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

Lin, Hong; Kenney, S. Ray; Phillips, Genevieve K; Simpson, Denise S.; Schroeder, Chad E.; Nöth, Julica; Romero, Elsa; Swanson, Scarlett; Waller, Anna; Strouse, J. Jacob; Carter, Mark; Chigaev, Alexandre; Ursu, Oleg; Oprea, Tudor I.; Hjelle, Brian; Golden, Jennifer E.; Aubé, Jeffrey; Hudson, Laurie G.; Buranda, Tione; Sklar, Larry A.; Wandinger‐Ness, Angela

doi:10.1074/jbc.m112.435941

Cited by 144 publications

(131 citation statements)

References 70 publications

(77 reference statements)

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“…1 and 2). To exert finer temporal control over Cdc42 function, we employed the Cdc42 inhibitor, ML141, which specifically inhibits Cdc42 nucleotide dissociation and activation (Hong et al, 2013), and reduced active Cdc42 levels in sea urchin embryos as measured with a Cdc42 pull-down assay (Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

Sepúlveda-Ramírez

Toledo-Jacobo

Henson

et al. 2018

Developmental Biology

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In the sea urchin embryo, gastrulation is characterized by the ingression and directed cell migration of primary mesenchyme cells (PMCs), as well as the primary invagination and convergent extension of the endomesoderm. Like all cell shape changes, individual and collective cell motility is orchestrated by Rho family GTPases and their modulation of the actomyosin cytoskeleton. And while endomesoderm specification has been intensively studied in echinoids, much less is known about the proximate regulators driving cell motility. Toward these ends, we employed anti-sense morpholinos, mutant alleles and pharmacological inhibitors to assess the role of Cdc42 during sea urchin gastrulation. While inhibition of Cdc42 expression or activity had only mild effects on PMC ingression, PMC migration, alignment and skeletogenesis were disrupted in the absence of Cdc42, as well as elongation of the archenteron. PMC migration and patterning of the larval skeleton relies on the extension of filopodia, and Cdc42 was required for filopodia in vivo as well as in cultured PMCs. Lastly, filopodial extension required both Arp2/3 and formin actin-nucleating factors, supporting models of filopodial nucleation observed in other systems. Together, these results suggest that Cdc42 plays essential roles during PMC cell motility and organogenesis.

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Section: Resultsmentioning

confidence: 99%

Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

Sepúlveda-Ramírez

Toledo-Jacobo

Henson

et al. 2018

Developmental Biology

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“…[49] This compound impairs nucleotide binding by several small GTPases with low nanomolar inhibitory constants (K i = 12.9-19.7 nm) in vitro.I na ni dentical setup,C ID29950007 (ML141, 2)w as identified as aC dc42-selective noncompetitive allosteric inhibitor (Figure 3a). [50] In this case,t he binding of 2 locks Cdc42 in an inactive conformation, thereby inducing nucleotide release and inhibition of Cdc42-mediated cellular functions such as filopodia formation or cell migration (Figure 3b). Furthermore, 2 proved useful for combination with colony-stimulation factor (G-CSF) in the mobilization of hematopoietic stem and progenitor cells.…”

Section: Interference With Nucleotide Bindingmentioning

confidence: 98%

“…However, the experiment involves an ucleotideremoving gel-filtration step,s oi td oes not clearly show whether the inhibitor can compete with cellular nucleotide concentrations. [71] To achieve cell penetration, SML-10-70-1(5), acaged phosphoramidate derivate of 4,was synthesized, which impairs Akt and ERK phosphorylation in aH 358 cell line at ac oncentration of 100 mm and proliferation in KRas G12C mutant H358 and H23 cells with ah alf-maximum effective concentration (EC 50 )o f2 7mm and 48 mm,r espectively.I nterestingly, 5 also shows antiproliferative effects in A549 cells,w hich contain aG 12S mutation, thus indicating additional nonselective modes of action. [59] By using at ethering approach, another set of covalent inhibitors was identified that make use of the G12C mutation as ac hemically targetable anchor.…”

Section: Mutant-selective Targeting Through Small Moleculesmentioning

confidence: 99%

“…However,s everal high-throughput screens (HTSs) identified compounds interfering with GTPase nucleotide binding in vitro. [49][50][51] By using ab ead-based flow cytometry assay [52] to test 300 000 compounds,CID1067700 (1)was identified as adirect competitor for nucleotide binding (Figure 3a). [49] This compound impairs nucleotide binding by several small GTPases with low nanomolar inhibitory constants (K i = 12.9-19.7 nm) in vitro.I na ni dentical setup,C ID29950007 (ML141, 2)w as identified as aC dc42-selective noncompetitive allosteric inhibitor (Figure 3a).…”

Section: Interference With Nucleotide Bindingmentioning

confidence: 99%

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Direct Modulation of Small GTPase Activity and Function

et al. 2015

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Small GTPases are a family of GDP-/GTP-binding proteins that serve as biomolecular switches inside cells to control a variety of essential cellular processes. Aberrant function and regulation of small GTPases is associated with a variety of human diseases, thus rendering these proteins highly interesting targets in drug discovery. However, this class of proteins has been considered "undruggable", as intensive decade-long efforts did not yield clinically relevant direct modulators of small GTPases. Recently, the targeting of small GTPases has gained fresh impetus through the discovery of novel transient cavities on the protein surfaces and the application of new targeting strategies. Besides Ras proteins, other small GTPases have attracted increased attention since improved biological insight in combination with novel targeting strategies identified them as promising targets in drug discovery. This Review gives an overview of relevant aspects of the superfamily of small GTPases and summarizes recent progress and perspectives for the direct modulation of these challenging targets.

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“…Y-27632 specifically inhibits the RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK), one of the downstream RhoA effectors (42,43). ML141 is a selective and allosteric inhibitor for Cdc42 (44), and AZA1 inhibits the activation of Cdc42 and Rac1 (45). NSC23766 inhibits the Rac1-specific GEF but has no effect on RhoA and Cdc42 activity (46,47).…”

Section: Resultsmentioning

confidence: 99%

Graf1 Controls the Growth of Human Parainfluenza Virus Type 2 through Inactivation of RhoA Signaling

Ohta

Goto

Matsumoto

et al. 2016

J Virol

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Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes activation of RhoA, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 colocalized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited, and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1 and that Graf1 inhibits hPIV-2 growth through RhoA inactivation. IMPORTANCE Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causesRhoA activation, which is suppressed by Graf1. Graf1 colocalizes with viral RNP (vRNP) in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited hPIV-2 growth through RhoA inactivation. Rho GTPases are members of the Ras superfamily of 20-to 30-kDa small GTPases. They are highly conserved in eukaryotes and act as molecular switches to regulate essential cellular functions. To date, at least 22 members of the Rho GTPases have been identified in mammalian cells (1, 2). The most well characterized members, namely, RhoA, Cdc42, and Rac1, affect a variety of cellular activities, including actin reorganization, apoptosis, intracellular trafficking, and cell polarity (1-5). Rho GTPases regulate cellular activities by coordinating with other host proteins such as focal adhesion kinase (FAK) and Akt. It is important for viruses to establish an environment that facilitates their growth by controlling these cellular activities. Rho GTPases and their related proteins affect the life cycles of some viruses, including respiratory syncytial virus (RSV) (6, 7), Ebola virus (8), vesicular stomatitis virus (8), Epstein-Barr virus (9), influenza A virus (IAV) (10, 11), and rotavirus (12). The relationship between herpesvirus and Rho GTPases has been well investigated (13). We previously reported that Rho activation promotes syncytium formation induced by human parainfluenza virus type 2 (hPIV-2) (14). However, it remains unknown whe...

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

Cited by 144 publications

References 70 publications

Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

Direct Modulation of Small GTPase Activity and Function

Graf1 Controls the Growth of Human Parainfluenza Virus Type 2 through Inactivation of RhoA Signaling

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