A novel small-molecule screening strategy identifies mitoxantrone as a RhoGTPase inhibitor

Abstract

RhoGTPases are GDP/GTP molecular switches that control a wide variety of cellular processes, thereby contributing to many diseases, including cancer. As a consequence, there is great interest in the identification of small-molecule inhibitors of RhoGTPases. In the present paper, using the property of GTP-loaded RhoGTPases to bind to their effectors, we describe a miniaturized and robust assay to monitor Rac1 GTPase activation that is suitable for large-scale high-throughput screening. A pilot compound library screen revealed that the topoisomerase II poison MTX (mitoxantrone) is an inhibitor of Rac1, and also inhibits RhoA and Cdc42 in vitro. We show that MTX prevents GTP binding to RhoA/Rac1/Cdc42 in vitro. Furthermore, MTX strongly inhibits RhoGTPase-mediated F-actin (filamentous actin) reorganization and cell migration. Hence, we report a novel biochemical assay yielding the identification of RhoGTPase inhibitors and we present a proof-of-concept validation with the identification of MTX as a novel pan-RhoGTPase inhibitor.

ROBO1, a tumor suppressor and critical molecular barrier for localized tumor cells to acquire invasive phenotype: Study in African‐American and Caucasian prostate cancer models

“…Our interpretation of this data is that the loss of ROBO1 lifts restrictions over DOCK1 that in turn activates Rac1. There have been attempts to develop Rac1 inhibitors for cancer treatment; however, homology with RhoGTPase family is a major limitation to develop Rac1‐specific drug . The clinically significant finding of this study is that suppression of DOCK1 was observed to reduce the activity of Rac1 in indolent CaP cells, therefore identifying DOCK1 suppression and ROBO1‐reactivation as future strategies to treat metastatic CaP in African‐Americans.…”

“…There have been attempts to develop Rac1 inhibitors for cancer treatment; however, homology with RhoGTPase family is a major limitation to develop Rac1-specific drug. 34 The clinically significant finding of this study is that suppression of DOCK1 was observed to reduce the activity of Rac1 in indolent CaP cells, therefore identifying DOCK1 suppression and ROBO1-reactivation as future strategies to treat metastatic CaP in African-Americans. E-cadherin is reported to maintain the intercellular junctions and cytoskeleton stability by tight physical interactions with catenin molecules.…”

Direct Modulation of Small GTPase Activity and Function

“…[100] Compound 24 impairs GTP loading of RhoA/Rac1/Cdc42 in vitro,Factin reorganization, and cell migration in aw ound-healing assay.H owever,a se ach member of the Rho-GTPase family exerts defined functions by regulating distinct signaling pathways,s elective inhibitors instead of pan inhibitors are desired.…”

Mitoxantrone, More than Just Another Topoisomerase II Poison

“…As members of the kinome bind and hydrolyze ATP, Rho (Ras homology) GTPases analogously hydrolyze GTP to cycle between “on” (GTP bound) and “off” (GDP bound) states . Through this simple cyclical process, Rho GTPases function as master regulators of a wide variety of cellular processes including motility, morphogenesis, and proliferation . Members of the Rho GTPase family represent appealing targets for therapeutic intervention as their deregulation has been described in multiple diseases including cancer .…”

“…Through this simple cyclical process, Rho GTPases function as master regulators of a wide variety of cellular processes including motility, morphogenesis, and proliferation . Members of the Rho GTPase family represent appealing targets for therapeutic intervention as their deregulation has been described in multiple diseases including cancer . A recent screening campaign for the identification of small molecule inhibitors of GTP‐loaded Rac1, a well‐characterized member of the Rho GTPase family, revealed mitoxantrone as a valid hit in vitro (Tables and ) .…”