Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein–protein complexes

doi:10.1073/pnas.0700773104

Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein–protein complexes

*Institut National de la Santé et de la Recherche Médicale, U554 and
^†Université Montpellier 1 et 2, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5048, Centre de Biochimie Structurale, 34090 Montpellier, France;
^‡Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France; and
^§Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique–Unité Mixte de Recherche 6097, 660 Route des Lucioles, 06560 Valbonne, France

Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved May 7, 2007 (received for review February 1, 2007)

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.

Footnotes

^¶To whom correspondence should be addressed. E-mail: cherfils{at}lebs.cnrs-gif.fr
Author contributions: J.V. and M.Z. contributed equally to this work; J.V., M.Z., H.B., J.-C.Z., A.P., C.A.R., J.C., and A.C. designed research; J.V., M.Z., H.B., J.-C.Z., A.P., C.A.R., and A.C. performed research; B.G. contributed new reagents/analytic tools; J.V., M.Z., H.B., J.-C.Z., A.P., P.C., C.A.R., J.C., and A.C. analyzed data; and J.C. and A.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700773104/DC1.
Abbreviations:

SMG,

small GTP-binding protein;

GEF,

guanine nucleotide exchange factor;

BFA,

Brefeldin A;

mGTP,

methylanthraniloyl GTP;

MDCK,

Madin–Darby canine kidney.