title: Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes.
abstract: Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U554, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5048, Centre de Biochimie Structurale, 34090 Montpellier, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Viaud
authors.forename: Mahel
authors.initials: M
authors.lastname: Zeghouf
authors.forename: Hélène
authors.initials: H
authors.lastname: Barelli
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Zeeh
authors.forename: André
authors.initials: A
authors.lastname: Padilla
authors.forename: Bernard
authors.initials: B
authors.lastname: Guibert
authors.forename: Pierre
authors.initials: P
authors.lastname: Chardin
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Royer
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cherfils
authors.forename: Alain
authors.initials: A
authors.lastname: Chavanieu
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D003198:Computer Simulation; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000378:metabolism; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D000814:Aniline Compounds; D001562:Benzimidazoles; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C521938:LM11 compound; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D034741:RNA, Small Interfering; C066755:Sec7 guanine nucleotide exchange factors; C102474:cytohesin-2; D006153:Guanosine Diphosphate; D020126:Brefeldin A; D020823:ADP-Ribosylation Factor 1