Article

pubs.acs.org/biochemistry

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide

Exchange Factors with Small Molecules: Evidence of Unique
Inhibitory Proles
Sarah Benabdi, Francois Peurois, Agata Nawrotek, Jahnavi Chikireddy, Tatiana Caneque,,,
Takao Yamori,,@ Isamu Shiina,# Yoshimi Ohashi, Shingo Dan, Raphael Rodriguez,,,
Jacqueline Cherls,*, and Mahel Zeghouf*,

Laboratoire de Biologie et Pharmacologie Appliquee CNRS, Ecole Normale Superieure Paris-Saclay, 61 avenue du president Wilson,
94235 Cachan, France

Institut Curie, PSL Research University, Chemical Cell Biology group, 26 rue dUlm, 75248 Paris Cedex 05, France

CNRS UMR3666, 75005 Paris, France

INSERM U1143, 75005 Paris, France

Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550,
Japan
#
Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo 162-8601, Japan
*
S Supporting Information

ABSTRACT: Arf GTPases and their guanine nucleotide

exchange factors (ArfGEFs) are major regulators of membrane
trac and organelle structure in cells. They are associated with
a variety of diseases and are thus attractive therapeutic targets
for inhibition by small molecules. Several inhibitors of unrelated
chemical structures have been discovered, which have shown
their potential in dissecting molecular pathways and blocking
disease-related functions. However, their specicity across the
ArfGEF family has remained elusive. Importantly, inhibitory
responses in the context of membranes, which are critical
determinants of Arf and ArfGEF cellular functions, have not
been investigated. Here, we compare the eciency and
specicity of four structurally distinct ArfGEF inhibitors,
Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella
and Rickettsia RalF). Inhibition was assessed by uorescence kinetics using pure proteins, and its modulation by membranes was
determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family
resemblance, each inhibitor has a specic inhibitory prole. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729
inhibits all GEFs, the strongest eects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding
domain in Legionella RalF reveals a strong inhibitory eect of BFA that is not measured on its GEF domain alone. This study
demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf
pathways by these inhibitors to best guide their use and development as therapeutic agents.

S mall GTPases regulate many aspects of cell logistics,

including signaling, dynamics of the acting cytoskeleton,
and membrane trac.1 Common to all small GTPases is their
ability to function as molecular switches, by alternating between
inactive GDP-bound and active GTP-bound forms.2 GDP/
GTP alternation is nely regulated by families of guanine
nucleotide exchange factors (GEFs), which stimulate the
dissociation of GDP, and families of GTPase-activating proteins
(GAPs), which stimulate GTP hydrolysis.3 A direct conse-
quence of the roles of small GTPases and their regulators in the
regulation of pivotal molecular nodes in the cell is that they
have been linked with a plethora of human diseases, including
cancers4 and infections.5 Extensive eorts are therefore being
made to discover and develop chemical inhibitors. However,
this has remained a challenge, notably because the functions of
small GTPases are mostly based on coordinated protein
protein and proteinmembrane interactions and their com-
plexes exhibit highly dynamic structures lacking well-dened
pockets, all of which are considered poorly druggable features.6
In addition, individual GTPases are often involved in
Received: July 23, 2017
Revised: August 22, 2017
Published: August 31, 2017

2017 American Chemical Society 5125 DOI: 10.1021/acs.biochem.7b00706

Biochemistry 2017, 56, 51255133
Biochemistry Article

Figure 1. Small molecules and proteins used in this study. (A) Chemical structures of the compounds. (B) Small GTPases and GEF constructs used
for the in vitro nucleotide exchange assay in solution (left) and in the presence of a membrane (right). Numbers refer to the rst and last residues of
each construct. (C) SDSPAGE analysis of the recombinant proteins. The associated Figure S2 shows control inhibition assays performed with the
related small GTPase Rac1 and its GEF Dock5 and DLS of liposome integrity in the presence of the inhibitors.

concurrent processes through the combination of related
regulators,7 whereby even on-target inhibition can lead to
detrimental o-pathway eects.
Arf GTPases constitute a subfamily of small GTPases with
regulatory functions in most major aspects of membrane trac
and organelle structure in the cell.8,9 Their roles in diseases
have expanded over the past decade, notably in cancer,1012
inammation,13 and infections.1416 In metazoans, they are
activated by 15 dierent GEFs (ArfGEF hereafter) that bear a
conserved catalytic Sec7 domain that stimulates GDP/GTP
exchange.17 The Sec7 domain is decorated with various other
domains, the nature of which denes ArfGEF subfamilies.18 An
additional ArfGEF, RalF, is secreted by the bacterial pathogens
5126
Legionella pneumophila and Rickettsia prowazekii to activate Arf
GTPases in infected cells.14 In addition to GEFs, membranes
play a pivotal role in the activation of Arf GTPases. These
GTPases depart from canonical small GTPases by a built-in
structural mechanism whereby they couple their activation by
GTP to their recruitment to membranes.19 This mechanism
uses a myristoylated N-terminal helix, which constitutes the
major site of interaction of Arf GTPases with membranes, and
it is controlled by ArfGEFs to ensure that Arf-GTP is associated
with membranes.20,21 ArfGEFs are themselves regulated by
membranes through membrane-binding domains: members of
the cytohesin, EFA6, and BRAG subfamilies carry a
phospholipid-binding pleckstrin homology (PH) domain that
DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133
Biochemistry
follows their Sec7 domain; large Golgi ArfGEFs have
membrane-binding elements in the N- and C-termini of their
Sec7 domain; and RalF proteins have a membrane-binding
domain uniquely found in bacteria that is also involved in
autoinhibition.22
The rst discovered GEF inhibitor is Brefeldin A (BFA), a
natural compound that inhibits Arf functions at the Golgi.23,24
The biochemical and structural mechanism of BFA has been
deciphered, leading to the rst direct demonstration that small
GTPases and their GEFs are druggable targets despite their
complex structural landscape.21,25,26 BFA binds at the interface
between Arf and its GEFs, thereby stalling the Arf-GDP/
ArfGEF intermediate that initiates the nucleotide exchange
reaction in an abortive conformation. This mode of action
allows it to target only the large Golgi ArfGEFs by recognizing
a tyrosine in the Arf-binding site, which is substituted by a
phenylalanine in BFA-insensitive ArfGEFs, and to be inactive
toward the Arf6 isoform.27 Remarkably, various chemicals of
unrelated structure have since been discovered to inhibit
ArfGEFs, using a variety of screening strategies. The interfacial
mechanism of BFA inspired an in silico screen that produced
LM11, a chemical compound that inhibits Arf1 and cytohesins
in vitro and in cells.28 Another inhibitor of cytohesins, SecinH3,
was discovered using an RNA aptamer displacement in vitro
screen.29 A uorometric GEF assay-based screen identied
NAV-2729, which blocks spontaneous activation of Arf6 and its
activation by cytohesins and BRAG.12 Other inhibitors have
been discovered by phenotypic screens. Golgicide A was
identied from a high-throughput luciferase-based screen for
small molecules that inhibit intracellular toxin transport in host
cells; it causes Golgi and TGN dispersal by targeting GBF1, an
ArfGEF that functions at the Golgi.30 Another inhibitor of
GBF1 is LG186;31 LG186 is a derivative of Exo2, which was
originally identied through an image-based phenotypic screen
for perturbation of exocytosis.32 M-COPA (also called AMF-
26) was identied on the basis of the similarity of its growth
inhibitory prole toward human cancer cell lines with that of
BFA; it dispersed GBF1 in a manner reminiscent of that of BFA
and was proposed to inhibit GBF1 specically.33
ArfGEF inhibitors have already a long history in the
elucidation of molecular pathways in the cell.34 For instance,
BFA was instrumental in deciphering the molecular basis of
membrane dynamics at the Golgi.35 Golgicide A identied a
pivotal role for GBF1 in maintaining bidirectional transport in
the cell.30 SecinH3 revealed the role of cytohesins and Arfs in
the response to insulin.29,36 LM11 pointed to a role of Arf1 in
breast cancer cell invasion through formation of focal
adhesions.37 ArfGEF inhibitors also contributed to identifying
potential therapeutic pathways that could be manipulated to
combat diseases. For example, mouse models of inammation
exposed to SecinH3 had reduced vascular permeability, a
process leading to inammation, which suggested that
cytohesins and the Arf6 GTPase are valid targets for the
treatment of inammatory conditions.13 Likewise, several
ArfGEF inhibitors were shown to have antitumoral activities.
NAV-2729 reduced the level of uveal melanoma cell
proliferation and tumorigenesis in a mouse model.12 M-
COPA led to tumor regression in mice xenografts.33,38 BFA,
Golgicide A, SecinH3, and LM11 markedly reduced the
incidence of stem cell tumors in Drosophila by blocking lipid
droplet usage.11
These studies highlighted Arf GTPases and their regulators
as attractive candidates for therapeutic intervention. Small
5127
Article
molecule inhibitors constitute important tools for developing
drugs to manipulate them in disease-relevant settings. However,
using these inhibitors for deciphering molecular mechanisms in
disease-related pathways faces several challenges. First, all
ArfGEFs share a highly conserved Sec7 catalytic domain,17
which raises the issue drug specicity. Second, inhibition
eciency in vitro has been mostly analyzed in solution, which
underestimates GEF eciencies by one to several orders of
magnitude.3942 Quantitative in vitro analysis of inhibitors
using puried proteins is an ecient approach to address these
questions.43 In this study, we used uorescence kinetics-based
assays with puried Arf GTPases and representative human and
bacterial ArfGEFs to establish the inhibitory proles of four
inhibitors of unrelated structure, BFA, SecinH3, M-COPA, and
NAV-2729 (Figure 1A). The eect of membranes was
investigated for three of these inhibitors, by incorporating
articial membranes in the kinetics assays and by using
lipidated GTPases and ArfGEF constructs that contain a
membrane-binding segment. This study reveals that each small
molecule has a specic inhibitory prole. It also uncovers an
unexpected sensitivity of LpRalF to BFA that was detected only
in the presence of membranes. This analysis should enable an
accurate interpretation of the eect of these inhibitors in
dissecting the biology of Arf GTPases and their GEFs and
facilitate their further development.
MATERIALS AND METHODS
Chemicals. GDP, GTP, and N-methylanthraniloyl GTP
(mGTP) were from Jena Bioscience. BFA was from Sigma and
SecinH3 from Tocris Bioscience. M-COPA was synthesized as
described in ref 33. NAV-2729 was synthesized as described in
Supporting Information (spectra shown in Figure S1).
Theoretical solubilities were predicted using the U.S. Environ-
mental Protection Agencys EPISuite [compiled on Chem-
spider (http://www.chemspider.com)].
Protein Expression and Purication. Protein constructs
used in this study are shown in Figure 1B. N-Terminally
truncated bovine 17Arf1 (identical to the human form) and
human 13Arf6 were expressed and puried and loaded with
GDP prior to use as described in ref 27. Myristoylated full-
length Arf1 (myrArf1 hereafter) was obtained by co-expression
with yeast N-myristoyl transferase in Escherichia coli and
puried as described in ref 44. Human BRAG2Sec7, ARNOSec7,
BIG1Sec7, and EFA6Sec7 were produced in E. coli and puried as
described in refs 39, 45, 27, and 41, respectively. Human
BRAG2Sec7PH, ARNOSec7PH, and EFA6Sec7PH were expressed in
E. coli and puried as described in refs 39, 41, and 46,
respectively. RalF proteins from L. pneumophila (LpRalF) and
R. prowazekii (RpRalF) and their isolated Sec7 domains were
produced and puried as described in refs 42 and 47. Human
Rac1 and the GEF domain of mouse Dock5 were obtained as
described in ref 48. Human BIG1DHSec7 was expressed in insect
cells and puried as described in ref 49. All proteins were highly
pure as assessed by SDSPAGE (Figure 1C). Protein
concentrations were measured using their respective absorption
coecient at 280 nm after centrifugation to remove any
precipitate.
Liposome Preparation. All lipids were purchased from
Avanti Polar Lipids. Experiments carried out with human
ArfGEFs were performed with liposomes containing 48%
phosphatidylcholine (PC), 20% phosphatidylethanolamine
(PE), 30% phosphatidylserine (PS), and 2% phosphatidylino-
sitol 4,5-diphosphate [PI(4,5)P2]. Experiments with LpRalF
DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133
Biochemistry
and RpRalF were performed with liposomes containing 40%
PC, 19% PE, 25% PS, 1% PI(4,5)P2, and 15% cholesterol. All
liposomes were prepared and extruded through 200 nm
polycarbonate lters as described previously.39 Liposomes
were stored at room temperature and used within 2 days.
Nucleotide Exchange Assays. Nucleotide exchange
kinetics were monitored by the change in tryptophan
uorescence that follows the conformational change from Arf-
GDP to Arf-GTP (excitation and emission wavelengths of 292
and 340 nm, respectively). In some experiments with BFA and
liposomes, kinetics were monitered using Forster resonance
energy transfer (FRET) between tryptophan and mGDP (292
and 440 nm, respectively), which gives equivalent kinetics.43
Fluorescence measurements were performed with a Cary
Eclipse uorimeter (Varian) at 37 C, under continuous
stirring in a total volume of 800 L. Experiments in solution
used 1 M N-terminally truncated Arf-GDP and 100 nM
ArfGEF, except for RpRalF and LpRalF that were used at 400
nM. Protein and inhibitors (or DMSO for controls) were
incubated in HKM buer [50 mM HEPES (pH 7.4), 120 mM
potassium acetate, 1 mM MgCl2, and 1 mM DTT] for 2 min at
37 C before initiation of the reaction with 100 M GTP.
Exchange assays with liposomes were performed as described
above using 0.4 M myrArf1 in the presence of 100 M
liposomes. ArfGEF concentrations were 5 nM for
BRAG2Sec7PH, 10 nM for ARNOSec7PH, EFA6Sec7PH, and LpRalF,
and 100 nM for RpRalF and BIG1DHSec7. Exchange rates (kobs)
were determined from monoexponential ts over the entire
kinetics and expressed as a percentage of control activity. This
approach was preferred to initial rate velocity analysis, which
can be misestimated if compounds absorb light or have intrinsic
uorescence. All exchange reactions were performed in at least
triplicate, and means are given the standard deviation (SD).
Dynamic Light Scattering Analysis (DLS). The distribu-
tion of liposome sizes was analyzed by DLS using a DynaPro
NanoStarTM instrument (Wyatt Technology). Sets of 10
autocorrelation curves were acquired at 37 C at the end of
each exchange assay in a disposable cuvette (Eppendorf). The
mean radius was determined with DYNAMICS (Wyatt
Technology) assuming that the size distribution is a simple
Gaussian function.
RESULTS
Strategy. Two well-established (BFA and SecinH3) and
two recently discovered (M-COPA and NAV-2729) structurally
unrelated inhibitors were selected for this study (Figure 1A).
To analyze the eciency of the inhibitors, we used highly
puried small GTPases and GEFs (Figure 1B,C) and measured
their inhibition by uorescence kinetics, which reports
accurately and quantitatively on nucleotide exchange inhib-
ition.43 We selected representative guanine nucleotide exchange
factors from all ArfGEF subfamilies, including a large Golgi
ArfGEF, BIG1, three endocytic ArfGEFs, ARNO (a cytohesin),
EFA6, and BRAG2, and two ArfGEFs from pathogenic bacteria,
L. pneumophila and R. prowazekii RalF. We used dierent Arf
and ArfGEF constructs for the solution and membrane
experiments, because membrane-binding elements are inhib-
itory in Arf and in several ArfGEFs. For experiments performed
in solution, we used the isolated Sec7 domain of the ArfGEFs
and Arf constructs lacking their myristoylated N-terminal helix
(17Arf1 and 13Arf6, respectively, hereafter), which is
autoinhibitory in solution and requires direct interaction with
membranes for inhibition release.19,22 For experiments
5128
Article
performed on membranes, we used lipidated full-length Arf1,
which carries a myristate lipid at its N-terminus. Membrane-
binding elements in ArfGEF constructs used in liposome-based
assays were the PH domains for ARNO, BRAG2, and EFA6,
the DCB-HUS domain for BIG1, and the C-terminal capping
domain for RalF proteins. The PH domain of ARNO50 and the
capping domain of RalF proteins42 are autoinhibitory, while the
PH domains of BRAG239,40 and EFA641 and the DCD-HUS
domains of BIG122 are not. The concentrations of GEFs were
selected to support eciencies at which inhibitory eects could
be accurately measured. Arf1 was used as a reference in all
experiments, except in the case of EFA6, which activates Arf1
only in the presence of membranes,41 in which case 13Arf6
was used for the solution assays. Inhibitors were used at xed
concentrations at which signicant inhibition of at least one
ArfGEF could be measured. All inhibitors were assayed for
nonspecic eects in two control experiments. First, eects
outside the Arf/ArfGEF targets were assayed on the small
GTPase Rac1 and its specic GEF Dock5 as described in ref 48.
None exhibited a signicant eect on the Dock5-mediated Rac1
activation (Figure S2A). Second, nonspecic eects on
liposomes were monitored by using DLS, which indicated
that none of the compounds induced aggregation or disruption
of liposomes (Figure S2B).
Inhibition of Human and Bacterial ArfGEFs by BFA in
Solution and on Membranes. The natural fungal toxin BFA
is the best described ArfGEF inhibitor to date. BFA inhibits
ArfGEFs by an uncompetitive mechanism,25 in which it
stabilizes the Arf-GDP/ArfGEF complex in an abortive
conformation that cannot proceed to nucleotide dissocia-
tion.21,26 Its eciency toward eukaryotic ArfGEFs has been
quantied in solution assays, showing that it inhibits specically
large Golgi ArfGEFs such as BIG1, while PH domain-
containing ArfGEFs are resistant (for example, see ref 27).
We extended this analysis to the Sec7 domains of Legionella and
Rickettsia RalF (Figure 2A and Figure S3A). We observed that
Rickettsia RalF is inhibited by BFA, whereas Legionella RalF is
resistant, which is consistent with the presence of a Tyr residue
in the active site of the former and a Phe residue in the latter.
Next, we determined the eciency of BFA toward human and
bacterial ArfGEFs in the presence of liposomes (Figure 2B and
Figure S3B). BIG1DHSec7 and full-length Rickettsia RalF were
strongly inhibited, consistent with the sensitivities of their Sec7
domains in solution, while BRAG2Sec7PH and ARNOSec7PH,
whose Sec7 domains are resistant in solution, were only slightly
inhibited. Activation of myrArf1 by EFA6Sec7PH was also
insensitive to BFA, extending previous observations that it
does not inhibit the activation of myrARF6 by EFA6.51
Surprisingly, full-length LpRalF was strongly inhibited in this
assay, at odds with the resistance of its Sec7 domain. This was
not an indirect eect due to autoinhibition because ARNO,
which is autoinhibited by its PH domain, remained resistant to
BFA on membranes. Thus, the presence of the membrane-
binding domain in LpRalF creates a sensitivity that is not
encoded by its Sec7 domain alone. Together, this analysis
shows that BFA is a strong inhibitor of Golgi ArfGEFs and
Rickettsia RalF in solution and on membranes and uncovers an
overlooked inhibition of Legionella RalF that could not be
detected with the Sec7 domain alone in solution.
Inhibition of Human and Bacterial ArfGEFs by
SecinH3 in Solution and on Membranes. SecinH3 inhibits
cytohesins in vitro29 and has been widely used as a proxy for
the involvement of cytohesins in cellular processes.13,36,52
DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133
Biochemistry
Figure 2. Analysis of the inhibitory eciency of BFA (50 M) on
human and bacterial GEFs. (A) Sensitivity of bacterial RalF proteins to
BFA in solution. kobs rates are expressed as a percentage of the rate
obtained with DMSO. The right panel shows a representative
nucleotide kinetics curve. (B) Sensitivity of human and bacterial
ArfGEFs to BFA with liposomes. All assays were performed with
myr
Arf1. The right panel shows a representative nucleotide kinetics
curve. kobs values are expressed as a percentage of the rate obtained
with DMSO. Values are the mean of triplicates SD. The associated
Figure S3 shows representative nucleotide kinetics curves for the
experiments shown here.
Currently, its specicity toward other human ArfGEFs has been
only investigated for EFA6Sec7, which showed that this GEF is
not a target of the drug in vitro.29 We analyzed its eciency on
human and bacterial ArfGEFs in solution and with liposomes.
SecinH3 appeared to be insoluble at concentrations as low as
15 M, as judged by a noisy autouorescence signal of the
compound alone that stabilized after prolonged stirring (not
shown); therefore, all experiments were performed at 5 M, a
concentration close to its theoretical solubility where
autouorescence was no longer observed. At this concentration,
SecinH3 had a moderate inhibitory eect on ARNO in
solution, which was slightly higher with liposomes (Figure 3A
and Figure S4A). No inhibition was observed for other human
and bacterial GEFs, whether measurements were performed in
solution or in the presence of membranes (Figure 3B and
Figure S4B). Because ARNO activates all Arf isoforms,
including Arf1 and Arf6,46 we determined the eciency of
SecinH3 at blocking activation of Arf6. We observed that
SecinH3 inhibited 13Arf6 with an eciency slightly higher
than that of 17Arf1 (Figure 3A). This analysis conrms that
SecinH3 inhibits cytohesins specically, and this eect does not
depend on which Arf GTPase is the substrate, with the caveat
that the maximal inhibitory eect that could be measured (30%
of inhibition of ARNO toward 13Arf6 in solution or myrArf1
on membranes) was limited by its poor solubility.
Inhibition of Human and Bacterial ArfGEFs by M-
COPA in Solution and on Membranes. M-COPA inhibits
the growth of cancer cell lines with a prole that resembles that
of BFA, and it causes the dispersal of the large Golgi ArfGEF
GBF1, suggesting that this ArfGEF is a relevant mechanistic
target in cells.33,53,54 Direct analysis of this inhibitor toward
5129
Article
Figure 3. Inhibition analysis of SecinH3 (5 M) in solution and on
membranes. (A) Sensitivity of human and bacterial ArfGEFs to
SecinH3 in solution. All experiments were performed with 17Arf1,
except for the EFA6Sec7 experiment in which 13Arf6 was used. The
right panel shows a representative nucleotide kinetics curve. (B)
Sensitivity of human and bacterial ArfGEFs to SecinH3 with
liposomes. All assays were performed with myrArf1. The right panel
shows a representative nucleotide kinetics curve. kobs values are
expressed as described in the legend of Figure 2. The associated Figure
S4 shows representative nucleotide kinetics curves for the experiments
shown here.
puried Arf GTPases and ArfGEFs has not been reported. A
concentration of 16 M provided ecient inhibition of a
related Golgi ArfGEF, BIG1, in the solution GEF assay. M-
COPA inhibited all Sec7 domains in solution at this
concentration, with eciencies ranging from 20 to 60%
inhibition (Figure 4A and Figure S5A). All ArfGEF constructs
containing a membrane-binding domain were also inhibited in
the presence of liposomes, with slightly higher eciencies (30
80% inhibition) (Figure 4B and Figure S5B). There was no
clear correlation between the eciency of inhibition in solution
and with liposomes. For example, the eect of M-COPA was
strongest for BIG1 in solution and for LpRalF with liposomes
and weakest for EFA6 in solution and for BRAG2 with
liposomes. Our data thus indicate that M-COPA is a pan-
ArfGEF inhibitor and that its inhibitory prole is dierent from
that of BFA, despite their similar patterns of inhibition of
cancer cell growth and dispersal of the Golgi.33,54 The activity
of M-COPA toward GBF1 could not be assessed directly,
because this ArfGEF has resisted so far our attempts to produce
an active construct. However, a direct and potent inhibition of
GBF1 can be predicted from the inhibitory activity of M-COPA
on all ArfGEFs tested in our assay.
Inhibition of Human and Bacterial ArfGEFs by NAV-
2729. NAV-2729 was recently described as an inhibitor of the
spontaneous activation of Arf6, and it also inhibited the
activation of Arf6 by its GEFs, ARNO and BRAG2, in
solution.12 NAV-2729 was synthesized as described in
Supporting Information. We analyzed the inhibitory prole of
NAV-2729 at a concentration of 25 M, which was reported to
result in almost total inhibition of spontaneous and GEF-
stimulated Arf6 activation in vitro.12 Under the conditions used
DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133
Biochemistry
Figure 4. Inhibition analysis of M-COPA (15 M) in solution and on
membranes. (A) Sensitivity of human and bacterial ArfGEFs to M-
COPA in solution. All experiments were performed with 17Arf1,
except for the EFA6Sec7 experiment in which 13Arf6 was used. The
right panel shows a representative nucleotide kinetics curve. (B)
Sensitivity of human and bacterial ArfGEFs to M-COPA with
liposomes. All assays were performed with myrArf1. The right panel
shows a representative nucleotide kinetics curve. kobs values are
expressed as in Figure 2. The associated Figure S5 shows
representative nucleotide kinetics curves for the experiments shown
here.
in our assays, NAV-2729 inhibited spontaneous nucleotide
exchange of 13Arf6 by 15% (Figures 5A and FIgure S6A).
To determine the eciency of NAV-2729 in GEF-stimulated
nucleotide exchange, we rst used BRAG2Sec7PH, which is highly
active toward 13Arf6 in solution.39,40 NAV-2729 inhibited the
activation of 13Arf6 by BRAG2Sec7PH by 25% (Figure 5A and
Figure S6A). 17Arf1 has no measurable spontaneous
nucleotide exchange. Alternatively, GDP/GTP exchange in
17Arf1 can be observed in the absence of a GEF by addition
of EDTA, which displaces the bound Mg2+ ion. Under these
conditions, NAV-2729 had no eect on the spontaneous
activation of 17Arf1. Surprisingly, activation of 17Arf1 by
BRAG2Sec7PH was inhibited by NAV-2729, and the eciency
was markedly higher than that for Arf6 (50%). In a dose
response experiment, nucleotide exchange rates were reduced
by 50% by 10 M NAV-2729 for 17Arf1 while 50% inhibition
was not achieved even at 25 M NAV-2729 for 13Arf6
(Figure 5B). It should be noted that IC50 values could not be
determined with this assay because the inhibitor was predicted
to be highly insoluble above this concentration (using
Chemspider).
Next, we analyzed the specicity of NAV-2729 in solution
toward the Sec7 domains of human ArfGEFs using 17Arf1 as
a substrate (except for EFA6, for which the substrate was
13Arf6) with a concentration of 25 M, at which strong
inhibition was observed (Figure 5C and Figure S6B). Potent
inhibition was seen for the Sec7 domain of BRAG2 (60%), and
a moderate inhibition of 20% was seen for all other Sec7
domains that were evaluated. This is consistent with the
observation that knockdown of BRAG2, but not ARNO,
phenocopied the reduced rate of proliferation of uveal
5130
Article
Figure 5. Inhibition analysis of NAV-2729. (A) Eect of NAV-2729 on
spontaneous and BRAG2Sec7PH-stimulated activation of 17Arf1 and
13Arf6. (B) Dose response of NAV-2729 toward the activation of
17Arf1 and 13Arf6 by BRAG2Sec7PH. (C) Sensitivity of human and
bacterial Sec7 domains to NAV-2729 (25 M) in solution. The right
panel shows a representative nucleotide kinetics curve. kobs values are
expressed as described in the legend of Figure 2. The associated Figure
S6 shows representative nucleotide kinetics curves for the experiments
shown here.
melanoma cells resulting from treatment with NAV-2729.12
Finally, we sought to analyze the eect of NAV-2729 in the
presence of liposomes. No matter which GEF was used, we
observed complex kinetics that precluded further investigation.
This eect was not due to liposome aggregation or disruption
(Figure S2B) and will require further investigation. Together,
this analysis reveals that NAV-2729 inhibits the activation of
both Arf1 and Arf6 by their GEFs, with a higher eciency for
BRAG2 and for Arf1.
DISCUSSION
In this study, we determined the eciency of four unrelated
chemical compounds at inhibiting six ArfGEFs of human and
bacterial origin, based on uorescence kinetics assays with
highly puried proteins. Our analysis conrms that all inhibitors
are active at inhibiting directly at least one ArfGEF, and it
reveals that each of them has a unique inhibitory prole despite
the similarity of the Arf and ArfGEF targets included in this
study (Table 1 and Table S1). Notably, family-wide character-
ization of SecinH3 conrms that it targets only cytohesins,
although its inhibitory eciency is rather low regardless of
whether the Sec7 domain in solution or the full-length protein
with liposomes was used. Our analysis also identies M-COPA
as a potent pan-ArfGEF and shows that NAV-2729 inhibits all
DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133
Biochemistry Article

Table 1. Family-wide Inhibition Proles of ArfGEF analysis indicates that inhibitors that target the Sec7 domain
Inhibitorsa can in general be assayed for specicity by using a simple setup
with soluble versions of the small GTPases and the isolated
BFA SecinH3 M-COPA NAV-2729
Sec7 domain of their GEFs. However, the inhibition by BFA of
sol. mb. sol. mb. sol. mb. sol. full-length LpRalF, but not its Sec7 domain alone, underlines
BRAG2 0b + 0 0 ++ ++ +++ the potential of small molecules to target the exchange
ARNO 0b + + ++ ++ +++ + mechanism as a whole by incorporating elements provided by
EFA6 0b 0 0 0 ++ + ++ appended regulatory domains.
BIG1 +++b +++ 0 0 +++ +++ + Caveats in the interpretation of the eciencies and
LpRalF 0 +++ + + ++ +++ + specicities of inhibitors can lead to misleading results.55
RpRalF +++ +++ 0 0 +++ +++ + First, most inhibitors except BFA are poorly soluble as
a
Percentages of inhibition were evaluated from kobs values determined predicted from their theoretical solubilities, which can lead to
without and with the inhibitor as indicated. Legend: sol., in solution; misinterpretation if the inhibitors are used at the concentrations
mb., in the presence of 100 M liposomes; 0, < 10% inhibition; +, 10 above full solubility. Direct measurement of the actual solubility
20% inhibition; ++, 2040% inhibition; +++, >40% inhibition. is dicult, and caution is required to avoid solubility issues.
b
Determined in ref 27. The percentages of inhibition can be found This was exemplied in our study of SecinH3, which exhibited
in Table S1. a bizarre autouorescence signal. This signal stabilized upon
prolonged stirring of the sample, pointing to a solubility issue
ArfGEFs with a higher eciency toward BRAG2. We note that that was resolved by decreasing its concentration from 15 to 5
the concentration at which we observed inhibition of ArfGEFs M, which is close to its predicted solubility. At this
by M-COPA is higher than those that resulted in growth concentration, specic interference with cytohesins but no
defects or dispersal of the Golgi, which were in the range of other ArfGEFs was observed, consistent with previous
0.11 M.33,54 A possible explanation is that M-COPA is more studies,29 although this was at the cost of maximal inhibition,
ecient on GBF1 than on any of the ArfGEFs included in this which did not exceed 30%. Another approach would be to
analysis. Alternatively, our observation that M-COPA is a monitor the inhibitory response at increasing inhibitor
potent inhibitor of other ArfGEFs raises the possibility that M- concentrations; a discontinuity with the appearance of a
COPA also aects the activity of ArfGEFs other than GBF1, plateau of inhibition can suggest that the solubility limit has
each with a moderate eciency but with eects that add up to been reached. Finally, analysis of the kinetics using initial
yield bioactivity. The observation that NAV-2729 also inhibits velocities or single points can incorrectly estimate the inhibitory
several ArfGEFS, with BRAG2 being the most aected, suggests eects for inhibitors that have intrinsic uorescence or
that it may also function by small and cumulative eects on absorbance at the wavelengths used to measure the kinetics.
several ArfGEFs to achieve bioactivity. Modeling the kinetics over the entire duration of the assay
All inhibitors were ecient at inhibiting the activation of (ideally to the plateau) alleviates such issues. With these
Arf1, which was used as a reference in most experiments. technical precautions in mind, it is desirable to measure the
Inhibitory eects were also measured on Arf6 for all inhibitors, eciencies and specicities of small molecules using puried
except for BFA. Notably, our family-wide analysis showed that proteins as an important initial step toward validating them as
NAV-2729 is a dual Arf1/Arf6 inhibitor and is more eective tools for cellular or chemical biology.
toward Arf1 than Arf6 is; this is in contrast to a previous report Mounting evidence indicates that Arf GTPases and their
that found that it targets only Arf6.12 On the basis of these GEFs are pivotal regulatory nodes in major physiological
observations, it is likely that the other Arf family members functions such as insulin signaling,29,36 vascular stability,13 or
(Arf3Arf5) not assayed in this study are also inhibited by lipolysis11 and in pathologies such as cancers1012 and
these small molecules with similar ArfGEF specicity proles. infections.1416 Small molecule inhibitors are therefore urgently
Because membranes are integral components of the needed to interrogate the biology of these pathways,34 and their
activation of Arf GTPases by their GEFs and productive accurate biochemical characterization is an important step
interaction between Arf GTPases and GEFs takes place on toward the identication of which target(s) they engage to yield
membranes,22 we also compared the eciencies of the bioactivity.56 Our family-wide inhibitory proles of ArfGEF
inhibitors using lipidated Arf GTPases and ArfGEF constructs inhibitors will provide increased accuracy for future analysis of
comprising a membrane-binding domain (Table 1 and Table molecular pathways involved in diseases and help attain
S1). Surprisingly, membranes had in general a moderate eect therapeutic goals. Ultimately, this knowledge will feed the
on the eciency and selectivity of inhibition. A notable development of drugs that modulate Arf and ArfGEF
exception was Legionella RalF. The Sec7 domain of Legionella interactions and of other biotechnology applications.57,58
RalF was resistant to BFA in solution, as is expected from the
presence of a discriminating Phe residue in its active site. In
contrast, the full-length protein, whose activity can be detected

*
ASSOCIATED CONTENT
S Supporting Information
only in the presence of membranes,42 was strongly inhibited by The Supporting Information is available free of charge on the
BFA. On the basis of the presence of aromatic residues in the ACS Publications website at DOI: 10.1021/acs.bio-
inhibitory capping domain that mimic equivalent residues of chem.7b00706.
Arf, a plausible model is one in which BFA binds at the
interface between the Sec7 and capping domains to stabilize Chemical synthesis of NAV-2729; analysis of nonspecic
RalF in an autoinhibited conformation. Thus, inhibition of eects; representative kinetics curves for the experiments
LpRalF by BFA would be an intramolecular variation of the with BFA, SecinH3, M-COPA, and NAV-2729; and a
interfacial mechanism whereby BFA inhibits Golgi ArfGEFs by table of percentages of inhibition (PDF)
trapping an abortive Arf/Sec7 complex.21 Together, our SMILES molecular formula strings (CSV)
5131 DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133
Biochemistry
AUTHOR INFORMATION
Corresponding Authors
*E-mail: jacqueline.cherls@ens-paris-saclay.fr.
*E-mail: mahel.zeghouf@ens-paris-saclay.fr.
ORCID
Tatiana Caneque: 0000-0002-1110-0643
Isamu Shiina: 0000-0002-8749-2540
Mahel Zeghouf: 0000-0003-3686-2510
Present Address
@ transformed stem cells in adult Drosophila. Nature 538, 109113.
T.Y.: Pharmaceuticals and Medical Devices Agency, Shin-
Kasumigaseki Building, Tokyo 100-0013, Japan.
Author Contributions
S.B. and F.P. contributed equally to this work.
Funding
This work was supported by grants from the Fondation pour la
Recherche Medicale, the Institut National du Cancer, and the
CNRS to J. Cherls and from the Institut National du Cancer
to R.R.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
The authors are grateful to Lionel Duarte, Yann Ferrandez,
Alexandra Joubert, and Lurlene Akendengue (ENS Paris-
Saclay) for protein purications. The authors thank TOCRIS
Bioscience for performing quality controls on SecinH3.
ABBREVIATIONS
BFA, Brefeldin A; DCB-HUS, dimerization and cyclophilin-
binding homology upstream of sec7 domain; DLS, dynamic
light scattering; DMSO, dimethyl sulfoxide; EDTA, ethyl-
enediaminetetraacetic acid; FRET, Forster resonance energy
tranfer; GAP, GTPase-activating protein; GDP, guanosine
diphosphate; GEF, guanine nucleotide exchange factor; GTP,
guanosine triphosphate; IC50, half-maximal inhibitory concen-
tration; LpRalF, L. pneumophila RalF protein; PC, phosphati-
dylcholine; PE, phosphatidylethanolamine; PH, pleckstrin
homology; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate;
PS, phosphatidylserine; RpRalF, R. prowazekii RalF protein;
TGN, trans-Golgi network; SDSPAGE, sodium dodecyl
sulfatepolyacrylamide gel electrophoresis.
REFERENCES
(1) Takai, Y., Sasaki, T., and Matozaki, T. (2001) Small GTP-binding
proteins. Physiol. Rev. 81, 153208.
(2) Wittinghofer, A., and Vetter, I. R. (2011) Structure-function
relationships of the G domain, a canonical switch motif. Annu. Rev.
Biochem. 80, 943971.
(3) Cherfils, J., and Zeghouf, M. (2013) Regulation of small GTPases
by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269309.
(4) Vigil, D., Cherfils, J., Rossman, K. L., and Der, C. J. (2010) Ras
superfamily GEFs and GAPs: validated and tractable targets for cancer
therapy? Nat. Rev. Cancer 10, 842857.
(5) Aktories, K. (2011) Bacterial protein toxins that modify host
regulatory GTPases. Nat. Rev. Microbiol. 9, 487498.
(6) Arkin, M. R., Tang, Y., and Wells, J. A. (2014) Small-molecule
inhibitors of protein-protein interactions: progressing toward the
reality. Chem. Biol. 21, 11021114.
(7) Cherfils, J., and Zeghouf, M. (2011) Chronicles of the GTPase
switch. Nat. Chem. Biol. 7, 493495.
(8) DSouza-Schorey, C., and Chavrier, P. (2006) ARF proteins: roles
in membrane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347358.
5132
Article
(9) Donaldson, J. G., and Jackson, C. L. (2011) ARF family G
proteins and their regulators: roles in membrane transport, develop-
ment and disease. Nat. Rev. Mol. Cell Biol. 12, 362375.
(10) Morishige, M., Hashimoto, S., Ogawa, E., Toda, Y., Kotani, H.,
Hirose, M., Wei, S., Hashimoto, A., Yamada, A., Yano, H., Mazaki, Y.,
Kodama, H., Nio, Y., Manabe, T., Wada, H., Kobayashi, H., and Sabe,
H. (2008) GEP100 links epidermal growth factor receptor signalling
to Arf6 activation to induce breast cancer invasion. Nat. Cell Biol. 10,
8592.
(11) Singh, S. R., Zeng, X., Zhao, J., Liu, Y., Hou, G., Liu, H., and
Hou, S. X. (2016) The lipolysis pathway sustains normal and
(12) Yoo, J. H., Shi, D. S., Grossmann, A. H., Sorensen, L. K., Tong,
Z., Mleynek, T. M., Rogers, A., Zhu, W., Richards, J. R., Winter, J. M.,
Zhu, J., Dunn, C., Bajji, A., Shenderovich, M., Mueller, A. L.,
Woodman, S. E., Harbour, J. W., Thomas, K. R., Odelberg, S. J.,
Ostanin, K., and Li, D. Y. (2016) ARF6 Is an Actionable Node that
Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma. Cancer
Cell 29, 889904.
(13) Zhu, W., London, N. R., Gibson, C. C., Davis, C. T., Tong, Z.,
Sorensen, L. K., Shi, D. S., Guo, J., Smith, M. C., Grossmann, A. H.,
Thomas, K. R., and Li, D. Y. (2012) Interleukin receptor activates a
MYD88-ARNO-ARF6 cascade to disrupt vascular stability. Nature 492,
252255.
(14) Nagai, H., Kagan, J. C., Zhu, X., Kahn, R. A., and Roy, C. R.
(2002) A bacterial guanine nucleotide exchange factor activates ARF
on Legionella phagosomes. Science 295, 679682.
(15) Selyunin, A. S., Sutton, S. E., Weigele, B. A., Reddick, L. E.,
Orchard, R. C., Bresson, S. M., Tomchick, D. R., and Alto, N. M.
(2011) The assembly of a GTPase-kinase signalling complex by a
bacterial catalytic scaffold. Nature 469, 107111.
(16) Wesolowski, J., Weber, M. M., Nawrotek, A., Dooley, C. A.,
Calderon, M., St, St. Croix, C. M., Hackstadt, T., Cherfils, J., and
Paumet, F. (2017) Chlamydia Hijacks ARF GTPases To Coordinate
Microtubule Posttranslational Modifications and Golgi Complex
Positioning. mBio 8, e02280-16.
(17) Cox, R., Mason-Gamer, R. J., Jackson, C. L., and Segev, N.
(2004) Phylogenetic analysis of Sec7-domain-containing Arf nucleo-
tide exchangers. Mol. Biol. Cell 15, 14871505.
(18) Casanova, J. E. (2007) Regulation of Arf activation: the Sec7
family of guanine nucleotide exchange factors. Traffic 8, 14761485.
(19) Pasqualato, S., Renault, L., and Cherfils, J. (2002) Arf, Arl, Arp
and Sar proteins: a family of GTP-binding proteins with a structural
device for front-back communication. EMBO Rep. 3, 10351041.
(20) Goldberg, J. (1998) Structural basis for activation of ARF
GTPase: mechanisms of guanine nucleotide exchange and GTP-
myristoyl switching. Cell 95, 237248.
(21) Renault, L., Guibert, B., and Cherfils, J. (2003) Structural
snapshots of the mechanism and inhibition of a guanine nucleotide
exchange factor. Nature 426, 525530.
(22) Nawrotek, A., Zeghouf, M., and Cherfils, J. (2016) Allosteric
regulation of Arf GTPases and their GEFs at the membrane interface.
Small GTPases 7, 283296.
(23) Helms, J. B., and Rothman, J. E. (1992) Inhibition by brefeldin
A of a Golgi membrane enzyme that catalyses exchange of guanine
nucleotide bound to ARF. Nature 360, 352354.
(24) Donaldson, J. G., Finazzi, D., and Klausner, R. D. (1992)
Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine
nucleotide onto ARF protein. Nature 360, 350352.
(25) Peyroche, A., Antonny, B., Robineau, S., Acker, J., Cherfils, J.,
and Jackson, C. L. (1999) Brefeldin A acts to stabilize an abortive
ARF-GDP-Sec7 domain protein complex: involvement of specific
residues of the Sec7 domain. Mol. Cell 3, 275285.
(26) Mossessova, E., Corpina, R. A., and Goldberg, J. (2003) Crystal
structure of ARF1*Sec7 complexed with Brefeldin A and its
implications for the guanine nucleotide exchange mechanism. Mol.
Cell 12, 14031411.
(27) Zeeh, J. C., Zeghouf, M., Grauffel, C., Guibert, B., Martin, E.,
Dejaegere, A., and Cherfils, J. (2006) Dual specificity of the interfacial
DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133
Biochemistry
inhibitor brefeldin a for arf proteins and sec7 domains. J. Biol. Chem.
281, 1180511814.
(28) Viaud, J., Zeghouf, M., Barelli, H., Zeeh, J. C., Padilla, A.,
Guibert, B., Chardin, P., Royer, C. A., Cherfils, J., and Chavanieu, A.
(2007) Structure-based discovery of an inhibitor of Arf activation by
Sec7 domains through targeting of protein-protein complexes. Proc.
Natl. Acad. Sci. U. S. A. 104, 1037010375.
(29) Hafner, M., Schmitz, A., Grune, I., Srivatsan, S. G., Paul, B.,
Kolanus, W., Quast, T., Kremmer, E., Bauer, I., and Famulok, M.
(2006) Inhibition of cytohesins by SecinH3 leads to hepatic insulin
resistance. Nature 444, 941944.
(30) Saenz, J. B., Sun, W. J., Chang, J. W., Li, J., Bursulaya, B., Gray,
N. S., and Haslam, D. B. (2009) Golgicide A reveals essential roles for
GBF1 in Golgi assembly and function. Nat. Chem. Biol. 5, 157165.
(31) Boal, F., Guetzoyan, L., Sessions, R. B., Zeghouf, M., Spooner,
R. A., Lord, J. M., Cherfils, J., Clarkson, G. J., Roberts, L. M., and
Stephens, D. J. (2010) LG186: An inhibitor of GBF1 function that
causes Golgi disassembly in human and canine cells. Traffic 11, 1537
1551.
(32) Feng, Y., Jadhav, A. P., Rodighiero, C., Fujinaga, Y.,
Kirchhausen, T., and Lencer, W. I. (2004) Retrograde transport of
cholera toxin from the plasma membrane to the endoplasmic
reticulum requires the trans-Golgi network but not the Golgi apparatus
in Exo2-treated cells. EMBO Rep. 5, 596601.
(33) Ohashi, Y., Iijima, H., Yamaotsu, N., Yamazaki, K., Sato, S.,
Okamura, M., Sugimoto, K., Dan, S., Hirono, S., and Yamori, T.
(2012) AMF-26, a novel inhibitor of the Golgi system, targeting ADP-
ribosylation factor 1 (Arf1) with potential for cancer therapy. J. Biol.
Chem. 287, 38853897.
(34) Mishev, K., Dejonghe, W., and Russinova, E. (2013) Small
molecules for dissecting endomembrane trafficking: a cross-systems
view. Chem. Biol. 20, 475486.
(35) Jackson, C. L. (2002) Brefeldin A revealing the fundamental
principles governing membrane dynamics and protein transport.
Subcell. Biochem. 34, 233272.
(36) Fuss, B., Becker, T., Zinke, I., and Hoch, M. (2006) The
cytohesin Steppke is essential for insulin signalling in Drosophila.
Nature 444, 945948.
(37) Schlienger, S., Ramirez, R. A., and Claing, A. (2015) ARF1
regulates adhesion of MDA-MB-231 invasive breast cancer cells
through formation of focal adhesions. Cell. Signalling 27, 403415.
(38) Ohashi, Y., Okamura, M., Hirosawa, A., Tamaki, N., Akatsuka,
A., Wu, K. M., Choi, H. W., Yoshimatsu, K., Shiina, I., Yamori, T., and
Dan, S. (2016) M-COPA, a Golgi Disruptor, Inhibits Cell Surface
Expression of MET Protein and Exhibits Antitumor Activity against
MET-Addicted Gastric Cancers. Cancer Res. 76, 38953903.
(39) Aizel, K., Biou, V., Navaza, J., Duarte, L. V., Campanacci, V.,
Cherfils, J., and Zeghouf, M. (2013) Integrated conformational and
lipid-sensing regulation of endosomal ArfGEF BRAG2. PLoS Biol. 11,
e1001652.
(40) Jian, X., Gruschus, J. M., Sztul, E., and Randazzo, P. A. (2012)
The pleckstrin homology (PH) domain of the Arf exchange factor
Brag2 is an allosteric binding site. J. Biol. Chem. 287, 2427324283.
(41) Padovani, D., Folly-Klan, M., Labarde, A., Boulakirba, S.,
Campanacci, V., Franco, M., Zeghouf, M., and Cherfils, J. (2014)
EFA6 controls Arf1 and Arf6 activation through a negative feedback
loop. Proc. Natl. Acad. Sci. U. S. A. 111, 1237812383.
(42) Folly-Klan, M., Alix, E., Stalder, D., Ray, P., Duarte, L. V.,
Delprato, A., Zeghouf, M., Antonny, B., Campanacci, V., Roy, C. R.,
and Cherfils, J. (2013) A novel membrane sensor controls the
localization and ArfGEF activity of bacterial RalF. PLoS Pathog. 9,
e1003747.
(43) Zeeh, J. C., Antonny, B., Cherfils, J., and Zeghouf, M. (2008) In
vitro assays to characterize inhibitors of the activation of small G
proteins by their guanine nucleotide exchange factors. Methods
Enzymol. 438, 4156.
(44) Franco, M., Chardin, P., Chabre, M., and Paris, S. (1995)
Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide
exchange at physiological Mg2+ levels. J. Biol. Chem. 270, 13371341.
5133
Article
(45) Beraud-Dufour, S., Robineau, S., Chardin, P., Paris, S., Chabre,
M., Cherfils, J., and Antonny, B. (1998) A glutamic finger in the
guanine nucleotide exchange factor ARNO displaces Mg2+ and the
beta-phosphate to destabilize GDP on ARF1. EMBO J. 17, 3651
3659.
(46) Peurois, F., Veyron, S., Ferrandez, Y., Ladid, I., Benabdi, S.,
Zeghouf, M., Peyroche, G., and Cherfils, J. (2017) Characterization of
the activation of small GTPases by their GEFs on membranes using
artificial membrane tethering. Biochem. J. 474, 12591272.
(47) Folly-Klan, M., Sancerne, B., Alix, E., Roy, C. R., Cherfils, J., and
Campanacci, V. (2015) On the use of Legionella/Rickettsia chimeras
to investigate the structure and regulation of Rickettsia effector RalF. J.
Struct. Biol. 189, 98104.
(48) Vives, V., Cres, G., Richard, C., Busson, M., Ferrandez, Y.,
Planson, A. G., Zeghouf, M., Cherfils, J., Malaval, L., and Blangy, A.
(2015) Pharmacological inhibition of Dock5 prevents osteolysis by
affecting osteoclast podosome organization while preserving bone
formation. Nat. Commun. 6, 6218.
(49) Ramaen, O., Joubert, A., Simister, P., Belgareh-Touze, N.,
Olivares-Sanchez, M. C., Zeeh, J. C., Chantalat, S., Golinelli-Cohen, M.
P., Jackson, C. L., Biou, V., and Cherfils, J. (2007) Interactions
between conserved domains within homodimers in the BIG1, BIG2,
and GBF1 Arf guanine nucleotide exchange factors. J. Biol. Chem. 282,
2883428842.
(50) DiNitto, J. P., Delprato, A., Gabe Lee, M. T., Cronin, T. C.,
Huang, S., Guilherme, A., Czech, M. P., and Lambright, D. G. (2007)
Structural basis and mechanism of autoregulation in 3-phosphoinosi-
tide-dependent Grp1 family Arf GTPase exchange factors. Mol. Cell 28,
569583.
(51) Franco, M., Peters, P. J., Boretto, J., van Donselaar, E., Neri, A.,
DSouza-Schorey, C., and Chavrier, P. (1999) EFA6, a sec7 domain-
containing exchange factor for ARF6, coordinates membrane recycling
and actin cytoskeleton organization. EMBO J. 18, 14801491.
(52) Li, J., Malaby, A. W., Famulok, M., Sabe, H., Lambright, D. G.,
and Hsu, V. W. (2012) Grp1 plays a key role in linking insulin
signaling to glut4 recycling. Dev. Cell 22, 12861298.
(53) Shiina, I., Umezaki, Y., Ohashi, Y., Yamazaki, Y., Dan, S., and
Yamori, T. (2013) Total synthesis of AMF-26, an antitumor agent for
inhibition of the Golgi system, targeting ADP-ribosylation factor 1. J.
Med. Chem. 56, 150159.
(54) Ignashkova, T. I., Gendarme, M., Peschk, K., Eggenweiler, H.
M., Lindemann, R. K., and Reiling, J. H. (2017) Cell survival and
protein secretion associated with Golgi integrity in response to Golgi
stress-inducing agents. Traffic 18, 530544.
(55) Arrowsmith, C. H., Audia, J. E., Austin, C., Baell, J., Bennett, J.,
Blagg, J., Bountra, C., Brennan, P. E., Brown, P. J., Bunnage, M. E.,
Buser-Doepner, C., Campbell, R. M., Carter, A. J., Cohen, P.,
Copeland, R. A., Cravatt, B., Dahlin, J. L., Dhanak, D., Edwards, A. M.,
Frederiksen, M., Frye, S. V., Gray, N., Grimshaw, C. E., Hepworth, D.,
Howe, T., Huber, K. V., Jin, J., Knapp, S., Kotz, J. D., Kruger, R. G.,
Lowe, D., Mader, M. M., Marsden, B., Mueller-Fahrnow, A., Muller, S.,
OHagan, R. C., Overington, J. P., Owen, D. R., Rosenberg, S. H.,
Roth, B., Ross, R., Schapira, M., Schreiber, S. L., Shoichet, B.,
Sundstrom, M., Superti-Furga, G., Taunton, J., Toledo-Sherman, L.,
Walpole, C., Walters, M. A., Willson, T. M., Workman, P., Young, R.
N., and Zuercher, W. J. (2015) The promise and peril of chemical
probes. Nat. Chem. Biol. 11, 536541.
(56) Schurmann, M., Janning, P., Ziegler, S., and Waldmann, H.
(2016) Small-Molecule Target Engagement in Cells. Cell Chem. Biol.
23, 435441.
(57) Zhu, Y., Xiao, T., Lei, S., Zhou, F., and Wang, M. W. (2015)
Application of chemical biology in target identification and drug
discovery. Arch. Pharmacal Res. 38, 16421650.
(58) Milroy, L. G., Grossmann, T. N., Hennig, S., Brunsveld, L., and
Ottmann, C. (2014) Modulators of protein-protein interactions. Chem.
Rev. 114, 46954748.
DOI: 10.1021/acs.biochem.7b00706
Biochemistry 2017, 56, 51255133