Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2013 Feb 4;110(8):3155–3160. doi: 10.1073/pnas.1212324110

Small-molecule inhibitors targeting G-protein–coupled Rho guanine nucleotide exchange factors

Xun Shang a, Fillipo Marchioni a, Chris R Evelyn a, Nisha Sipes a, Xuan Zhou a, William Seibel b, Matthew Wortman b, Yi Zheng a,1
PMCID: PMC3581902  PMID: 23382194

Abstract

The G-protein–mediated Rho guanine nucleotide exchange factor (GEF)–Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein–regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM Kd and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein–coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG–RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme–substrate pair of GEF–RhoA at their binding interface that leads to enhanced efficacy and specificity.

Rho family GTPases are intracellular signaling molecules that regulate cytoskeleton organization, gene expression, cell cycle progression, cell motility, and other cellular processes (1–4). The activities of many Rho family members hinge upon a delicate balance between the GTP-bound, active state and the GDP-bound, inactive state, which is subject to tight regulation in cells in response to physiologic and pathologic signals. Rho guanine nucleotide exchange factors (GEFs) represent the major class of activating enzymes of Rho GTPases by serving to relay a variety of signals to catalyze GDP/GTP exchange of specific Rho GTPases. To date, >80 RhoGEFs have been discovered that regulate the activities of over a dozen Rho GTPases in mammals (5). The overabundance of RhoGEFs vs. Rho GTPase substrates allows the GEFs to function in a tissue/cell type- and signaling pathway-specific manner, even though multiple RhoGEFs may possess the activating potential for a given Rho GTPase.

Consistent with a broad association of abnormal Rho GTPase activities in human cancers, a number of RhoGEFs have been reported to be overexpressed and/or hyperactivated in cancer cells, and they may be causal for tumor cell invasion and/or proliferation (6). The DH-PH domains shared among the DBL RhoGEF family, which constitutes the major class of RhoGEFs (7), have been characterized as the critical binding and catalytic motif required for Rho GTPase activation, and in-depth knowledge of the mechanism of DH-PH–mediated GEF reaction has been obtained in the last two decades (8–10). Among the DBL-like RhoGEFs, the heterotrimeric G-protein–regulated LARG, p115RhoGEF, and PDZRhoGEF form a unique RhoGEF subfamily that transduce signals from Gα12/13-coupled chemokine or mitogen receptors to RhoA through their RGS and DH-PH domains (11–15). The Gα12/13–RhoGEF–RhoA signaling cascade has been proposed as a useful target in cancer or neurologic diseases (16).

Small-molecule chemicals, as a major structural class of drugs, are broadly pursued in targeting oncoproteins and their signaling pathways. However, only proteins containing suitable hydrophobic pockets are considered druggable (17–20), which significantly limits the scope of drug discovery effort. In the Gα12/13–RhoGEF–RhoA signaling cascade, the G proteins and Rho GTPases are considered difficult to target in part because they have globular structures with limited druggable surface areas. Here, we present evidence that G-protein–coupled RhoGEFs constitute valid targets and can be specifically targeted at the catalytic site of DH-PH domains by chemical compounds. Furthermore, we demonstrate a strategy to target RhoA signaling by dual inhibitors of the GEF–RhoA pair of enzyme–substrate interface, to achieve improved efficacy and specificity, expanding the traditional “one target, one drug” approach in rational drug design.

Results

Targeting the RhoA Interactive Site of LARG via Virtual Screening.

We used data from the crystal structures of LARG–RhoA complex (21) [Protein Data Bank (PDB) ID code 1X86] to identify a relatively stable interaction site (low β factor in the crystal structure). At this interaction site, RhoA protrudes into a concavity on the surface of LARG between residues N975 and R986; therefore, we hypothesized that a small molecule that bound in that concavity would reduce or prevent RhoA binding to LARG. From the docking screen of more than 4 million compounds in the ZINC library (22, 23), the top scoring 49 chemicals predicted to bind to the LARG binding concavity were tested for their ability to inhibit the binding interaction between the DH-PH domain module of LARG and RhoA. Purified DH-PH domains of LARG, expressed as the (His)6-tagged proteins in Escherichia coli, were incubated with RhoA in the presence of a given compound at 100 μM concentration. Among the compounds tested, Y16, which is predicted to dock at the DH-PH junction of LARG (Fig. 1 A and B), showed an activity in suppressing LARG binding to RhoA (Fig. 1C and Table S1). Y16 was effective in inhibiting LARG binding to RhoA at 10 μM concentration under the pull-down assay conditions, and the activity was dose dependent (Fig. 1D). Y16 thus represents a candidate inhibitor of the LARG interactive site with RhoA.

Fig. 1.

Fig. 1.

Open in a new tab

Identification of Y16 as an inhibitor of G-protein–coupled Rho GEFs. (A) The chemical structure of Y16. (B) A simulated docking model of Y16 on LARG surface. (Left) Top view of the binding pocket of LARG bound to Y16. (Right) Top view of the predicted structural contacts of Y16 to RhoA. (C) The inhibitory effect of a panel of compounds predicted by virtual screening to dock on LARG DH-PH module was tested in a complex formation assay. (His)6-tagged LARG (1 μg) was incubated with GST alone, GST-RhoA (1 μg), or GST-Cdc42 (1 μg) immobilized on glutathione agarose beads in the presence or absence of the 1 mM indicated compounds. After an incubation at 4°C for 1 h, the beads-associated (His)6-LARG was detected by anti-His Western blotting. (D) Dose-dependent specific inhibition of LARG binding to RhoA by Y16. (His)6-tagged LARG (1 μg) was incubated with GST alone or GST-fused RhoA on glutathione agarose beads in a binding buffer containing different concentrations of Y16. The beads-associated (His)6-LARG was detected by anti-His Western blotting. (E) The inhibitory effects of Y16 on the interaction between RhoA and multiple RhoGEFs. NIH 3T3 cells transiently overexpressing GFP-p115 RhoGEF or Flag-PDZRhoGEF or stably overexpressing GST-DBL or Flag-LBC, were harvested and the cell lysates were subjected to the His-RhoA or GST-RhoA pull-down assay in the absence or presence of Y16 at the indicated concentrations. (F) Y16 has no effect on Cdc42 or Rac1 binding to their respective GEFs. (Top) Myc-tagged Tiam1 expressed in HEK293T cell lysates were incubated with GST alone or GST-Rac1 conjugated with glutathione agarose beads in the presence of increasing concentrations of Y16. The beads-associated myc-Tiam1 was probed by anti-myc Western blotting. (Middle) (His)6-tagged TrioN (1 μg) was incubated with GST alone or GST-Rac1 conjugated with glutathione agarose beads in the presence of increasing concentrations of Y16. The beads-associated (His)6-TrioN was detected by anti-His Western blotting. (Bottom) (His)6-tagged Cdc42 (1 μg) was incubated with GST alone or GST-Intersectin conjugated with glutathione agarose beads in the presence of increasing concentrations of Y16. The beads-associated (His)6-Cdc42 was detected by anti-His Western blotting.

Compound Y16 and Its Analogs Are a Class of Inhibitors of G-Protein–Coupled RhoGEFs.

To examine the specificity of Y16 among RhoA interactive molecules, we have carried out additional biochemical tests of binding interaction of RhoA with its GEFs, effectors, and RhoGAP in the present of Y16. These complex formation assays revealed that, although Y16 is active in inhibiting RhoA interaction with RhoGEFs p115 RhoGEF and PDZ RhoGEF, the other two RGS domain-containing RhoGEFs in addition to LARG, it does not affect RhoA interaction with DBL or LBC (Fig. 1E), both of which can readily activate RhoA. Furthermore, Y16 did not interfere with the binding of Cdc42 and Rac1 to their respective GEFs, intersectin and TrioN, respectively, nor RhoA binding with its effector/GAP molecules ROCKII, mDia, PKN, and p190RhoGAP (Fig. 1F and Fig. S1). These results provide biochemical evidence that Y16 is a selective inhibitor of the LARG-related G-protein–coupled RhoGEFs, capable of inhibiting the RhoGEF–RhoA interaction. A preliminary structure–activity relationship study of structural analogs of Y16, all bearing a phenylpyrazolidine scaffold, by testing their respective activity in inhibiting LARG binding to RhoA in vitro, found a very narrow SAR with Y16 as the most potent analog (Table S2). Even analog YA01, which differs only in a methyl group, showed a substantially reduced potency (27.1%) relative to Y16.

Biochemical Characterization of Y16 Interaction with LARG.

To define the mechanism of Y16 action, we have sought to determine the binding constant of Y16 to LARG, examine its effect on LARG-mediated GEF reaction, and map the possible site of Y16 interaction with LARG. First, a microscale thermophoresis analysis, which allows a sensitive detection of small-molecule binding to a protein target (24), was carried out by titrating the chemical to purified LARG DH-PH protein. This assay shows that Y16 binds to this catalytic fragment of LARG with a Kd of ∼76 ± 8 nM (Fig. 2A). As controls, Y16 does not bind to TrioN (a GEF for Rac1/RhoG) or Intersectin (a GEF for Cdc42) (Fig. S2). Second, to examine whether Y16 could inhibit RhoGEF-catalyzed guanine nucleotide-exchange reaction of RhoA, a GDP/GTP exchange assay was performed in the presence or absence of Y16. Y16 was able to inhibit the GDP dissociation from RhoA catalyzed by LARG dose dependently without affecting the GEF reactions of Rac1 and Cdc42 catalyzed by TrioN and Intersectin, respectively (Fig. 2B and Fig. S2). Third, to examine the structural residues of LARG involved in Y16 binding, LARG point mutants bearing Ala mutation around the predicted docking sites, i.e., E982, K979, and N983, of LARG, were tested for their binding affinities to Y16. Two of these residues are conserved among three G-protein–coupled RhoGEFs, but are mostly divergent from other DBL family RhoGEFs (Fig. 2C). The N983A mutant lost the binding ability to Y16 with a Kd > 500 μM, whereas the K979A and E982A mutants showed a reduced affinity with Kd values of 0.47 and 2.1 μM, respectively (Fig. 2D). As a control, the V1099A mutant of LARG, a mutant bearing a mutation outside the predicted docking pocket, was not affected in Y16 binding (Kd = 79 ± 49 nM). Overall, these results suggest a relative tight binding of Y16 to the unique site in the DH-PH domains of LARG to impinge on the GEF activation reaction of the RhoA substrate.

Fig. 2.

Fig. 2.

Open in a new tab

Biochemical characterization of Y16 interaction with Rho GEF. (A) Y16 was effective in inhibiting RhoA GDP/GTP exchange reaction stimulated by LARG in a dose-dependent manner. Increasing concentrations of G04 or Y16 were included in the exchange buffer as indicated. (B) Microscale thermophoresis analysis of Y16 binding to LARG. Purified proteins including RhoA and LARG were first labeled with Alexa 647 fluorescence dye. G04 or Y16 was titrated between 0.76 and 25,000 nM to the constant amount of labeled proteins (100 nM). Data are representative of three independent experiments. (C) Amino acid sequence alignment of LARG (975–987) with PDZRhoGEF, p115RhoGEF, DBL, LBC, and Vav1. (D) Microscale thermophoresis analysis of Y16 binding to LARG mutants. Purified LARG mutants were first labeled with Alexa 647 fluorescence dye. Y16 was titrated at increasing concentrations. Data are representative of three independent experiments.

Y16 Specifically Inhibits RhoA Activity in Cells.

To examine whether the lead RhoGEF inhibitor Y16 is effective in specifically suppressing RhoA activity in cells, fibroblast cells grown in serum-free media were treated with Y16 in different concentrations, followed by stimulation with 10% (vol/vol) calf serum. As shown in Fig. 3A, Y16 could inhibit RhoA-GTP formation induced by serum dose dependently and was specific for RhoA because it did not affect the activities of Cdc42 and Rac1 in the same cells (Fig. 3B). In addition, Y16 efficiently inhibited serum or SDF-1α–induced phospho-MLC and phospho-FAK formation (Fig. 3C), which are downstream of RhoA. Previously, it has been established that the serum component lysophosphatidic acid (LPA) elicits a signaling cascade through G-protein–coupled RhoGEFs and RhoA to regulated cell cytoskeleton organizations (1–3). To further evaluate the ability of Y16 to inhibit RhoA-mediated cell functions, we next examined actin cytoskeleton structures of cells stimulated by serum or LPA in the absence or presence of Y16. Fig. 3D shows that, in the presence of Y16, both stress fiber and focal complex of the cells were significantly reduced, whereas Fig. S3 shows that the Rac1-mediated lamellipodia and Cdc42-mediated filapodia under the stimulation by PDGF and Bradykinin, respectively, were not affected. Given the implicated role of RhoA in actin cytoskeleton organization and adhesion (1–3), these results demonstrate that Y16 is active in specifically inhibiting cellular RhoA-GTP and RhoA-mediated signaling function.

Fig. 3.

Fig. 3.

Open in a new tab

Cellular efficacy and specificity of Y16 in suppressing RhoA activity. (A and B) NIH 3T3 cells were treated with Y16 at the indicated concentrations for 24 h in serum-free media. Cells were subsequently stimulated with 10% calf serum for 15 min and were subjected to GST-Rhotekin or GST-PAK1 effector domain pull-down assays, and the activities of RhoA, Cdc42, and Rac1 were examined. Relative amounts of GTP-bound form of the GTPases were quantified by densitometry measurements and normalized to those of the unstimulated cells. (C) Y16 inhibited the serum or/and SDF-1α–induced phospho-MLC and phospho-FAK activities. Western blots are of p-MLC and p-FAK and relevant controls of NIH 3T3 cells treated with Y16 at indicated concentrations in serum-free media and subsequently stimulated by 10% calf serum or/and 100 ng/mL SDF1-α for 10 min. (D) The effect of Y16 on cell stress fiber and focal complex was assessed in Swiss 3T3 cells. Cells were treated with 30 μM Y16 in serum-free media, subsequently stimulated with 10% calf serum for 10 min, and stained with rhodamine–phalloidin for F-actin and anti-vinculin for focal adhesion complexes. Images shown are representative of more than 100 cells examined. (E) Y16 does not affect the constitutively active RhoA Q63L mutant-induced actin stress fiber or focal adhesion formation. NIH 3T3 cells were transfected with constitutively active RhoA Q63L mutant or a control vector and subsequently were grown on tissue culture dish in a serum-free media in the presence of 50 μM Y16. After 24-h starvation, cells were stimulated with 10% calf serum for 30 min and were stained with rhodamine–phalloidin to reveal F-actin structure and anti-vinculin antibody for focal adhesion.

To further examine the specificity of Y16 in cells, we put Y16 to a test of cells expressing constitutively active RhoA or ROCKII mutant that produces F-actin–based responses independent of endogenous RhoGEF activity. As shown in Fig. 3E and Fig. S4, Y16 did not alter actin stress fiber or focal adhesion complex formation induced by a constitutively active RhoA mutant Q63L, or a constitutively active ROCKII. Furthermore, although Y16 was capable of inhibiting serum-induced phospho-MLC formation, an event mediated by RhoA activity in NIH 3T3 cells (Fig. 3C), it did not interfere with phosphor-MLC in cells overexpressing RhoA Q63L mutant (Fig. S5). Under the assay conditions, Y16 appeared to be noncytotoxic as it did not affect the survival status of the cells (Fig. S6). These results provide further evidence that the effect of Y16 toward GEF-RhoA signaling is likely specific in cells.

Y16 Acts Synergistically with Rhosin/G04 in Inhibiting LARG–RhoA Interaction and RhoA Activity.

We previously identified a chemical compound, Rhosin/G04, that specifically binds to RhoA protein and inhibits RhoA activity in diverse physiological and pathological systems (25). Because Rhosin/G04 specifically interacts with RhoA at the GEF recognition site while Y16 binds to LARG at the RhoA recognition site, we further hypothesized that dual targeting by the two inhibitors may have a synergistic effect on the GEF–RhoA interaction as they act on the same interface of GEF–RhoA binding, and consequently, on RhoA activity and signaling functions. We thus examined the in vitro and cellular effects when Rhosin/G04 and Y16 were used together. First, when Rhosin/G04 and Y16 were combined together, the working concentrations required for effective inhibition of RhoA binding to LARG were significantly decreased (IC50 of ∼1 μM compared with ∼10 and ∼5 μM of G04 or Y16 alone, respectively) (Fig. 4A), suggesting that Rhosin/G04 and Y16 can synergistically inhibit RhoA–LARG binding interaction. Second, applying Rhosin/G04 and Y16 together to NIH 3T3 cells potently inhibited RhoA activity (Fig. 4B). At 2.5 μM each, Rhosin and Y16 inhibited ∼50% RhoA-GTP content, and at 5 μM each ∼80% RhoA-GTP stimulated by serum, which were much more potent than the effect of Rhosin/G04 or Y16 acting alone (∼80% inhibition at 30 μM). Even under a higher concentration of Y16 and Rhosin/G04 combination (50 μM each) when endogenous RhoA-GTP content was effectively suppressed, no effect on Rac1-GTP or Cdc42-GTP content in cells was observed (Fig. 4C), suggesting a high degree of specificity of the inhibitors. Importantly, the Rhosin/G04 and Y16 combination was able to completely abolish the RhoA-medicated cell stress fiber formation at 2.5 μM concentration of each of the inhibitors, whereas it required over 10 μM of Rhosin/G04 or Y16 alone to show such a cellular effect (Fig. 4D). These results suggest that Y16 and Rhosin/G04 can act synergistically to inhibit RhoA activity.

Fig. 4.

Fig. 4.

Open in a new tab

Synergistic effects of Y16 with Rhosin on GEF–RhoA interaction and RhoA activity. (A) Dose-dependent specific inhibition of LARG binding to RhoA by Y16, G04, or G04+Y16 combination at varying concentrations. (B) Effects of Y16, G04, or G04+Y16 combination in suppressing RhoA activity in NIH 3T3 cells. NIH 3T3 cells were treated with Y16, G04, or G04+Y16 combination at the indicated concentrations for 24 h in serum-free media. Cells were subsequently stimulated with 10% calf serum for 15 min and were subjected to GST-Rhotekin effector domain pull-down assays, and the activities of RhoA were examined. Relative amounts of GTP-bound form of the GTPases were quantified by densitometry measurements and normalized to those of the unstimulated cells. (C) Effect of Y16, G04, or G04+Y16 combination on Cdc42 and Rac1 activities. NIH 3T3 cells were treated with Y16, G04, or G04+Y16 combination at the indicated concentrations for 24 h in serum-free media. Cells were subsequently stimulated with 10% calf serum for 15 min and were subjected to GST-Rhotekin and GST-PAK1 effector domain pull-down assays, and the activities of RhoA, Cdc42, and Rac1 were examined. Relative amounts of GTP-bound form of the GTPases were quantified by densitometry measurements and normalized to those of the nontreated cells. (D) Y16, G04, or G04+Y16 combination selectively blocks cellular response to LPA-stimulated actin stress fiber formation. NIH 3T3 cells were treated with Y16, G04, or G04+Y16 combination of the indicated concentrations in serum-free DMEM medium prior to stimulation with LPA (20 ng/mL) for 10 min. The cells were stained with rhodamine–phalloidin to reveal F-actin structures.

Y16 Effectively Inhibits the Growth, Migration, and Invasion Activities of Breast Cancer Cells.

Aberrant RhoGEF activity has been associated with cancer cell hyperproliferative, migration, and invasive characteristics (6, 7). Next, we investigated the effect of Y16 on the growth, migration, and invasion, as well as mammosphere formation, activities of breast cancer cells, properties associated with tumorigenic potential (26). Y16 could significantly inhibit MCF7 breast cancer cell growth, migration, and invasion, and the RhoA inhibitor Rosin/G04 (Fig. 5 A–C). The Y16-treated MCF7 cells also yielded smaller size and reduced number of mammospheres dose dependently (Fig. 5E). Thus, Y16 is capable of suppressing breast cancer cell behaviors.

Fig. 5.

Fig. 5.

Open in a new tab

Targeting RhoA activity by Y16 and Rhosin in breast cancer cells. (A) Y16, G04, or G04+Y16 combination inhibits MCF7 breast cell growth. MCF7 cells were plated at 1.5 ×104/24 well in the presence of Y16, G04, or G04+Y16 combination. Cell numbers were determined at the indicated times. (B) Y16, G04, or G04+Y16 combination inhibits MCF7 cell migration. MCF7 cells were subjected migration assays in the presence of Y16, G04, or G04+Y16 combination. (C) Y16, G04, or G04+Y16 combination inhibits MCF7 cells invasion. The invasive activities were assayed in a Matrigel-coated transwell. (D) Y16, G04, or G04+Y16 combination inhibits RhoA and its downstream signaling activities in MCF7-derived mammospheres. (Upper) MCF7-derived mammospheres were treated with Y16, G04, or G04+Y16 combination at the indicated concentrations. Spheres were collected, and the RhoA activity was examined. Relative amounts of GTP-bound form of RhoA were quantified by densitometry measurements and normalized to those of the untreated cells. (Lower) Western blots are of p-MLC of MCF7-derived mammospheres. Y16, G04, or G04+Y16 combination inhibits MCF-7 cell-derived mammosphere formation. (E) Y16, G04, or G04+Y16 combination inhibits MCF-7 cell-derived mammosphere formation. MCF-7 cells were treated with Y16, G04, or G04+Y16 combination at the indicated concentrations for 24 h in serum-free media. MCF7 cells were dissociated to single cells with trypsin and cultured for 10 d at the density of 2 × 104/mL in suspension in the media containing G04 or Y16 at the indicated concentration. Photographs were taken after a 10-d culture. Images shown are representative of 5–10 fields containing a total of at least 100 spheres, which were chosen randomly.

To examine whether Y16 could worked synergistically with Rhosin/G04 to more effectively suppress cancer cell activities, we examined a combination of Y16 with Rhosin/G04 at micromolar working concentrations. Combined treatment of Y16 and Rhosin/G04 at 5 μM each could effectively inhibit cell growth, migration, invasion, and mammosphere formation activities (Fig. 5 A–E and Fig. S7), showing a clear synergy over the effects when each inhibitor was used alone. Accompanying the manifested cell activity inhibitions, RhoA-GTP and downstream signaling of RhoA, measured by p-MLC level, showed a dose-dependent reduction in the Rhosin/G04- or Y16-treated mammospheres (Fig. 5D). Although Rhosin/G04 or Y16 administration alone caused ∼50% inhibition of RhoA activity at 10 μM, combined Rhosin/G04 and Y16 reached ∼70% inhibition of RhoA-GTP or the downstream p-MLC when each was at 2.5 μM (Fig. 5D). These results clearly indicate that Y16 and Rhosin/G04, each capable of selectively inhibiting one target of the RhoGEF–RhoA enzyme–substrate pair, can act synergistically to inhibit RhoA activity and RhoA-regulated breast cancer cell behaviors.

Discussion

Rho family GTPases are critical intracellular signal transducers involved in diverse cell signal transduction processes from cell adhesion molecules, growth factor receptors, and G-protein–coupled receptors. The DBL family GEFs are the major class of activators for Rho GTPases including RhoA, Cdc42, and Rac1, and are known to possess transforming activity (6–10). The three DBL family members, LARG, PDZRhoGEF, and p115 RhoGEF, represent a distinct RhoGEF subfamily characterized by the presence of a RGS domain at their N termini that binds to the heterotrimeric Gα12/13 proteins, and also the RhoGEF module, DH-PH domains, that directly signals to RhoA (11–15). The Gα12/13–RhoGEF–RhoA pathway has been implicated in cancer cell migration, invasion, and proliferation, and is proposed to be useful anticancer drug target.

Small-molecule chemicals are a class of attractive drug candidates and pharmacologic tools useful in targeting aberrant Rho GTPase signaling (27). Extensive structural and biochemical studies of the RhoGEF–RhoA interaction and GEF reaction have presented detailed information valuable for the design of small molecules specifically targeting the GEF–Rho GTPase axis. Because Rho GTPases, like Ras, are globular structures and lack useful grooves and pockets on their surface for high-affinity chemical binding (21), they are not considered as traditional “druggable” targets, which normally require deep hydrophobic pockets (17–20). However, the DH-PH module of DBL family RhoGEFs appears suitable for targeting by small-molecule inhibitors judged by the relatively stable concavity formed at an interaction site between LARG residues N975 and R986, which are required for Rho GTPase substrate recognition and catalysis (5). Furthermore, as enzymes, RhoGEFs may serve as effective targets because a partial blockade of the GEF activity could result in an amplified suppression of downstream Rho GTPase signal flows. Thus, we have used the mechanistic information of GEF–Rho interaction to pursue a rational design strategy by structure-based simulation and docking screen for chemicals targeting the site of a G-protein–coupled RhoGEF enzyme essential for Rho activation, i.e., the DH-PH junction of LARG.

Y16, the lead compound, docks with a favorable energy into the C terminus of LARG DH domain junction site with PH domain, a region critical for RhoA recognition and catalysis. Y16 and its analogs show a specific inhibitory activity of LARG. Significantly, Y16 displayed a specificity against the heterotrimeric G-protein–regulated RhoGEFs, i.e., LARG, p115RhoGEF, and PDZRhoGEF, with a measured binding constant of ∼76 nM Kd, and did not interfere with the action of other RhoA-activating DBL family RhoGEFs such as DBL and LBC. Mutagenesis studies of LARG indicate that the Y16 binding site is located in the C terminus of the DH domain, a region essential for RhoA interaction. In cells, Y16 displays a selective inhibition of RhoA-GTP formation and RhoA-mediated F-actin stress fiber and focal adhesion formation. Importantly, the cellular inhibitory effects of Y16 can be rescued by the expression of a constitutively active RhoA or ROK mutant that is capable of bypassing endogenous RhoA-GTP to elicit downstream signaling. These biochemical and cell-biological properties indicate that Y16 is a G-protein–coupled RhoGEF-selective inhibitor. Although the molecular details of the specificity of Y16 toward LARG and related RhoGEFs remain unclear, it appears that the highly conserved structures of the LARG subfamily G-protein–coupled RhoGEFs contain unique residues corresponding to K979 and N983 of LARG to allow a distinction of Y16 and analogs from other DBL family DH-PH containing GEFs. To more clearly define the mechanism of action, it will be a priority in future studies to obtain a structure of Y16 or its derivative in complex with LARG GEF domains. In vitro, Y16 binds to its target, LARG, with ∼80 nM affinity but it takes higher than micromolar concentrations for it to be effective in cells. This is not unlike most kinase/enzyme inhibitors that are in use therapeutically: in addition to target binding affinity, additional factors such as transmembrane delivery/cell uptake efficacy, biostability, and nonspecific absorption/binding by serum or cellular proteins may contribute to its efficacy in cells.

To directly target RhoA, we recently have identified a lead chemical inhibitor, Rhosin/G04, which contains two aromatic chemical fragments tethered by a flexible linker and may dock into two separate shallow grooves sandwiching the GEF recognition site of RhoA between the switch I and switch II regions (25). Rhosin/G04 displays a micromolar binding affinity to RhoA and can inhibit GEF binding to RhoA (25). However, partly due to the relative low binding affinity, over 30 μM concentration of Rhosin/G04 is required to effectively inhibit RhoA activity in cells, leaving more to be desired for improvement. Given that Y16 and Rhosin are a pair of inhibitors designed to recognize the opposite interactive surfaces between LARG and RhoA, we reasoned that Y16 and Rhosin may work synergistically to inhibit LARG–RhoA signaling because the GEF and RhoA interact transiently and dynamically at defined intracellular locations. Indeed, we found that, when being applied together, Y16 and Rhosin yielded a synergistic potency in suppressing LARG–RhoA interaction in vitro and RhoA activation in cells. This synergy is expected to further enhance specificity, in addition to potency, for inhibiting the GEF–RhoA signaling pathway and shows an advantage in an approach of “one interactive site, two drugs” in rationally devising inhibitors interfering with an enzyme–substrate interface. Subsequently, we show in a breast cancer cell model that a combined application of Y16 and Rhosin is advantageous at more effectively inhibiting RhoA activity and tumor sphere growth.

Overall, our work presents an attempt in translating the mechanistic information of G-protein–mediated GEF–Rho GTPase signaling to rational design of chemical inhibitors useful for studying the physiological and pathologic roles of G-protein and Rho GTPase signaling. The studies also demonstrate a strategy of two inhibitors for one enzyme–substrate interaction is useful for selective targeting of signaling cascades deemed of “low druggability.” This approach may have broad implications for drug discovery efforts targeting diverse signaling networks.

Materials and Methods

Virtual Screening.

The atomic interactions between RhoA and LARG were obtained from PDB ID 1X86 (21). The concave surface to either side RhoA Trp58 meets these criteria and was chosen as the binding site for virtual screening (SI Materials and Methods).

Microscale Thermophoretic Analysis.

A NanoTemper Monolith Instrument (NT.015) was used for measuring thermophoresis (SI Materials and Methods).

Statistical Analysis.

All experimental data were analyzed and compared for statistically significant differences by two-tailed Student t test. Data are presented as the averaged values ± SDs, where applicable.

Additional experimental procedures are described in SI Materials and Methods.

Supplementary Material

Supporting Information
supp_110_8_3155__index.html (7.3KB, html)

Acknowledgments

This work was supported in part by National Institutes of Health Grants R01 CA141341, R01 CA150547, and P30 DK090971.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.A.C. is a guest editor invited by the Editorial Board.

References

  • 1.Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature. 2002;420(6916):629–635. doi: 10.1038/nature01148. [DOI] [PubMed] [Google Scholar]
  • 2.Ridley AJ. Rho family proteins: Coordinating cell responses. Trends Cell Biol. 2001;11(12):471–477. doi: 10.1016/s0962-8924(01)02153-5. [DOI] [PubMed] [Google Scholar]
  • 3.Zohn IM, Campbell SL, Khosravi-Far R, Rossman KL, Der CJ. Rho family proteins and Ras transformation: The RHOad less traveled gets congested. Oncogene. 1998;17(11 Reviews):1415–1438. doi: 10.1038/sj.onc.1202181. [DOI] [PubMed] [Google Scholar]
  • 4.Jaffe AB, Hall A. Rho GTPases: Biochemistry and biology. Annu Rev Cell Dev Biol. 2005;21:247–269. doi: 10.1146/annurev.cellbio.21.020604.150721. [DOI] [PubMed] [Google Scholar]
  • 5.Rossman KL, Der CJ, Sondek J. GEF means go: Turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6(2):167–180. doi: 10.1038/nrm1587. [DOI] [PubMed] [Google Scholar]
  • 6.Vigil D, Cherfils J, Rossman KL, Der CJ. Ras superfamily GEFs and GAPs: Validated and tractable targets for cancer therapy? Nat Rev Cancer. 2010;10(12):842–857. doi: 10.1038/nrc2960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zheng Y. Dbl family guanine nucleotide exchange factors. Trends Biochem Sci. 2001;26(12):724–732. doi: 10.1016/s0968-0004(01)01973-9. [DOI] [PubMed] [Google Scholar]
  • 8.Hart MJ, Eva A, Evans T, Aaronson SA, Cerione RA. Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product. Nature. 1991;354(6351):311–314. doi: 10.1038/354311a0. [DOI] [PubMed] [Google Scholar]
  • 9.Cerione RA, Zheng Y. The Dbl family of oncogenes. Curr Opin Cell Biol. 1996;8(2):216–222. doi: 10.1016/s0955-0674(96)80068-8. [DOI] [PubMed] [Google Scholar]
  • 10.Hart MJ, et al. Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product. J Biol Chem. 1994;269(1):62–65. [PubMed] [Google Scholar]
  • 11.Chikumi H, et al. Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential. Oncogene. 2004;23(1):233–240. doi: 10.1038/sj.onc.1207012. [DOI] [PubMed] [Google Scholar]
  • 12.Hart MJ, et al. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Science. 1998;280(5372):2112–2114. doi: 10.1126/science.280.5372.2112. [DOI] [PubMed] [Google Scholar]
  • 13.Fukuhara S, Murga C, Zohar M, Igishi T, Gutkind JS. A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J Biol Chem. 1999;274(9):5868–5879. doi: 10.1074/jbc.274.9.5868. [DOI] [PubMed] [Google Scholar]
  • 14.Fukuhara S, Chikumi H, Gutkind JS. RGS-containing RhoGEFs: The missing link between transforming G proteins and Rho? Oncogene. 2001;20(13):1661–1668. doi: 10.1038/sj.onc.1204182. [DOI] [PubMed] [Google Scholar]
  • 15.Fukuhara S, Chikumi H, Gutkind JS. Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho. FEBS Lett. 2000;485(2–3):183–188. doi: 10.1016/s0014-5793(00)02224-9. [DOI] [PubMed] [Google Scholar]
  • 16.Lappano R, Maggiolini M. G protein-coupled receptors: Novel targets for drug discovery in cancer. Nat Rev Drug Discov. 2011;10(1):47–60. doi: 10.1038/nrd3320. [DOI] [PubMed] [Google Scholar]
  • 17.Verdine GL, Walensky LD. The challenge of drugging undruggable targets in cancer: Lessons learned from targeting BCL-2 family members. Clin Cancer Res. 2007;13(24):7264–7270. doi: 10.1158/1078-0432.CCR-07-2184. [DOI] [PubMed] [Google Scholar]
  • 18.Hopkins AL, Groom CR. The druggable genome. Nat Rev Drug Discov. 2002;1(9):727–730. doi: 10.1038/nrd892. [DOI] [PubMed] [Google Scholar]
  • 19.Hopkins AL, Groom CR. Target analysis: A priori assessment of druggability. Ernst Schering Res Found Workshop. 2003;42(42):11–17. doi: 10.1007/978-3-662-05314-0_2. [DOI] [PubMed] [Google Scholar]
  • 20.Russ AP, Lampel S. The druggable genome: An update. Drug Discov Today. 2005;10(23–24):1607–1610. doi: 10.1016/S1359-6446(05)03666-4. [DOI] [PubMed] [Google Scholar]
  • 21.Kristelly R, Gao G, Tesmer JJ. Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor. J Biol Chem. 2004;279(45):47352–47362. doi: 10.1074/jbc.M406056200. [DOI] [PubMed] [Google Scholar]
  • 22.Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG. ZINC: A free tool to discover chemistry for biology. J Chem Inf Model. 2012;2012(Jun):15. doi: 10.1021/ci3001277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Krieger E, Darden T, Nabuurs SB, Finkelstein A, Vriend G. Making optimal use of empirical energy functions: Force-field parameterization in crystal space. Proteins. 2004;57(4):678–683. doi: 10.1002/prot.20251. [DOI] [PubMed] [Google Scholar]
  • 24. Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1:100. [DOI] [PubMed]
  • 25.Shang X, et al. Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases. Chem Biol. 2012;19(6):699–710. doi: 10.1016/j.chembiol.2012.05.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Dontu G, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003;17(10):1253–1270. doi: 10.1101/gad.1061803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA. 2004;101(20):7618–7623. doi: 10.1073/pnas.0307512101. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
supp_110_8_3155__index.html (7.3KB, html)
1212324110_pnas.201212324SI.pdf (945.5KB, pdf)

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES