Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe

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.This work was supported by National Science Foundation (NSF) Grant MCB0956027 and National Institutes of Health Grant R03 MH081231-01 from the Molecular Libraries Program (to A. W. N.); University of New Mexico Center for Molecular Discovery Molecular Libraries Probe Production Centers (UNMCMD MLPCN) National Institutes of Health Grants U54MH084690 and R01HL081062 (to L. A. S.); UNM National Center for Research Resources (NCRR) Grant 5P20RR016480 (to L. G. H.); National Institutes of Health Grant R21 CA170375-01 through the NCI (to A. W. N., L. G. H., and J. E. G.); National Institutes of Health Grants NS066429 and AI092130 (to T. B.); and University of Kansas Specialized Chemistry Center (KUSCC) MLPCN National Institutes of Health Grant U54HG005031 (to J. A.)

Teaching Under Pressure

The purpose of this comparative case study was to investigate, explore, and describe the methods and practices of secondary instrumental music education in a public school setting. Two sites, a Rhode Island public school and a Rhode Island community music school, were chosen for observation. Two major ensembles, a jazz band and a concert band, were observed at each site on four different occasions. Observations were organized by the National Standards of Music Education, although the study did not focus on whether or not the programs “met” the standards. Data was also placed into the category of non-musical factors. Observations and analysis found that the non-musical factors, and themes that emerged from those factors, were the largest noticeable difference between the two sites. These factors also contributed to the disparity in the two sites abilities to address the standards. Non-musical factors such as scheduling and interruptions were the major issues facing the public school site, including split rehearsal times between ensembles and missed rehearsals due to assemblies. Both sites had significant gaps in their addressing of the standards, although the community music school met more of the standards. This may just be a result of the increase in time spent in rehearsal. This research opens the questions of whether or not the constraints facing public school music educators are hindering their ability to fully educate their students. Research can also be performed to gauge the perceptions that secondary instrumental music educators have on the standards

Endocrine late effects in childhood cancer survivors

Endocrine complications are highly prevalent in childhood cancer survivors. Approximately 50% of survivors will experience at least one hormonal disorder over the course of their lives. Endocrine complications often are observed in survivors previously treated with radiation to the head, neck, or pelvis. We provide an overview the most common endocrine late effects seen in survivors, including hypothalamic-pituitary dysfunction, primary thyroid dysfunction, obesity, diabetes mellitus, metabolic syndrome, and decreased bone mineral density. Primary gonadal injury is discussed elsewhere in this series. Given a variable latency interval, a systematic approach where individuals are periodically screened on the basis of their risk factors can help to improve health outcomes by prompt diagnosis and treatment of evolving endocrinopathies. These recommendations must be revised in the future given changes and improvements in cancer treatment over time

Endocrine late effects in childhood cancer survivors

Endocrine complications are highly prevalent in childhood cancer survivors. Approximately 50% of survivors will experience at least one hormonal disorder over the course of their lives. Endocrine complications often are observed in survivors previously treated with radiation to the head, neck, or pelvis. We provide an overview the most common endocrine late effects seen in survivors, including hypothalamic-pituitary dysfunction, primary thyroid dysfunction, obesity, diabetes mellitus, metabolic syndrome, and decreased bone mineral density. Primary gonadal injury is discussed elsewhere in this series. Given a variable latency interval, a systematic approach where individuals are periodically screened on the basis of their risk factors can help to improve health outcomes by prompt diagnosis and treatment of evolving endocrinopathies. These recommendations must be revised in the future given changes and improvements in cancer treatment over time

Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases

<div><p>Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library^® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses—using the rotationally constrained carboxylate in R-naproxen—led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and efficacy in the treatment of several epithelial cancer types on account of established human toxicity profiles and novel activities against Rho-family GTPases.</p></div

R-naproxen inhibits Rac and Cdc42 activation in response to growth factor stimulus of cells.

<p>(<b>A-B</b>) GLISA effector binding assays were used to quantify Rac1 and Cdc42 GTPase activities in Swiss 3T3 fibroblast cell lysates following preincubation for varying times (15 min or 1 h) or with varying concentrations of R-naproxen, S-naproxen or 6MNA, with or without (+/-) 100 ng/ml EGF stimulation. In panel A, R-naproxen and 6-MNA were used at 300 μM and time of exposure varied as indicated. One-way ANOVA and Dunnett’s multiple comparison test shows select R-Naproxen samples significantly (*p<0.05, ***p<0.001) different from EGF stimulated controls. In panel B, drug doses ranged from 10–300 μM as indicated. CID2950007 is a selective inhibitor of Cdc42 and served as a positive control. N = 3–8. One-way ANOVA and Dunnett’s multiple comparison test did not identify significant differences relative to EGF-stimulated controls. (<b>C-D</b>) Flow cytometric G-trap assay was used to quantitatively assess dose dependent inhibition of Rac1 and Cdc42 activation in HeLa cells following 2 h pre-treatment with R-naproxen, S-naproxen or 6MNA (30–1000 μM) and 2 min EGF stimulation (100 ng/ml). The inhibition curves were fitted to the sigmoidal dose-response equation in GraphPad Prism 5. Quantification of three independent measurements are plotted ± SEM.</p

R-naproxen acts via a COX-independent mechanism.

<p>OvCa433 cells were left untreated (BSA only) or incubated with or without 300 μM of S-naproxen or R-naproxen for 48 h. Thereafter, stimulation was for 10 min with EGF where indicated. Cell lysates were resolved by SDS-PAGE and immunoblotted for phosphorylated EGFR (pEGFR) or phosphorylated ERK (pERK). Phosphorylation of EGFR and ERK were determined in the presence of S-naproxen (SN) or R-naproxen (RN), without (BSA) or with EGF (EGF). Shown are western blots probed with phospho-specific antibodies for (<b>A</b>) pEGFR and (<b>C</b>) pERK relative to immunoblots for total EGFR or ERK proteins. Bar graphs show quantification of each phosphoprotein by densitometry and normalized to total protein (measured either by immunoblot or via Coomassie staining) (<b>B</b>) pEGFR/total protein and (<b>D</b>) pERK/total protein or total ERK. N = 2 One-way ANOVA and Tukey’s multiple comparison test shows EGF-stimulated samples +/- drug values significantly (*p<0.05, **p<0.01, ***p<0.001) different from BSA controls as indicated on the graph. Unstimulated samples +/- drug were not statistically different when compared pairwise and the same was true for pairwise comparisons of stimulated samples +/- drug.</p