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1.  Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe* 
The Journal of Biological Chemistry  2013;288(12):8531-8543.
Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases.
Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays.
Conclusion: The identified compound is a highly Cdc42-selective inhibitor.
Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research.
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.
PMCID: PMC3605667  PMID: 23382385
Cdc42; Cytoskeleton; GTPase; Integrin; Migration
2.  Receptor protein tyrosine phosphatases are novel components of a polycystin complex 
Biochimica et biophysica acta  2010;1812(10):1225-1238.
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, but the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling.
PMCID: PMC3156852  PMID: 21126580
polycystins; tyrosine kinase; tyrosine phosphatase; adherens junctions; primary cilium; G-protein coupled signaling
3.  Adult Human CD133/1+ Kidney Cells Isolated from Papilla Integrate into Developing Kidney Tubules 
Biochimica et biophysica acta  2011;1812(10):1344-1357.
Approximately 60,000 patients in the US are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin+ and CD133/1+ cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1+ cells. Isolated CD133/1+ papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1+ progenitors from the papilla and cortex, became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules.
PMCID: PMC3166446  PMID: 21255643
kidney disease; ADPKD; regenerative medicine; renopoietic; mesenchymal stem cell; Tamm-Horsfall/uromodulin; metanephric organ culture; xanthosine
4.  Identification of a Small GTPase Inhibitor using a High-Throughput Flow Cytometry Bead-Based Multiplex Assay 
Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small molecule inhibitors. We describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different GST-tagged small GTPases were bound to glutathione beads each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This novel multiplex assay allowed us to screen a library of almost 200,000 compounds and identify over 1,200 positive compounds, which were further verified by dose response analyses, using 6 to 8-plex assays. After the elimination of false positive and negative compounds, several small molecule families with opposing effects on GTP-binding activity were identified. Here we detail the characterization of MLS000532223, a general inhibitor that prevents GTP-binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high throughput screening (HTS) via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.
PMCID: PMC3433230  PMID: 20008126
Ras; Rab and Rho GTPases; actin cytoskeleton; bead-based multiplex assay; flow cytometry; fluorescent GTP binding
5.  Rab7 Mutants Associated with Charcot-Marie-Tooth Disease Exhibit Enhanced NGF-Stimulated Signaling 
PLoS ONE  2010;5(12):e15351.
Missense mutants in the late endosomal Rab7 GTPase cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth disease type 2B (CMT2B). As yet, the pathological mechanisms connecting mutant Rab7 protein expression to altered neuronal function are undefined. Here, we analyze the effects Rab7 CMT2B mutants on nerve growth factor (NGF) dependent intracellular signaling in PC12 cells. The nerve growth factor receptor TrkA interacted similarly with Rab7 wild-type and CMT2B mutant proteins, but the mutant proteins significantly enhanced TrkA phosphorylation in response to brief NGF stimulation. Two downstream signaling pathways (Erk1/2 and Akt) that are directly activated in response to phospho-TrkA were differentially affected. Akt signaling, arising in response to activated TrkA at the plasma membrane was unaffected. However Erk1/2 phosphorylation, triggered on signaling endosomes, was increased. Cytoplasmic phospho-Erk1/2 persisted at elevated levels relative to control samples for up to 24 h following NGF stimulation. Nuclear shuttling of phospho Erk1/2, which is required to induce MAPK phosphatase expression and down regulate signaling, was greatly reduced by the Rab7 CMT2B mutants and explains the previously reported inhibition in PC12 neurite outgrowth. In conclusion, the data demonstrate a mechanistic link between Rab7 CMT2B mutants and altered TrkA and Erk1/2 signaling from endosomes.
PMCID: PMC3000344  PMID: 21151572

Results 1-5 (5)