Figure 9.1. Inhibition of Rac GTPase function. (A) Approaches for blocking Rac function. Various small molecule inhibitors of Rac function have been described or considered. These include inhibitors of Rac post-translational modification. Rac terminates in a CAAX tetrapeptide sequence (C = Cys, A = aliphatic amino acid, X = Leu). This CAAX motif signals for three sequential post-translational modifications that convert the cytosolic, inactive Rac GTPase to a plasma membrane-associated protein. Geranylgeranyltransferase I (GGTaseI) catalyzes addition of the C20 geranylgeranyl isoprenoid to the Cys residue of the CAAX motif, followed by Rac converting enzyme 1 (Rce1)-catalyzed proteolytic removal of the AAX residues, and isoprenylcysteine carboxyl methyltransferase (Icmt)-catalyzed carboxyl methylation of the now terminal geranylgeranylated cysteine residue. Rac cycles between an inactive GDP-bound and an active GTP-bound state that is regulated by GTPase activating proteins (RhoGAPs) and guanine nucleotide exchange factors (RhoGEFs). Rac-GTP binds preferentially to a large spectrum of functionally diverse effectors (E) that regulate cytoplasmic signaling networks. GGTaseI inhibitors (GGTIs) block all CAAX-signaled modifications, rendering Rac cytosolic and inactive. Cysmethynil blocks the final CAAX modification step by inhibiting Icmt. NSC23766 inhibits RacGEF activation of Rac, whereas EHT 1864 impairs Rac-GTP formation and prevents Rac binding and activation of downstream effectors. (B) Structure of EHT 1864.

Figure 9.2. Fluorescence-based assays used to monitor EHT 1864 activity. (A) Addition of EHT 1864 to Rac1MantGDP complexes causes loss of the bound nucleotide. Rac1 (2 μM) preloaded with mantGDP was incubated in 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM MgCl₂. At the desired time, either EDTA or EHT 1864 was added to a final concentration of 10 mM and 50 μM, respectively. Changes in fluorescence were followed at λ_ex = 290 nm and λ_em = 440, where a decrease in fluorescence represents a loss in FRET between Trp 56 of Rac1 and the mant group, and therefore represents loss of mant nucleotide into solution. (B) EHT 1864 inhibits nucleotide loading at high concentrations. Incubation of 2 μM Rac1 with excess inhibitor prevents mant nucleotide loading that is stimulated by the addition of excess EDTA, as compared to in the absence of inhibitor. Exchange was followed on a SpectroMax Gemini at λ_ex = 290 nm, λ_em = 440 nm. An increase in fluorescence represents the binding of mant nucleotide to the Rho GTPase. (C) The EHT 1864 inhibitor is inherently fluorescent. Excitation and emission spectra were collected for the inhibitors, and optimal λ_ex and λ_em were found to be 360 and 440 nm, respectively. Data were collected using 10 μM inhibitor in a 20 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, and 50 mM NaCl buffer. (D) Binding curve for the interaction of EHT 1864 and Rac1. Incremental 1-μl volumes of Rac buffer were added to a 1 μM solution of EHT 1864 (both Rac and EHT 1864 were in 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 1 mM MgCl₂). The Rac solution also contained 1 μM EHT 1864. Increases in anisotropy, reflecting increases in Racinhibitor formation, were followed at λ_ex = 360 nm and λ_em = 440 nm. Data were fitted to a binding curve, from which a K_D can be estimated.

Figure 9.3. EHT 1864 is effective in specifically inhibiting PDGF-induced lamellipodia formation. (A) PDGF-induced actin reorganization is inhibited by EHT 1864. After overnight serum starvation in growth medium alone or supplemented with 5 μM EHT 1864 or EHT 8560 for the last 4 h of incubation, NIH/3T3 cultures were treated with 5 ng/ml PDGF for 15 min and then fixed, and actin filaments were visualized with Alexa-phalloidin. Scale bar represents 20 μm. Results shown are representative of three independent experiments. (B) Quantitation of data shown in A. Graphic representation of the percentage of PDGF-stimulated cells with lamellipodia in the presence or absence of EHT 1864 or 8560 and quantified on 100 cells for each condition. Results shown are the mean of three independent experiments; error bars indicate standard error of the mean. (C) EHT 1864 inhibits Rac1 interaction with Pak-RBD in vivo. NIH/3T3 cells were transiently cotransfected with expression constructs encoding Rac1(61L) or GFP-Pak-RBD. Cells were then maintained in growth medium supplemented with 0.5% calf serum and incubated with EHT 1864 for specific time periods. Cells were then washed and fixed for analysis on an inverted fluorescent microscope (λ_ex = 480 nm λ_em = 520 nm).

Figure 9.4. EHT 1864 blocks oncogenic Ras-stimulated cell transformation. (A) Expression level of H-Ras(61L) in stably transfected NIH/3T3 cells. The expression of HA epitope-tagged H-Ras(61L) protein was detected by blot analysis using an anti-HA antibody (clone 3F10; Roche Diagnostics). Blot analysis for β-actin (clone AC15; Sigma-Aldrich) was also done to verify equivalent total protein loading. (B) EHT 1864 inhibition of Ras-induced formation of foci of transformed cells. NIH/3T3 cells stably transfected with the empty pCGN-hygro (vector) or encoding H-Ras(61L) were plated and allowed to reach confluency. Cells were cultured in 10% serum growth medium, either alone or supplemented with 5 μM EHT 1864. The appearance of foci of transformed cells was quantitated 14 days after plating. Cells were then fixed and stained with crystal violet. (C) Quantitation of data shown in B. Data shown are representative of two independent experiments, each performed in duplicate.

Figure 9.5. EHT 1864 partially inhibits oncogenic Ras-induced anchorage-independent growth of NIH/3T3 fibroblasts. (A) Single-cell suspensions of NIH/3T3 cells stably transfected with the empty pCGN-hygro (vector) plasmid or encoding H-Ras(61L) were cultured in a soft agar medium in the presence or absence of 5 μM EHT 1864, and the appearance of colonies of proliferating cells was monitored 16 days later. (B) Quantitation of data shown in A. Data shown are representative of two independent experiments, each performed in triplicate.