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.

cystin-1, the product of the major gene mutated in autosomal dominant polycystic kidney disease (ADPKD), has been shown to associate with multiple epithelial cell junctions. Our hypothesis is that polycystin-1 is an important protein for the initial establishment of cell-cell junctions and maturation of the cell and that polycystin-1 localization is dependent on the degree of cell polarization. Using laser-scanning confocal microscopy and two models of cell polarization, polycystin-1 and desmosomes were found to colocalize during the initial establishment of cell-cell contact when junctions were forming. However, colocalization was lost in confluent monolayers. Parallel morphological and biochemical evaluations revealed a profound mispolarization of desmosomal components to both the apical and basolateral domains in primary ADPKD cells and tissue. Studies of the intermediate filament network associated with desmosomes showed that there is a decrease in cytokeratin levels and an abnormal expression of the mesenchymal protein vimentin in the disease. Moreover, we show for the first time that the structural alterations seen in adherens and desmosomal junctions have a functional impact, leaving the ADPKD cells with weakened cell-cell adhesion. In conclusion, in this paper we show that polycystin-1 transiently colocalizes with desmosomes and that desmosomal proteins are mislocalized as a consequence of polycystin-1 mutation.

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Epithelial ovarian cancer is the major cause of gynecologic malignancy deaths. Because of their roles in cell adhesion and migration, Rho family GTPases have been suggested as potential therapeutic targets in human cancers. We identify the Rac1 and Cdc42 GTPases as relevant targets in papillary serous and endometriod tumors. Cdc42 is overexpressed in primary human ovarian tumors and cancer cell lines, and a novel splice variant Rac1b is upregulated in tumors of advanced stage and grade. GTPase activities in primary ascites are 3 to 6-fold higher than in cultured cells. R-Naproxen was identified by high throughput screening of a Prestwick compound library as a select non-steroidal anti-inflammatory drug (NSAID) from 23 tested that selectively targets Rac1 and Cdc42 in a bead-based assay using purified proteins. The drug is demonstrated to have positive benefit against cell behaviors required for ovarian cancer dissemination and metastasis using both cell lines and primary human tumor cell isolates. Human ovarian cells show slowed cell proliferation, as well as impaired migration, adhesion and invadopodia formation. Other NSAIDs with structural similarity (S-naproxen and 6-methoxy naphthalene acetic acid) lack these properties, while a specific Rac inhibitor NSC 23766 mimics the effects. Molecular docking shows R-Naproxen can bind the GDP-bound, but not GTP-bound Rac1, suggesting it may act by stabilizing Rac and Cdc42 in the inactive state. R-Naproxen has potential for rapid translation and efficacy in the treatment of metastatic ovarian cancer on account of FDA approval and novel activities against Rho-family GTPases. Funding for this study was generously provided by NIH grants U54MH074425, U54MH084690, R03MH081231, P30CA118100 and UNM Cancer Center FIG.0990MD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2011-LB-214