Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange

Small Molecules Increase a Catalytically Active Form of Human SOS1-Mediated Nucleotide Exchange on Ras.

Our laboratory has recently reported small molecules that bind to Ras and inhibit a catalytically active form of human SOS1 (SOS^cat) -catalyzed nucleotide exchange (8). During these studies, we also identified molecules from a related chemical series that have the opposite effect and increase the rate of nucleotide exchange in vitro (Fig. 1). Compound 1, a 3-(4-aminopiperidinyl)methyl-indole with an attached glycine, weakly increased SOS^cat-catalyzed nucleotide exchange, which was indicated by an increase in the exchange of BODIPY-GDP for unlabeled GTP. To improve the activity of these molecules, we synthesized additional compounds based on the aminopiperidine indole core. The addition of a tryptophan resulted in compound 2, which activated nucleotide exchange in a concentration-dependent manner (Fig. 1 B and E) and was more potent than 1 (Table S1). The addition of a methyl or halide group to position 5 of the indole (compounds 3 or 4, respectively) produced an additional increase in nucleotide exchange activation and lower EC₅₀ values (Table S1). Compound 4 increased SOS^cat-catalyzed nucleotide exchange with an EC₅₀ of 14 μM (Fig. 1 C and E). Replacement of the methylene linker between the indole and piperidine ring with a carbonyl resulted in a complete loss of activity (compound 5) (Fig. 1 D and E). Unlike our previously reported inhibitors of nucleotide exchange (8), the structure–activity relationship of this series did not correlate with direct binding to Ras (Table S1). It was, therefore, important to understand how these molecules function at the molecular level.

Compounds Activate Nucleotide Exchange in an SOS-Dependent Manner That Does Not Involve Ras Binding to the Allosteric Site of SOS.

To determine how the compounds activate nucleotide exchange, we examined previously reported GEF-independent and -dependent mechanisms. No increase in intrinsic nucleotide exchange on Ras was observed on addition of up to 400 μM compound 4 (Fig. 2A), indicating that chelation of magnesium or destabilization of bound nucleotide is not responsible for the activity (18, 19). Comparison of the exchange rates between intrinsic and SOS^cat-catalyzed exchange revealed that compound 4 activates nucleotide exchange in an SOS-dependent manner (Fig. 2B).

Crystal structures and biochemical experiments have identified an allosteric Ras binding site on SOS that increases its catalytic activity (14, 15). Indeed, titration of Ras^Y64A, a mutant form of Ras that binds to the allosteric site of SOS but does not undergo nucleotide exchange (15), resulted in a concentration-dependent increase in the rate of SOS^cat-catalyzed nucleotide exchange. GTP-bound Ras^Y64A was more effective than GDP-bound Ras^Y64A at stimulating nucleotide exchange (EC₅₀ = 0.74 μM), consistent with its role as the preferred binding partner for the allosteric site of SOS (Fig. 2 C and D) (20). Under the same conditions, addition of 100 μM compound 4 produced an intermediate rate of nucleotide exchange, suggesting that the compound effect on nucleotide exchange is physiologically relevant compared with the activation resulting from Ras binding at the allosteric site (Fig. 2E).

To test whether binding of Ras to the allosteric site on SOS is required for the compound-mediated increase in nucleotide exchange, we used two previously reported mutants, SOS^cat-W729E and SOS^{cat-L687E/R688A}, as well as the longer SOS^{Dbl homology–pleckstrin homology (DH-PH)-cat} construct to prevent Ras binding to the allosteric site (20). Consistent with reported data, SOS^cat-W729E, SOS^{cat-L687E/R688A}, and SOS^DH-PH-cat had slightly slower basal nucleotide exchange rates than WT SOS^cat (Fig. S1). The addition of 100 μM compound 4 to either SOS^cat-W729E or SOS^{cat-L687E/R688A} resulted in activation of nucleotide exchange, suggesting that compound-mediated activation does not require Ras binding to the allosteric site (Fig. 2F and Fig. S1). Nucleotide exchange reactions catalyzed by the longer construct of SOS containing both the autoinhibitory DH-PH and catalytic domains, SOS^DH-PH-cat, revealed that compound 4 is also capable of activating nucleotide exchange catalyzed by autoinhibited SOS (Fig. 2F and Fig. S1). Probing allosteric Ras binding and compound in combination revealed that compound 4 can further activate SOS-catalyzed nucleotide, even in the presence of saturating levels of GTP-bound Ras^Y64A (Fig. 2G). These data strongly support the hypothesis that these compounds activate nucleotide exchange through a distinct mechanism, which can be elicited regardless of the presence or absence of Ras bound at the allosteric site.

Addition of 100 μM compound 2 to nucleotide exchange reactions catalyzed by murine Ras-GRF1, an alternate Ras-GEF, resulted in no activation, suggesting that these compounds maintain a degree of specificity for SOS (Fig. S2A). This finding is consistent with a sequence alignment of the CDC25 domains of SOS1 and Ras-GRF1, which have a 30% overall identity (Fig. S2B).

Although not an activator, Brefeldin A inhibits GEF-catalyzed nucleotide exchange by acting as an interfacial inhibitor of a GEF–GTPase interaction (21). Under our conditions, the decrease in fluorescence observed on nucleotide release from Ras would not preclude the formation of a dead end Ras:SOS complex. To examine this possibility, we tested the ability of compound 4 to activate nucleotide exchange using a range of Ras (50 nM to 8 μM) and SOS^cat (25 nM to 3.5 μM) concentrations. A similar activation was observed at high Ras:SOS ratios, which would require multiple catalytic turnovers (Fig. S3 A and B). EC₅₀ values remained consistent irrespective of Ras or SOS^cat concentrations (Fig. S3 C and D). Compound 4 also activated nucleotide exchange using unlabeled Ras followed by the addition of a mixture of SOS^cat and BODIPY-GTP (Fig. S3E). GDP release, intermediate complex formation, and BODIPY-GTP association must occur to observe an increase in fluorescence here, which was described previously (7). Activation of nucleotide exchange under these conditions supports the conclusion that these compounds activate the full process of nucleotide exchange, unlike interfacial GEF:GTPase inhibitors.

Compounds That Activate Nucleotide Exchange Bind to a Pocket Identified in the Ras:SOS:Ras Complex.

We obtained X-ray structures of multiple compounds bound to the H-Ras:SOS^cat:H-Ras^Y64A(GppNHp) ternary complex (Ras:SOS:Ras) (Fig. 3 and Table S2). The ligand-bound Ras:SOS:Ras complex structures were obtained using both soaking and cocrystallization methods under multiple conditions and in different crystal packing lattices. The H-Ras isoform was found to crystallize more readily in this complex than K-Ras. K-Ras and H-Ras have no residue changes within close proximity to the binding pocket, suggesting that the compounds are not likely to be specific for activating one isoform of Ras over another.

The crystal structures revealed that the compounds bind to the Ras:SOS:Ras complex in a hydrophobic pocket that is formed by the CDC25 domain of SOS adjacent to the Switch II (SwII) region of Ras (Fig. 3 A and B). The pocket is formed principally by the αE and αF helices of the SOS catalytic domain, which are connected by an exposed helical turn involving P894 (22). The remainder of the pocket is formed by residues from the coiled region and helical turn connecting the αD and αE helices of SOS. Some of the residues of SOS that form the pocket (e.g., N879, Y884, and H905) have previously been reported to directly interact with Ras (22). Notably, R73, located in the SwII region of Ras at the catalytic site of SOS, forms a stacking interaction with Y884 and interacts with the backbone carbonyl of N879 (Fig. 3B). Importantly, N879 and Y884 form the anterior wall of the binding pocket (Fig. 3B) and provide a direct link from the compound to the SwII region of Ras, which is critical for binding to the catalytic domain of SOS (23).

The structure–activity relationships of aminopiperidine indole-based compounds can be rationalized from the X-ray cocrystal structures. All compounds bind in a similar fashion, with the core indole occupying the most hydrophobic portion of the pocket (Fig. 3 C–E). The NH of the core indole forms a hydrogen bond at the bottom of the pocket with the backbone carbonyl of M878, whereas the aminopiperidine moiety is surface-exposed and rotated to D887. For compound 1, the terminal amine is oriented to the solvent (Fig. 3C). The tryptophan moiety of compound 2 folds back and occupies a hydrophobic pocket located at the helical turn formed by P894 (Fig. 3D). Compound 1, which lacks this tryptophan moiety, cannot access this pocket (Fig. 3C, arrow). The increased activity of compound 2 could be caused by the additional interactions made by the tryptophan moiety. Methyl substitution at position 5 of the core indole (compound 3) further improved compound activity (Table S1). The methyl group points to a space unoccupied by the unsubstituted indole of compound 2 (Fig. 3 D, arrow and E). Compound 4, the most active compound (Table S1), was unable to be crystallized because of limited compound solubility. However, it is hypothesized to bind similar to compound 3, with the chloro substitution occupying the same space as the methyl group of 3. Based on the crystal structures, FITC-conjugated derivatives of compounds 2 and 4 were designed. Saturation binding and competition experiments conducted with these probes indicate that improved compounds bind SOS with a higher affinity (Fig. S4 A and B and Table S1). These crystallographic and biochemical data suggests that the activity of the compounds is determined by their ability to optimally fill the pocket.

Amino Acid Substitution of Residues Within the Pocket Prevents Compound-Induced Activation of Nucleotide Exchange.

Residues in this pocket have been previously identified as being mutated in developmental RASopathy disorders. Two mutations in the CDC25 domain of SOS, E846K and P894R, cause Noonan Syndrome (24). E846K has been shown to profoundly perturb intracellular signaling and P894R slightly activates nucleotide exchange on Ras compared with WT SOS (25, 26). Of particular note, P894 forms the helical turn that defines the pocket occupied by the tryptophan moiety of compounds 2 and 3 (Fig. 3B), further supporting the hypothesis that the binding pocket occupied by the compounds in the cocrystal structures is important for the activation of Ras by SOS.

We used the crystal structures to design mutations that would be predicted to perturb compound binding. Nine mutants of SOS^cat (D887A, D887E, D887H, D887N, D887V, F890L, L901M, L901K, and H905M) were cloned, expressed, and purified. Mutations of F890, L901, and H905 were designed to reduce the space available at the bottom of the hydrophobic pocket, whereas mutations of D887 were used to determine the importance of this residue for binding (Fig. 3B). Nucleotide exchange rates were determined for each mutant form of SOS^cat from experiments conducted in the presence of DMSO or 100 μM compound 2 (Fig. 4B and Fig. S5). All mutant forms of SOS^cat catalyzed nucleotide exchange, confirming the proper folding and function of the mutant proteins (Fig. S5). Mutation of F890, L901, and H905 prevented compound-induced activation of nucleotide exchange, suggesting that compound activity is mediated predominantly by hydrophobic interactions in the pocket (Fig. 4). In contrast, mutation of D887 did not prevent the ability of compound 2 to activate nucleotide exchange (Fig. 4 B and C). These data strongly support the conclusion that this binding pocket is functionally important for the activation of Ras by SOS and responsible for the compound-mediated activation of SOS^cat-catalyzed nucleotide exchange.

Compounds Increase Ras-GTP and Perturb Ras Signaling in Cells.

HeLa cells treated with FITC-conjugated compound 4 and subsequently washed with PBS showed a strong intracellular fluorescence signal, confirming that these compounds are suitable for use in cell-based experiments (Fig. S6A). HeLa cells were treated for 15 min with DMSO, the inactive compound 5, or the active compounds 2 and 4 to assess the ability of these compounds to activate endogenous Ras. Ras-GTP levels were determined using a Ras binding domain pull-down assay. No increase in Ras-GTP levels was observed in cells treated with the inactive compound 5. In contrast, treatment with compound 4 resulted in a threefold increase in Ras-GTP levels (Fig. 5A), whereas compound 2 resulted in a smaller increase, consistent with the relative in vitro nucleotide exchange activity (Table S1). Treatment of HeLa cells with 100 μM compound 4 led to elevated Ras-GTP levels within 5 min that remained elevated for the entirety of a 30-min time course (Fig. 5B). These experiments show that the compounds activate nucleotide exchange in the cellular setting containing full-length endogenous SOS and Ras proteins.

We determined the effect of compounds 2–5 on Ras-mediated signaling in the MAPK and PI3K pathways. Treatment with compounds 2–4 causes a biphasic response in the MAPK pathway that is characterized by inhibition of extracellular signal-regulated kinase (ERK) phosphorylation at high compound concentration followed by a peak of increased ERK phosphorylation as compound concentration decreases (Fig. 5C). This signaling pattern is most evident with compounds 3 and 4. Because of the decreased potency of compound 2, only the increased ERK phosphorylation is visible in this concentration range. Compounds 2–4 also inhibit PI3K pathway signaling, which was evidenced by a decrease in phosphorylation of the protein kinase AKT. Importantly, the peak in ERK phosphorylation correlates with the IC₅₀ for inhibition of phosphorylation of AKT (Fig. 5C), suggesting that the two are regulated by the same underlying mechanism. As expected, the inactive compound 5 had no effect on ERK or AKT phosphorylation.

The biphasic response in ERK phosphorylation closely resembles the signaling induced by inhibitors of the B-Raf kinase in cells containing WT Raf (27). To investigate a similar paradoxical activation mechanism, we examined compound effect on Ras signaling in melanoma lines harboring well-characterized mutations in the Ras pathway. In the context of WT Ras (CHL-1) or N-Ras^Q61L (SK-MEL-2), the B-Raf inhibitor dabrafenib and compound 4 elicited a biphasic response in both mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and ERK phosphorylation (Fig. 5D). In MALME-3M cancer cells, which harbor a B-Raf^V600E mutation, dabrafenib was able to potently inhibit MEK and ERK phosphorylation, which was expected (Fig. 5D). Compound 4, however, had no effect on MEK or ERK phosphorylation, suggesting that this compound acts by a unique mechanism of action at the level of the Ras–SOS interaction, upstream of Raf kinase.

To further test the hypothesis that these compounds act at the level of the Ras–SOS interaction, serum-starved HeLa cells were pretreated with DMSO or 100 μM compound 4 and then stimulated with EGF. Compound 4 prevented EGF-induced activation of MEK and ERK; however, it had no effect on the activation of EGF receptor upstream of Ras, which was shown by an increase in tyrosine 1068 phosphorylation (Fig. 5E). These data support the conclusion that the compounds act at the level of the Ras–SOS interaction, downstream of EGF receptor and upstream of Raf, and establish a means to study acute Ras-mediated signaling using an approach that is distinct from other small molecules targeting this pathway.

Compounds 4 and 5 were assessed for their ability to affect cell growth and transformation. Consistent with the signaling observed in these cell lines, both WT (HeLa and CHL-1) and mutant (SK-MEL-2 and PANC-1) Ras harboring cancer cells showed a decrease in cell proliferation and anchorage-independent growth after treatment with compound 4 (Fig. 5 F and G and Fig. S6B). In contrast, the inactive compound 5 had little or no effect at concentrations up to 100 μM. This evidence suggests that compound binding to the Ras:SOS:Ras complex does not enhance cell growth but instead, may represent a mechanism to inhibit cell proliferation and transformation.

Protein Purification and X-Ray Crystallography.

For nucleotide exchange assays, recombinantly purified K-Ras^G12D (referred to as Ras; amino acids 1–169) and SOS^cat (amino acids 564–1049) containing the Ras exchanger motif and CDC25 domain were purified as described previously (8).

The H-Ras:SOS^cat:H-Ras^Y64A(GppNHp) complex was prepared as described previously (14). Protein:ligand complexes were prepared by adding a concentrated DMSO stock solution of the ligand to a final concentration of 2–5 mM. Additional detailed methods are in SI Materials and Methods.

Nucleotide Exchange Assays.

The rate of nucleotide dissociation was determined using Ras preloaded with BODIPY-GDP (excitation: 485, emission: 510; Life Technologies). Reactions were performed on a Hamamatsu Functional Drug Screening System 6000 with sequential additions of compound followed by GTP ± SOS^cat at 10 and 120 s to a well containing BODIPY-GDP–loaded Ras. Additional details are in SI Materials and Methods.

NMR, X-Ray Crystallography, and Cell-Based Experiments.

Details are in SI Materials and Methods.