title: Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.
abstract: 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.
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131; University of New Mexico (UNM) Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Lin
authors: L
authors: Hong
authors: Department of Pharmaceutical Sciences, University of New Mexico, Albuquerque, New Mexico 87131.
authors: S Ray
authors: SR
authors: Kenney
authors: Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Genevieve K
authors: GK
authors: Phillips
authors: University of Kansas Specialized Chemistry Center, Lawrence, Kansas 66047.
authors: Denise
authors: D
authors: Simpson
authors: University of Kansas Specialized Chemistry Center, Lawrence, Kansas 66047.
authors: Chad E
authors: CE
authors: Schroeder
authors: University of Kansas Specialized Chemistry Center, Lawrence, Kansas 66047.
authors: Julica
authors: J
authors: Nöth
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Elsa
authors: E
authors: Romero
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Scarlett
authors: S
authors: Swanson
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131; University of New Mexico (UNM) Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Anna
authors: A
authors: Waller
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131; University of New Mexico (UNM) Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico 87131.
authors: J Jacob
authors: JJ
authors: Strouse
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131; University of New Mexico (UNM) Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Mark
authors: M
authors: Carter
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Alexandre
authors: A
authors: Chigaev
authors: University of New Mexico (UNM) Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico 87131; Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico 87131; Department of Biochemistry and Molecular Biology, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Oleg
authors: O
authors: Ursu
authors: University of New Mexico (UNM) Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico 87131; Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico 87131; Department of Biochemistry and Molecular Biology, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Tudor
authors: T
authors: Oprea
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Brian
authors: B
authors: Hjelle
authors: University of Kansas Specialized Chemistry Center, Lawrence, Kansas 66047.
authors: Jennifer E
authors: JE
authors: Golden
authors: University of Kansas Specialized Chemistry Center, Lawrence, Kansas 66047; Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045.
authors: Jeffrey
authors: J
authors: Aubé
authors: Department of Pharmaceutical Sciences, University of New Mexico, Albuquerque, New Mexico 87131; Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Laurie G
authors: LG
authors: Hudson
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131.
authors: Tione
authors: T
authors: Buranda
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131; University of New Mexico (UNM) Center for Molecular Discovery, University of New Mexico, Albuquerque, New Mexico 87131; Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico 87131. Electronic address: lsklar@salud.unm.edu.
authors: Larry A
authors: LA
authors: Sklar
authors: Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131; Department of Pharmaceutical Sciences, University of New Mexico, Albuquerque, New Mexico 87131. Electronic address: wness@unm.edu.
authors: Angela
authors: A
authors: Wandinger-Ness
mesh_terms: D016475:3T3 Cells; D000494:Allosteric Regulation; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D039041:Integrin alpha4beta1 / Q000037:antagonists & inhibitors / Q000502:physiology; D051379:Mice; D015335:Molecular Probes / Q000494:pharmacology*; D009842:Oligopeptides / Q000378:metabolism; D010671:Phenylurea Compounds / Q000378:metabolism; D011485:Protein Binding; D011554:Pseudopodia / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology*; D029261:Sin Nombre virus / Q000502:physiology; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology*; D053586:Virus Internalization / Q000187:drug effects; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000096926:Benzenesulfonamides
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D039041:Integrin alpha4beta1; D015335:Molecular Probes; D009842:Oligopeptides; D010671:Phenylurea Compounds; D011720:Pyrazoles; D013449:Sulfonamides; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; C517048:4-((N'-2-methylphenyl)ureido)phenylalanyl-leucyl-alpha-aspartyl-valyl-prolyl-alanyl-alanyl-lysine; C581055:CID2950007; D000096926:Benzenesulfonamides; C581054:CID44216842; C107953:RAC1 protein, human; C066737:RHOA protein, human
references: J Biol Chem. 1996 Jun 7;271(23):13556-60
references: 8662891
references: Biochem J. 2000 Jun 1;348 Pt 2:241-55
references: 10816416
references: Nat Chem Biol. 2006 Jan;2(1):39-46
references: 16408091
references: Cancer Lett. 2003 Apr 10;193(1):99-107
references: 12691829
references: FEBS Lett. 2011 Jan 21;585(2):286-90
references: 21167153
references: J Biol Chem. 2001 Dec 28;276(52):48670-8
references: 11641394
references: Anal Biochem. 2008 Oct 15;381(2):258-66
references: 18638444
references: Oncogene. 2010 Jun 24;29(25):3715-22
references: 20418914
references: Microbiol Mol Biol Rev. 1999 Mar;63(1):54-105
references: 10066831
references: J Virol. 2003 Jan;77(2):1540-50
references: 12502867
references: Int J Oncol. 2012 May;40(5):1561-8
references: 22266952
references: J Biol Chem. 2005 Mar 25;280(12):11961-72
references: 15655247
references: Cell Signal. 2011 Sep;23(9):1415-23
references: 21515363
references: Curr Drug Targets. 2011 Jul 1;12(8):1202-15
references: 21561415
references: Nat Cell Biol. 2009 May;11(5):510-20
references: 19404330
references: J Cell Biol. 1983 Mar;96(3):857-65
references: 6682113
references: Traffic. 2010 Oct;11(10):1272-9
references: 20633244
references: Nature. 2006 May 25;441(7092):424-30
references: 16724053
references: Hepatology. 2003 Dec;38(6):1458-67
references: 14647057
references: Nature. 2002 Dec 12;420(6916):629-35
references: 12478284
references: Blood. 2011 Nov 3;118(18):4841-52
references: 21562048
references: Proc Natl Acad Sci U S A. 2004 May 18;101(20):7618-23
references: 15128949
references: Methods Biochem Anal. 2005;46:1-265
references: 16350889
references: J Biol Chem. 2007 Jul 20;282(29):21497-506
references: 17537723
references: Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2800-5
references: 14973186
references: Mol Biol Cell. 2005 Jul;16(7):3223-35
references: 15872091
references: Mol Biol Cell. 2006 Nov;17(11):4675-85
references: 16914516
references: Small GTPases. 2011 Nov 1;2(6):310-313
references: 22545228
references: Methods Mol Biol. 2012;757:3-14
references: 21909902
references: PLoS One. 2010 Sep 24;5(9):e13006
references: 20886051
references: Mol Cell Biol. 1995 Apr;15(4):1942-52
references: 7891688
references: Clin Cancer Res. 2004 Jul 15;10(14):4799-805
references: 15269155
references: Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976-80
references: 2690082
references: J Biol Chem. 2000 Aug 18;275(33):25299-307
references: 10843989
references: Cell. 1995 Apr 7;81(1):53-62
references: 7536630
references: J Biol Chem. 1996 Apr 26;271(17):10149-53
references: 8626575
references: Nature. 2003 Feb 13;421(6924):753-6
references: 12610628
references: J Immunol. 2007 Aug 1;179(3):1796-802
references: 17641046
references: Oncogene. 2011 May 26;30(21):2420-32
references: 21258406
references: Science. 1993 Jun 25;260(5116):1934-7
references: 8316833
references: BMC Cell Biol. 2003 Feb 24;4:2
references: 12600279
references: Methods Mol Biol. 2012;827:253-70
references: 22144280
references: J Virol. 2005 Jan;79(2):1191-206
references: 15613346
references: Science. 1998 Jan 23;279(5350):509-14
references: 9438836
references: Biochemistry. 1998 Apr 14;37(15):5296-304
references: 9548761
references: Proc Natl Acad Sci U S A. 2009 May 19;106(20):8221-6
references: 19416857
references: J Immunol. 2006 May 1;176(9):5418-25
references: 16622009
references: J Biol Chem. 2011 Jan 28;286(4):2375-81
references: 21115489
references: Anal Sci. 2001 Jan;17(1):155-60
references: 11993654
references: Oncogene. 2010 Apr 22;29(16):2427-40
references: 20118984
references: Cell. 1996 Nov 1;87(3):519-29
references: 8898204
references: Oncogene. 2010 May 13;29(19):2760-71
references: 20228844
references: J Cell Biol. 2003 Apr 28;161(2):417-27
references: 12707305
references: Immunity. 2004 Jan;20(1):25-35
references: 14738762
references: Exp Cell Res. 1995 Jun;218(2):499-507
references: 7796885
references: Br J Dermatol. 2007 Dec;157(6):1212-6
references: 17970806
references: Leuk Res. 1985;9(1):157-65
references: 3157837
references: Cell. 2007 Jun 1;129(5):865-77
references: 17540168
references: J Virol. 2009 Oct;83(19):9759-72
references: 19640983
references: Front Biosci (Landmark Ed). 2011 Jun 01;16:2561-71
references: 21622195
references: Nat Cell Biol. 2000 Oct;2(10):E191-6
references: 11025683
references: J Biol Chem. 2001 Nov 2;276(44):40606-13
references: 11514549
references: Anal Biochem. 2001 Apr 1;291(1):109-17
references: 11262163
references: Trends Cell Biol. 2006 Oct;16(10):522-9
references: 16949823
references: Semin Cell Dev Biol. 2011 Feb;22(1):57-68
references: 21147240
references: Biochem Soc Trans. 2011 Dec;39(6):1606-11
references: 22103495
references: Genome Biol. 2002;3(2):REVIEWS0002
references: 11864373
references: Science. 1996 Feb 16;271(5251):981-3
references: 8584934
references: J Biomol Screen. 2010 Jan;15(1):10-20
references: 20008126