Activation of Rho Family GTPases by Small Molecules

Abstract

Ras and Ras-related small GTPases are key regulators of diverse cellular functions that impact cell growth, survival, motility, morphogenesis, and differentiation. They are important targets for studies of disease mechanisms as well as drug discovery. Here, we report the characterization of small molecule agonists of one or more of six Rho, Rab, and Ras family GTPases that were first identified through flow cytometry-based, multiplexed high-throughput screening of 200000 compounds. The activators were categorized into three distinct chemical families that are represented by three lead compounds having the highest activity. Virtual screening predicted additional compounds with potential GTPase activating properties. Secondary dose-response assays performed on compounds identified through these screens confirmed agonist activity of 43 compounds. While the lead and second most active small molecules acted as pan activators of multiple GTPase subfamilies, others showed partial selectivity for Ras and Rab proteins. The compounds did not stimulate nucleotide exchange by guanine nucleotide exchange factors and did not protect against GAP-stimulated GTP hydrolysis. The activating properties were caused by a reversible stabilization of the GTP-bound state and prolonged effector protein interactions. Notably, these compounds were active both in vitro and in cell-based assays, and small molecule-mediated changes in Rho GTPase activities were directly coupled to measurable changes in cytoskeletal rearrangements that dictate cell morphology.

Figure 1

Structures of lead compounds from three chemical families of GTPase activators. HTS and virtual screening identify three families of small molecule GTPase activators, having the general structure section A–linker L–section B (outlined by boxes). The lead compounds representing the (a) nicotinic acid, (b) indole acid, and (c) salicylic acid analogue families are depicted.

Figure 2

Heat map and dose–response curves showing small molecule agonists active against GTPase targets. (a) Activity (−log EC₅₀) heat map of 43 compounds, representing three families (nicotinic acids, indole acids, and carboxylic acids), that activate GTP binding on the eight indicated GTPases. Blue reflects the best biological activity measured as increased in vitro nucleotide binding activity, whereas red reflects a lack of activity. CID numbers of compounds tested in dose–response assays are given (modified from Probe Report). CID numbers with asterisks indicate lead compounds from each of the three chemical families, and arrows indicate compounds with GTPase-selective activity. (b–d) Representative dose–response assays of the three lead compounds on six indicated GTPase targets. Assays were performed in the presence of 100 nM BODIPY-GTP and EDTA and varying concentrations of small molecule activators using proteins bound to red fluorescent glutathione beads of differing intensities. wt indicates wild type GTPase, and act indicates activated mutant GTPase. Mutants include H-RasG12V, Rac1Q61L, and Cdc42Q61L.

Figure 3

Small molecule activators increase nucleotide binding affinity, resulting in additional bound GTP over time and slowed dissociation of GTP. (a and b) Cdc42-conjugated beads were preincubated for 3 min with 1% DMSO and 10 μM activator 888706, respectively. Association binding of indicated concentrations of BODIPY-GTP to the Cdc42-conjugated beads was measured by following the fluorescence on the beads as a function of time. Cdc42-conjugated (c) and Rac1-conjugated (d) beads were preincubated with DMSO or 10 μM activator 888706. The fluorescence on the beads was measured as a function of time following the addition of 200 nM BODIPY-GTP. Dissociation of BODIPY-GTP was initiated via addition of GTP (200 μM final concentration) at 170 s (c) or 300 s (d). Plotted are mean channel fluorescence (MCF) values vs time in seconds, with the measured data as dots and lines for the calculated fits by one-state (DMSO) or two-state (with activator) binding models (see the text for details) (n = 2). Note that because of instrument setting differences, MCF values for Rac1-conjugated beads were higher. (e) Dissociation of BODIPY-GTP from Cdc42-conjugated beads was monitored after preincubation with 10 μM activator 888706 and 200 nM BODIPY-GTP binding for the indicated times. Plotted are MCF values vs time after the addition of 200 μM GTP.

Figure 4

Small molecule activators enhance effector protein binding but do not perturb GEF or GAP activities. (a and b) Dbs RhoGEF assay monitoring release of bound [³H]GDP from purified Cdc42 as detailed in Methods. Panel a shows the percentage of initial [³H]GDP remaining bound to Cdc42 (∼0.04 nM) (average of 3950 ± 475 cpm/sample at t₀) as a function of time after addition of excess unlabeled GTP in the presence of 200 nM Rho Dbs GEF alone, DMSO, or small molecule activators (n = 3). Panel b shows the best fit for GDP dissociation following additions of the Dbs GEF or activator compound determined using two-phase exponential decay (Y = P₁e^–K₁t + P₂e^–K₂t, where P₁ + P₂ = 100). Panel c shows [³H]GDP remaining bound to Cdc42 (∼0.04 nM) 20 min after addition of excess unlabeled GTP to samples, in the presence of the indicated activator and in the absence (−) or presence (+) of 200 nM Rho Dbs GEF. Samples treated with only DMSO served as negative controls (n = 2). (d) Purified Rac1 or Rho GTPases (17.8 μM) were incubated for 15 min in the presence of small molecule activators and 200 μM GTP prior to the addition of purified RhoGAP (5.5 μM). GTP hydrolysis was measured at 650 nm based on colorimetric detection of phosphate release after 20 min (n = 2). (e) PAK effector binding was used to measure Rac1 activation status. Purified His-Rac1 (80 ng) was briefly preincubated with compounds (0–12.5 μM); 125 nM GTPγS was added, and active Rac1 was isolated by binding to 5 μg GST-PAK-PBD immobilized on GSH beads. Bound active Rac1 was detected by immunoblotting. The top panel shows a representative immunoblot (previously shown in Figure 3A of Probe Report), and the bottom panel shows quantification of dose-dependent activation for three compounds (n = 1).

Figure 5

Small molecule activators lead to Rac1 and Cdc42 activation in cell-based assays. (a) Rac1 activation measured in an effector binding, bead-based flow cytometry assay. HeLa cells were serum starved overnight and then treated with DMSO (basal GTPase activity control – resting), activator compound, or 100 ng/mL EGF (stimulated positive control) for 2 min. Four activator compounds were tested at four concentrations each in the range of 0.1–100 μM (n = 2). Plotted are fold changes in Rac1-GTP levels in cells treated with compounds or EGF relative to those in resting cells. (b) Cdc42 activation measured in a GLISA. Swiss 3T3 cells were serum starved overnight and then treated with the indicated concentrations of the activator compounds. After serum starvation, control cells were treated with DMSO (vehicle) and stimulated with 100 ng/mL EGF for 2 min. Three lead compounds each were tested at 3 and 30 μM (n = 2). The baseline level Cdc42-GTP detected in resting cells was set to zero and subtracted from all values.

Figure 6

Small molecule activators induce Rho family GTPase-dependent morphologic changes in cells. (a) Resting RBL-2H3 mast cells were treated with DMSO (negative control, t₀), stimulated with ligand (DNP-BSA) to activate (positive control), or treated with two representative compounds (10 μM) from each of the three chemical families. Samples were fixed and stained with rhodamine phalloidin to visualize the actin cytoskeleton. Individual panels show x–y and x–z views of representative cells (n = 3). Bars are 10 μm. (b–g) Cell-based dose–response assays conducted across a dose range of 0.03–30 μM. Cell activation was quantified as an increase in cell area due to cell flattening. Four micrographs were counted for each condition taken at 40× magnification representing approximately 60–200 cells counted per condition. Untreated and DMSO-treated cells served as negative controls. DNP-BSA-stimulated cells provided a measure of maximal activation (n = 3).