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.protein-protein interactions ͉ inhibition ͉ GTPase ͉ guanine nucleotide exchage factor ͉ Arf1 factor

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

“…Kinetics Measurements of Nucleotide Exchange-All kinetics experiments were performed with a soluble Arf1 protein truncated N-terminal helix, which is not dependent on lipids for nucleotide exchange, and loaded with GDP before the experiments as previously described (35) To study the effect of His-AviTags on Arno/4M to equilibrium binding constant Arf1 binding, anisotropy binding profiles were obtained from the titration of Alexa488-labeled [⌬17]Arf1-GDP with Arno/4M bearing or not the His-AviTags (35).…”

“…Biotinylated [⌬17]Arf1 protein was expressed using a competent strain of E. coli BL21(DE3) transformed with a BirA pCDFDuet expression vector and purified as previously described (35). All polyhistidine tag-biotinylated Sec7 constructs were expressed and purified as followed.…”

“…[⌬17]Arf1 protein in its GDP form as well as Arno and Arno4M proteins were expressed and purified as previously described (32,35) except that for Sec7 domains used in nucleotide exchange tests the polyhistidine tag was cleaved using thrombin. Biotinylated [⌬17]Arf1 protein was expressed using a competent strain of E. coli BL21(DE3) transformed with a BirA pCDFDuet expression vector and purified as previously described (35).…”

Overexpression of ARF1 is associated with cell proliferation and migration through PI3K signal pathway in ovarian cancer

“…A role for ARF1 in the control of integrin-mediated cell adhesion was indicated by studies on Brefeldin A (BFA)-resistant Arf GEFs and it was shown that ARF1 mediated paxillin recruitment to the focal adhesions and potentiated rho-stimulated stress fiber formation in fibroblasts (15). furthermore, inhibition of Arf1 activation by targeting ARNO impaired ARNO-dependent migration of MDCK cells (16). In invasive breast cancer cells, stimulation of the epidermal growth factor receptor led to the activation of ARF1, the PI3K pathway, and ultimately cell migration and proliferation (17).…”

Les protéines ARF

“…Nous avons d'abord dûment noté que plus du tiers des sujets parkinsoniens étaient affligés d'un TCSP [9]. Nous avons ensuite observé que seuls les sujets parkinsoniens victimes d'un TCSP présentaient un ralentissement de l'activité EEG à l'éveil et une atteinte des fonctions cognitives détectée lors des tests neuropsychologiques [10,11]. Les sujets parkinsoniens non affectés de TCSP ne présentaient ni anomalies de l'EEG, ni atteintes des fonctions cognitives.…”