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. 2013 Mar 22;288(12):8531-8543.
doi: 10.1074/jbc.M112.435941. Epub 2013 Feb 4.

Characterization of a Cdc42 protein inhibitor and its use as a molecular probe

Lin Hong  1 , S Ray Kenney  2 , Genevieve K Phillips  3 , Denise Simpson  4 , Chad E Schroeder  4 , Julica Nöth  4 , Elsa Romero  5 , Scarlett Swanson  5 , Anna Waller  1 , J Jacob Strouse  1 , Mark Carter  1 , Alexandre Chigaev  5 , Oleg Ursu  6 , Tudor Oprea  6 , Brian Hjelle  5 , Jennifer E Golden  4 , Jeffrey Aubé  7 , Laurie G Hudson  8 , Tione Buranda  5 , Larry A Sklar  9 , Angela Wandinger-Ness  10
Affiliations

Characterization of a Cdc42 protein inhibitor and its use as a molecular probe

Lin Hong et al. J Biol Chem. .

Erratum in

  • J Biol Chem. 2014 Mar 7;289(10):6837

Abstract

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

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Figures

FIGURE 1.
FIGURE 1.
Two structural analogs selectively inhibit Cdc42 nucleotide binding activity. A, structures of CID2950007 and its analog CID44216842. B, the concentration effects of CID2950007 and CID44216842 on eight GTPases. The curves were fitted to the sigmoidal dose-response equation using GraphPad Prism. The percentage of response was calculated as the ratio of (sample MCF − negative control MCF)/(positive control MCF − negative control MCF) (where MCF is median channel fluorescence). For the positive control, DMSO instead of the compound was added; for the negative control, the compound was replaced with GTP at a concentration 5000-fold greater than BODIPY® FL GTP. The EC50 for Cdc42 wild type and Cdc42Q61L mutant were 2.1 and 2.6 μm, respectively, for CID2950007 and 1.0 and 1.2 μm, respectively, for CID44216842. C, the concentration effects of the two compounds on BODIPY® FL GDP binding to eight GTPases. The EC50 values for Cdc42 wild type and Cdc42Q61L mutant were 1.4 and 2.9 μm, respectively, for CID2950007 and 0.3 and 0.5 μm, respectively, for CID44216842. Data shown are representative of at least three independent sets of measurements with each set conducted in duplicate.
FIGURE 2.
FIGURE 2.
CID2950007 is an allosteric inhibitor of Cdc42. A, compound CID2950007 and GTP induce the dissociation of BODIPY® FL GTP through different mechanisms. After BODIPY® FL GTP bound to Cdc42, either 250 μm GTP (red) or 10 μm CID2950007 (purple) was added. The fluorescence curves were fitted to a single exponential equation to get dissociation rate constants (supplemental Equation 1). The concentration of GTP or CID2950007 did not affect the determination of the dissociation rate constant. B, the presence of CID2950007 changed both Bmax and Kd in the BODIPY® FL GTP equilibrium binding assay. C, CID2950007 inhibited BODIPY® FL GTP binding. An equal amount of buffer (green), 400 nm Cdc42 (red), 400 nm Cdc42 + 0.1% DMSO (blue), or 400 nm Cdc42 + 10 μm CID2950007 (purple) was added to a solution of 500 nm BODIPY® FL GTP. The inset shows the expanded view of the initial fluorescence change. D, analog CID44216842 has similar effects as CID 2950007 toward BODIPY® FL GTP binding to Cdc42. E, buffer was the same as that in A except that without EDTA, CID2950007 inhibited BODIPY® FL GTP binding at a slower rate. The inset shows the expanded view of the initial fluorescence change. F, CID2950007 inhibits BODIPY-GDP binding to Cdc42 similar to BODIPY-GTP binding. G and H, CID2950007 did not inhibit BODIPY® FL GTP binding to Ras (G) or Rac1 (H). Fluorescence intensity was normalized as percentage using the equilibrium reading as the maximum against which the percentage was obtained. Conditions were the same as that in A. I, model of the CID2950007 inhibition mechanism. After guanine nucleotide binds to Cdc42, CID2950007 associates with the complex and induces the dissociation of the guanine nucleotide. The resulting Cdc42 complex is locked in an inactive conformation. Experiments were performed in at least duplicates. Representative curves are shown.
FIGURE 3.
FIGURE 3.
CID2950007 did not show cytotoxicity in multiple cell lines. A and B, cell viability measured toward OVCA429 (A) and SKOV3ip (B) cells after a 4-day incubation at 0.1–10 μm using MTS assay. C–H, cell viability measured using luminescence assay. C, compound CID2950007 tested on Swiss 3T3 cells at concentrations of 0.5–10 μm for 24 h. D, compound CID2950007 tested on Vero E6 cells at concentrations of 0.5–10 μm for 48 h. E and F, compound CID2950007 tested on U937 ΔST cells up to 10 μm for 24 h (E) and up to 30 μm for 1 h (F). G and H, compound CID44216842 tested on U937 ΔST cells up to 10 μm for 24 h (G) and up to 30 μm for 1 h (H). A and B represent three independent trials with assays conducted in duplicate or triplicate. From C–H, experiments were conducted in at least triplicate. Means ± S.E. from all trials are plotted. For compound treatments at the experimental concentrations and time durations, there were no differences with p < 0.05 based on one-way ANOVA with Dunnett's post test and relative to either untreated control cells (A and B) or DMSO-treated cells (C–H). Statistical significance assessment was the same using either as the comparison group. RLU, relative light units.
FIGURE 4.
FIGURE 4.
Cdc42 activation was specifically inhibited by CID2950007 and its analog CID44216842 as demonstrated by the GLISA assay. A and B, Swiss 3T3 cells were serum-starved and then pretreated with compounds CID2950007 or CID44216842 at indicated concentrations as detailed under ”Experimental Procedures.“ Cells were subsequently stimulated with EGF to activate Cdc42 or Rac1 and compared with unstimulated cells. Control cells were mock-treated with 0.1% DMSO to account for compound diluent. After cell lysis, the amounts of active Cdc42 or Rac1 were quantified based on p21-activated protein kinase binding by GLISA. The following conditions were used: Cdc42, nine independent trials for CID2950007 and two independent trials for CID44216842 with duplicate or triplicate samples for each condition; Rac1, three independent trials for CID2950007 with duplicate or triplicate samples and one trial for CID44216842 with triplicate samples. C, Vero E6 cells were serum-starved, treated with CID2950007 or CID44216842, and stimulated with calpeptin to activate RhoA with one trial in triplicate. Immunofluorescence micrograph confirms RhoA activated stress fiber formation. Purified GTP-loaded Cdc42, Rac1, or RhoA control proteins were used to calculate the ng of activated GTPase. Means ± S.E. with EGF control subtracted from all trials are plotted. Asterisks denote p < 0.05 based on one-way ANOVA with Dunnett's post test and relative to EGF- or calpeptin-stimulated control cells.
FIGURE 5.
FIGURE 5.
CID2950007 decreased filopodia formation in 3T3 cells after bradykinin treatment. A, representative images of resting control cells, cells stimulated with 100 ng/ml bradykinin, and cells incubated with 10 μm CID2950007 for 1 h before the addition of bradykinin. Cells were permeabilized and stained as described under ”Experimental Procedures“ before the pictures were taken on a Zeiss LSM 510 Meta. Lower panels show 6.5-fold magnified views of boxed areas in top panels. The blue line shows tracing of filopodia for quantification. B, numbers of filopodia per cell. C, length of filopodia on each cell, 1 μm = 28.5 pixels. The experiment was repeated three times, and 30–60 cells were randomly imaged and analyzed. Mean lengths or number of filopodia ± S.E. from a single experiment are plotted. Asterisks denote p < 0.05 based on one-way ANOVA with Dunnett's post test relative to bradykinin-stimulated control cells.
FIGURE 6.
FIGURE 6.
CID2950007 inhibited the migration of OVCA429 (A) and SKOV3ip (B) cells in a dose-dependent manner. Cells were allowed to attach to the Boyden chambers. CID2950007 at indicated concentrations were added to the growth media. Membrane filters were imaged 48 h later. All assays represent three independent trials with three representative fields counted for each treatment. Means ± S.E. from all trials are plotted. Asterisks denote p < 0.05 based on one-way ANOVA with Dunnett's post test and relative to migration of untreated control cells. Statistical significance was the same when treated samples were compared with 0.1% DMSO treatment group.
FIGURE 7.
FIGURE 7.
CID2950007 inhibited Sin Nombre virus infection efficiency. CID2950007 at 10 μm was added to host Vero E6 cells either only during virus contact (60–75 min) followed by reincubation for 24–48 h in the absence of the compound or during virus contact and maintained throughout the culturing period after viral contact (24–48 h). The two conditions provide an indication of CID2950007 effects on virus entry alone as compared with effects on virus entry and replication. Viral RNA content was quantified after 24 or 48 h as described under ”Experimental Procedures.“ Viral infection experiments were repeated three times with 3–6 replicates per trial. Means ± S.E. from all trials are plotted. Asterisks denote p < 0.05 based on one-way ANOVA with Dunnett's post test and relative to infected control cells without compound treatment at the relevant time point. SNV, Sin Nombre virions.
FIGURE 8.
FIGURE 8.
CID2950007 inhibited VLA-4 activation in a dose-dependent manner. A, VLA-4 activation status is monitored in real time. LDV-FITC was first added to U937 FPRΔST cells to achieve resting state binding (Kd ∼12 nm) (25). Then, N-formyl peptide (N-fMLFF) was added to stimulate VLA-4 activation. When the ligand binding reached equilibrium, CID2950007 at the indicated concentrations or an equal volume of DMSO was added. Nonfluorescent LDV was added to dissociate remaining LDV-FITC. B, quantification of the CID2950007 inhibition effects. The inhibition percentage was calculated according to supplemental Equation 3. At least three independent trials were performed with each trial in duplicate. C, representative curves from one trial are shown. B and D, means ± S.E. from one trial are plotted. Asterisks denote p < 0.05 based on one-way ANOVA with Dunnett's post test and relative to DMSO treatment.
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