Approach to Targeting Ras

By: Carol A. Rouzer, VICB Communications
Published: February 27, 2014

Small molecules that bind to a complex of Ras and SOS modulate Ras activation and inhibit tumor cell growth.

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 guanine nucleotide exchange factors (GEFs) 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, facilitated by GTPase activating proteins, 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 factor 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 been met with limited success due to the lack of binding pockets on the protein. Now, Vanderbilt Institute of Chemical Biology member Stephen Fesik and his laboratory have taken a new approach to target Ras by discovering a small molecule that binds to a complex between Ras and the GEF SOS (son-of-sevenless homolog) [M. C. Burns et al. (2014) Proc. Natl. Acad. Sci. U.S.A., published online Feb. 18, DOI: 10.1073/pnas.1315798111].

Prior work in the Fesik lab had led to the discovery of small molecules that bind directly to Ras and inhibit nucleotide exchange [Q. Sun et al. (2012) Angew. Chem. Int. Ed. Engl., 51, 6140). During the course of this work, the investigators also discovered molecules, such as compound 1 (Figure 2), that increase the Ras nucleotide exchange rate stimulated by a catalytically active form of SOS (SOScat). Following up on this interesting finding, they synthesized analogs of the active compounds in search of molecules with greater potency. The results yielded active compounds 2, 3, and 4, and inactive compound 5, all of which contained the original aminopiperidine indole core structure and a tryptophan moiety (Figure 2).

Figure 2. Structures of molecules that potentiate SOS-mediated nucleotide exchange in Ras. Replacement of the glycine moiety of the aminopiperidine indole compound 1 yielded the more potent compound 2. Addition of indole substituents produced compounds 3 and 4 with greater increases in activity. Addition of a carbonyl to the linker between the indole and piperidine moieties produced inactive compound 5. Values are given for the percent increase in SOScat-mediated Ras nucleotide exchange obtained with 100 μM compound, and the EC50 for nucleotide exchange activation.

Mechanistic studies revealed that the active molecules had no effect on Ras’s intrinsic rate of guanine nucleotide exchange in the absence of SOScat, nor did they bind directly to Ras. Failure of the active compounds to enhance nucleotide exchange by Ras in the presence of the GEF Ras-GRF1 suggested that their activity was both SOS-dependent and SOS-selective.

SOS is unique among known GEFs in that it has two binding sites for Ras: a catalytic site that mediates nucleotide exchange and an allosteric site that increases the activity of the catalytic site. GTP-Ras is more effective than GDP-Ras as an allosteric activator of SOS. Studies using SOS mutants revealed that the allosteric site is not required for the activity of compound 4. Indeed, compound 4 could further stimulate nucleotide exchange mediated by wild-type SOScat even under conditions of maximal allosteric activation.

To better understand the mechanism of action of the nucleotide exchange-activating compounds, the Fesik group prepared co-crystal structures of the compounds with a ternary complex of H-Ras, SOScat, and H-RasY64A, a mutant form of Ras that binds to the allosteric site but does not undergo nucleotide exchange. The structures revealed that the compounds bind in a hydrophobic pocket that is formed between the CDC25 catalytic domain of SOS and the Switch II region of Ras (Figure 3). Mutations of residues in the SOScat portion of this pocket that were predicted to disrupt compound binding yielded proteins that were no longer sensitive to the stimulatory effect of the nucleotide exchange-activating compounds.


Figure 3. Results from X-ray crystallography of compounds with a ternary crystal structure of H-Ras, SOScat, and H-RasY64A. (A) Representation of the full crystal structure showing Compound 2 (green), SOScat (orange), H-RasY64A bound to a stable GTP analog (gray with the switch region in blue) in the allosteric site, and nucleotide-free H-Ras (gray with switch region in red) in the catalytic site. (B) Close-up of the hydrophobic binding pocket showing important residues. (C, D, E) Surface diagrams of compounds 1, 2, and 3, respectively, in the binding pocket. Reproduced with permission from M. C. Burns et al. (2014) Proc. Natl. Acad. Sci. U.S.A., published online Feb. 18, DOI: 10.1073/pnas.1315798111. Copyright 2014, Burns et al.

The investigators incubated HeLa cells with FITC-labeled compound 4. Subsequent detection of the fluorescent label in the cells demonstrated uptake of compound 4, indicating that it could be used for cell culture studies (Figure 4). The nucleotide exchange-stimulating activity of compound 4, led the researchers to hypothesize that it would stimulate Ras-dependent signaling in the cells. As expected, cells treated with compound 4 demonstrated a significant increase in GTP-Ras levels. However, when the investigators evaluated the effects of compound 4 on activation of the mitogen-activated protein kinase (MAPK) pathway, a downstream target of Ras, they found a biphasic response, with activation at lower compound concentrations and inhibition at high concentrations. Even more surprising was the finding that the downstream phosphoinositide 3-kinase (PI3-K) pathway was inhibited at all concentrations of compound 4 that they tested. When cells were treated with epidermal growth factor (EGF) to stimulate Ras-dependent signaling, compound 4 at concentrations of 100 μM suppressed MAPK activation though it had no effect on EGF-dependent responses upstream of Ras. As might be predicted from its inhibitory effect on Ras signaling, compound 4 inhibited cell proliferation and anchorage-independent growth of cancer cells that expressed either wild-type or mutant Ras (Figure 4).


Figure 4. (A) HeLa cells incubated with FITC-labeled compound 4 show uptake of the fluorescent label. DAPI stain shows the position of nuclei. (B) Compound 4 (red) inhibits cell proliferation (solid symbols) and anchorage independent growth (open symbols) in cells expressing wild-type Ras (HeLa and CHL-1) and in cells expressing mutant Ras (SK-MEL-2 and PANC-1). Inactive compound 5 (black) was much less effective. Reproduced with permission from M. C. Burns et al. (2014) Proc. Natl. Acad. Sci. U.S.A., published online Feb. 18, DOI: 10.1073/pnas.1315798111. Copyright 2014, Burns et al.

The results lay the foundation for further study of a new kind of Ras activity modulator that binds to Ras-protein complexes. Based on the stimulatory activity of the compounds on guanine nucleotide exchange, their ability to inhibit Ras signaling is somewhat unexpected. However, the researchers note that a similar biphasic effect of B-Raf inhibitors on MAPK signaling has been reported. B-Raf is a signaling kinase in the MAPK pathway that is downstream of Ras. The crystal structure data reveal important residues in SOS that make up the hydrophobic pocket formed when Ras interacts with the catalytic site of SOS. It is interesting to note that some of these residues have been identified as sites of mutation in patients suffering from hereditary defects in Ras signaling that lead to developmental disorders, such as Noonan syndrome. The structural data also provide key information that will guide further improvements in binding affinity as the Fesik lab continues its search for the next generation of these novel Ras activation modulators.







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