Rational design and characterization of a Rac GTPase-specific small molecule inhibitor

Abstract

The signaling pathways mediated by Rho family GTPases have been implicated in many aspects of cell biology. The specificity of the pathways is achieved in part by the selective interaction between Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. Here, we report a first-generation small-molecule inhibitor of Rac GTPase targeting Rac activation by GEF. The chemical compound NSC23766 was identified by a structure-based virtual screening of compounds that fit into a surface groove of Rac1 known to be critical for GEF specification. In vitro it could effectively inhibit Rac1 binding and activation by the Rac-specific GEF Trio or Tiam1 in a dose-dependent manner without interfering with the closely related Cdc42 or RhoA binding or activation by their respective GEFs or with Rac1 interaction with BcrGAP or effector PAK1. In cells, it potently blocked serum or platelet-derived growth factor-induced Rac1 activation and lamellipodia formation without affecting the activity of endogenous Cdc42 or RhoA. Moreover, this compound reduced Trio or Tiam1 but not Vav, Lbc, Intersectin, or a constitutively active Rac1 mutant-stimulated cell growth and suppressed Trio, Tiam1, or Ras-induced cell transformation. When applied to human prostate cancer PC-3 cells, it was able to inhibit the proliferation, anchorage-independent growth and invasion phenotypes that require the endogenous Rac1 activity. Thus, NSC23766 constitutes a Rac-specific small-molecule inhibitor that could be useful to study the role of Rac in various cellular functions and to reverse tumor cell phenotypes associated with Rac deregulation.

Fig. 1.

Identification of NSC23766 as an inhibitor of Rac1–Trio interaction. (A upper) The inhibitory effect of a panel of compounds predicted by virtual screening on Rac1 interaction with TrioN was tested in a complex formation assay. (His)₆-tagged TrioN (0.5 μg) was incubated with GST alone or nucleotide-free GST-Rac1 (2 μg) in the presence or absence of 1 mM indicated National Cancer Institute compound and 10 μl of suspended glutathione-agarose beads. After an incubation at 4°C for 30 min, the beads associated (His)₆-TrioN were detected by anti-His Western blotting. (A Lower) The effect of the compounds on Cdc42 binding to Intersectin was determined similarly; ≈1 μg of GST or GST-tagged Intersectin was incubated with the nucleotide-free, (His)₆-tagged Cdc42 (0.25 μg) under similar conditions. Data are representative of the results from four independent experiments. (B) A simulated docking model of NSC23766 on Rac1 surface. The docking model was generated with the flexx program and was visualized with viewerpro. (Left) A top view of the binding pocket of Rac1 bound to NSC23766. (Right) Predicted structural contacts of NSC23766 in the binding pocket of Trp⁵⁶. (C) The inhibitory effect of NSC23766 was examined with the Rac1L70A/S71A mutant binding to TrioN.

Fig. 2.

Dose-dependent specific inhibition of GEF interaction with Rac1 by NSC23766. (A) (His)₆-tagged TrioN (0.5 μg) was incubated with GST alone or nucleotide-free, GST-fused Cdc42 or Rac1 (2 μg) in the binding buffer containing different concentrations of NSC23766 and 10 μl of suspended glutathione-agarose. The beads associated (His)₆-TrioN were detected by anti-His Western blotting. (B) myc-tagged Tiam1 expressed in Cos-7 cell lysates were incubated with (His)₆-Rac1 in the presence of increasing concentrations of NSC23766. The association of Rac1 with Tiam1 was examined by anti-His blotting after anti-myc immunoprecipitation. (C) The AU-tagged PDZ-RhoGEF was expressed in Cos-7 lysates and incubated with GST or GST-RhoA in the presence of varying concentrations of NSC23766. The RhoA-associated PDZ-RhoGEF was probed with anti-AU antibody after affinity precipitation by glutathione agarose beads. (D) (His)₆-Rac1 loaded with GDP or GTPγS was incubated with GST-BcrGAP or GST-PAK1 (p21-binding domain) in the presence or absence of 200 μM NSC23766, and the interaction with GST-BcrGAP or GST-PAK1 was probed by anti-His blot after affinity precipitation by glutathione agarose beads.

Fig. 3.

NSC23766 was effective in specifically inhibiting Rac1 activation in cells. (A) The activation states of endogenous Rac1, Cdc42, and RhoA in NIH 3T3 cells with or without NSC23766 treatment were detected by the effector pull-down assays. At 80% confluency in the presence of 10% serum, NIH 3T3 cells in 100-mm dishes were treated with the indicated dosages of NSC23766 for 12 h. Cell lysates containing similar amounts of Rac1, Cdc42, or RhoA were incubated with the agarose-immobilized GST-PAK1, GST-WASP, or GST-Rhotekin, and the co-precipitates were subjected to anti-Rac1, Cdc42 or RhoA Western blot analysis to reveal the amount of GTP-bound Rho proteins. (B) The inhibitory effect of NSC23766 on the PDGF-stimulated Rac1 activation was determined by the GST-PAK1 pull-down assay. Serum-starved NIH 3T3 cells in the DMEM with different dosages of NSC23766 were treated with 10 nM PDGF for 2 min. (C) NSC23766 inhibited PDGF-stimulated lamellipodia formation. After overnight serum starvation in the presence or absence of 50 μM NSC23766, Swiss 3T3 cells were treated with 10 nM PDGF for the indicated time. The cells were fixed and stained with Rhodamine-labeled phalloidin. Results shown are representative of three independent experiments.

Fig. 4.

NSC23766 specifically inhibited Rac GEF-stimulated cell growth and transformation. (A) WT or L61Rac1-expressing NIH 3T3 cells were grown in 5% serum in the presence (–––) or absence (—) of 100 μM NSC23766. The cells were split in triplicate in six-well plates at a density of 5 × 10⁴ cells per well. The GTP-bound L61Rac1 and endogenous Rac1 of the L61Rac1-expressing cells were probed by GST-PAK1 pull-down after 12 h treatment with increasing concentrations of NSC23766. (B) WT or the GEF (Tiam1 or Lbc)-expressing NIH 3T3 cells were grown in 5% serum in the presence (–––) or absence (—) of 100 μM NSC23766, and the cell numbers were determined by daily cell counting. (C) Stable transfectant of Tiam1-expressing NIH 3T3 cells were cultured in 0.3% soft-agar medium for 14 days in the presence or absence of 100 μM NSC23766. The number and the morphology of the colonies were examined under a microscope. (D) Stable V12-H-Ras-expressing NIH 3T3 cells were assayed for the growth in the soft-agar medium in the presence or absence of 100 μM NSC23766. The colonies were scored 14 days after plating.

Fig. 5.

NSC23766 inhibited the proliferation, anchorage-independent growth and invasion of PC-3 prostate cancer cells. (A) PC-3 cells were grown in 5% calf serum supplemented with the indicated concentrations of NSC23766. The cells were split in triplicate in 96 wells at 1.5 × 10³ cells per well. Cell numbers were assayed by using CellTiter 96 AQueous cell proliferation assay kit in different days. (B) PC-3 and RWPE-1 prostate epithelial cells (1.25 × 10³ per well) were grown in 0.3% agarose in different doses of NSC23766, and the number of colonies formed in soft agar was quantified 12 days after plating. (C) PC-3 cells were placed in an invasion chamber for 24 h at 37°C in the absence or presence of 25 μM NSC23766.