title: Small-molecule inhibitors targeting G-protein-coupled Rho guanine nucleotide exchange factors.
abstract: The G-protein-mediated Rho guanine nucleotide exchange factor (GEF)-Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein-regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM K(d) and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein-coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG-RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme-substrate pair of GEF-RhoA at their binding interface that leads to enhanced efficacy and specificity.
authors: Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, Cincinnati, OH 45229, USA.
authors: Xun
authors: X
authors: Shang
authors: Fillipo
authors: F
authors: Marchioni
authors: Chris R
authors: CR
authors: Evelyn
authors: Nisha
authors: N
authors: Sipes
authors: Xuan
authors: X
authors: Zhou
authors: William
authors: W
authors: Seibel
authors: Matthew
authors: M
authors: Wortman
authors: Yi
authors: Y
authors: Zheng
mesh_terms: D000595:Amino Acid Sequence; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors*; D006801:Humans; D009361:Neoplasm Invasiveness / Q000517:prevention & control; D017386:Sequence Homology, Amino Acid
chemical_list: D020662:Guanine Nucleotide Exchange Factors
references: Oncogene. 2001 Mar 26;20(13):1661-8
references: 11313914
references: Nat Rev Drug Discov. 2011 Jan;10(1):47-60
references: 21193867
references: Nat Rev Cancer. 2010 Dec;10(12):842-57
references: 21102635
references: J Biol Chem. 2004 Nov 5;279(45):47352-62
references: 15331592
references: Proteins. 2004 Dec 1;57(4):678-83
references: 15390263
references: Drug Discov Today. 2005 Dec;10(23-24):1607-10
references: 16376820
references: J Biol Chem. 1994 Jan 7;269(1):62-5
references: 8276860
references: Nat Rev Mol Cell Biol. 2005 Feb;6(2):167-80
references: 15688002
references: Curr Opin Cell Biol. 1996 Apr;8(2):216-22
references: 8791419
references: Nat Rev Drug Discov. 2002 Sep;1(9):727-30
references: 12209152
references: FEBS Lett. 2000 Nov 24;485(2-3):183-8
references: 11094164
references: Clin Cancer Res. 2007 Dec 15;13(24):7264-70
references: 18094406
references: Chem Biol. 2012 Jun 22;19(6):699-710
references: 22726684
references: J Chem Inf Model. 2012 Jul 23;52(7):1757-68
references: 22587354
references: Proc Natl Acad Sci U S A. 2004 May 18;101(20):7618-23
references: 15128949
references: Nature. 2002 Dec 12;420(6916):629-35
references: 12478284
references: Oncogene. 1998 Sep 17;17(11 Reviews):1415-38
references: 9779988
references: Trends Biochem Sci. 2001 Dec;26(12):724-32
references: 11738596
references: Oncogene. 2004 Jan 8;23(1):233-40
references: 14712228
references: Trends Cell Biol. 2001 Dec;11(12):471-7
references: 11719051
references: Annu Rev Cell Dev Biol. 2005;21:247-69
references: 16212495
references: Nature. 1991 Nov 28;354(6351):311-4
references: 1956381
references: J Biol Chem. 1999 Feb 26;274(9):5868-79
references: 10026210
references: Science. 1998 Jun 26;280(5372):2112-4
references: 9641916
references: Genes Dev. 2003 May 15;17(10):1253-70
references: 12756227
references: Ernst Schering Res Found Workshop. 2003;(42):11-7
references: 12664532
references: Nat Commun. 2010 Oct 19;1:100
references: 20981028