Family-wide analysis of the inhibition of Arf guanine nucleotide

Subscriber access provided by Grand Valley State | University

Article

Family-wide analysis of the inhibition of Arf guanine nucleotide exchange factors with small molecules: evidence for unique inhibitory profiles. Sarah Benabdi, François Peurois, agata nawrotek, Jahnavi Chikireddy, Tatiana Cañeque, Takao Yamori, Isamu Shiina, Yoshimi Ohashi, Shingo Dan, Raphaël Rodriguez, Jacqueline Cherfils, and Mahel Zeghouf Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00706 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Family-wide analysis of the inhibition of Arf guanine nucleotide exchange factors with small molecules: evidence for unique inhibitory profiles. Sarah Benabdi,†, ‡ François Peurois,†, ‡ Agata Nawrotek, † Jahnavi Chikireddy, † Tatiana Cañeque,#,§,∥ Takao Yamori,⊥,π, Isamu Shiina,¶, Yoshimi Ohashi⊥, Shingo Dan⊥, Raphaël Rodriguez, #,§,∥ Jacqueline Cherfils,*, † and Mahel Zeghouf,*, †. †

Laboratoire de Biologie et Pharmacologie Appliquée CNRS / Ecole Normale Supérieure Paris-

Saclay, 61 avenue du président Wilson, 94235 Cachan, France. #

Institut Curie, PSL Research University, Chemical Cell Biology group, 26 rue d’Ulm, 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.

ACS Paragon Plus Environment

1

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

ABSTRACT: Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic 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 at dissecting molecular pathways and blocking disease-related functions. However, their specificity 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 compared the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA and NAV-2729, towards six ArfGEFs (human ARNO, EFA6, BIG1 and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence 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 specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs with a strongest effect against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF uncovers a strong inhibitory effect 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.

INTRODUCTION Small GTPases regulate many aspects of cell logistics, including signaling, dynamics of the acting cytoskeleton and membrane traffic (1). Common to all small GTPases is their ability to


2

Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

function as molecular switches, by alternating between inactive GDP-bound and active GTPbound forms (2). GDP/GTP alternation is finely 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 consequence 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 cancers (4) and infections (5). Extensive efforts 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 protein-membrane interactions and their complexes exhibit highly dynamic structures lacking well-defined pockets, all of which are considered poorly druggable features (6). In addition, individual GTPases are often involved in concurrent processes through the combination of related regulators (7), whereby even on-target inhibition can lead to detrimental off-pathway effects. Arf GTPases constitute a subfamily of small GTPases with regulatory functions in most major aspects of membrane traffic and organelle structure in the cell (8, 9). Their roles in diseases have expanded over the past decade, notably in cancer (10-12), inflammation (13) and infections (1416). In metazoans, they are activated by 15 different GEFs (ArfGEF hereafter) which bear a conserved catalytic Sec7 domain that stimulates GDP/GTP exchange (17). The Sec7 domain is decorated by various other domains, the nature of which defines ArfGEF subfamiles (18). An additional ArfGEF, RalF, is secreted by the bacterial pathogens 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


3

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 30

by GTP to their recruitment to membranes (19). This mechanism uses a myristoylated Nterminal 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 to 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 follows their Sec7 domain; large Golgi ArfGEFs have membranebinding elements in both N- and C-terminus of their Sec7 domain; and RalF proteins have a membrane-binding domain uniquely found in bacteria that is also involved in auto-inhibition (22). The first 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 first 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 towards 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 fluorometric GEF assaybased screen identified NAV-2729, which blocks spontaneous activation of Arf6 and its


4

Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

activation by cytohesins and BRAG (12). Other inhibitors have been discovered by phenotypic screens. Golgicide A was identified 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 identified through an image-based phenotypic screen for perturbation of exocytosis (32). M-COPA (also called AMF26) was identified based on the similarity of its growth inhibitory profile towards 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 specifically (33). ArfGEFs inhibitors have already a long history in elucidating 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 identified 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); and 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, mice models of inflammation exposed to SecinH3 had reduced vascular permeability, a process leading to inflammation, which suggested that cytohesins and the Arf6 GTPase are valid targets for the treatment of inflammatory conditions (13). Likewise, several ArfGEF inhibitors were shown to have anti-tumoral activities. NAV-2729 reduced uveal melanoma cell proliferation and tumorigenesis in a mouse model (12), M-COPA led to tumor regression in mice xenografts (33, 38), and BFA, Golgicide A, SecinH3 and LM11 markedly reduced stem-cell tumors in Drosophila by blocking lipid droplet usage (11).


5

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 30

These studies highlighted Arf GTPases and their regulators as attractive candidates for therapeutic intervention. Small 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. Firstly, all ArfGEFs share a highly conserved Sec7 catalytic domain (17), which raises the issue drug specificity. Secondly, inhibition efficiency in vitro has been mostly analyzed in solution, which underestimates GEF efficiencies by one to several orders of magnitude (39-42). Quantitative in vitro analysis of inhibitors using purified proteins is an efficient approach to address these questions (43). In this study, we used fluorescence kinetics-based assays with purified Arf GTPases and representative human and bacterial ArfGEFs to establish the inhibitory profiles of four inhibitors of unrelated structure, BFA, SecinH3, M-COPA and NAV-2729 (Figure 1A). The effect of membranes was investigated for three of these inhibitors, by incorporating artificial 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 specific inhibitory profile. 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 effect of these inhibitors to dissect the biology of Arf GTPases and their GEFs and facilitate their further development.

MATERIALS AND METHODS Chemicals. GDP, GTP and and N-methylanthraniloyl GTP (mGTP) are from Jena Bioscience. BFA is from Sigma and SecinH3 from Tocris Bioscience. M-COPA was synthesized as described in (33). NAV-2729 was synthesized as described in Figure S1. Theoretical solubilities


6

Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

were predicted using the US Environmental Protection Agency’s EPISuite™ (compiled on Chemspider http://www.chemspider.com).

Protein expression and purification. Protein constructs used in this study are shown in Figure 1B. N-terminally truncated bovine Δ17Arf1 (identical to human) and human Δ13Arf6 were expressed and purified and loaded with GDP prior to use as described in (27). Myristoylated fulllength Arf1 (myrArf1 hereafter) was obtained by co-expression with yeast N-myristoyl transferase in E. coli and purified as described in (44). Human BRAG2Sec7, ARNOSec7, BIG1Sec7 and EFA6Sec7 were produced in E. coli and purified as described in (39), (45), (27) and (41) respectively. Human BRAG2Sec7PH, ARNOSec7PH and EFA6Sec7PH were expressed in E. coli and purified as described in (39), (41) and (46), respectively. RalF proteins from Legionella pneumophila (LpRalF) and Rickettsia prowazekii (RpRalF) and their isolated Sec7 domains were produced and purified as described in (42, 47). Human Rac1 and the GEF domain of mouse Dock5 were obtained according to (48). Human BIG1DHSec7 was expressed in insect cells and purified as described in (49). All proteins were highly pure as assessed by SDS-PAGE (Figure 1C). Protein concentrations were measured using their respective absorption coefficient at 280 nm after centrifugation to remove any precipitate.

Liposomes preparation. All lipids were purchased from Avanti Polar Lipids. Experiments carried out with human ArfGEFs were done with liposomes containing 48% phosphatidylcholine (PC),

20%

phosphatidylethanolamine

(PE),

30%

phosphatidylserine

(PS)

and

2%

phosphatidylinositol-4,5-diphosphate (PI(4,5)P2). Experiments with LpRalF and RpRalF were done with liposomes containing 40% PC, 19% PE, 25% PS, 1% PI(4,5)P2 and 15% cholesterol.


7

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 30

All liposomes were prepared and extruded through 200 nm polycarbonate filters as described (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 fluorescence that follows the conformational change from Arf-GDP to Arf-GTP (excitation/emission wavelengths of 292/340 nm). In some experiments of BFA and liposomes, kinetics were monitered using Förster resonance energy transfer (FRET) between tryptophan and mGDP (292 nm/440 nm), which gives equivalent kinetics (43). Fluorescence measurements were done with a Cary Eclipse fluorimeter (Varian) at 37˚C, under continuous stirring in a total volume of 800 μL. Experiments in solution used 1 µM of N-terminally truncated Arf-GDP and 100 nM of ArfGEF, excepted for RpRalF and LpRalF that were used at 400 nM. Protein and inhibitors (or DMSO for controls) were incubated in HKM buffer (50 mM HEPES pH 7.4, 120 mM potassium acetate, 1 mM MgCl2 and 1 mM DTT) for 2 min at 37˚C before initiating the reaction with 100 µM GTP. Exchange assays with liposomes were done as above using 0.4 µM of myrArf1 in the presence of 100 µM of liposomes. ArfGEFs concentrations were 5 nM for BRAG2Sec7PH, 10 nM for ARNOSec7PH, EFA6Sec7PH and LpRalF, and 100 nM for RpRalF and for BIG1DHSec7. Exchange rates (kobs) were determined from monoexponential fits over the entire kinetics and expressed as percentage of control activity. This approach was preferred to initial rate velocity analysis, which can be misestimated if compounds absorb light or have intrinsic fluorescence. All exchange reactions were done at least in triplicate and means are given ± SD.

Dynamic light scattering analysis (DLS). The distribution of liposome sizes was analyzed by DLS using a DynaPro NanoStarTM instrument (Wyatt Technology). Sets of 10 autocorrelation


8

Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

curves were acquired at 37°C at the end of each exchange assay in a disposable cuvette (Eppendorf). The mean radius was determined with the software 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 NAV2729) structurally unrelated inhibitors were selected for this study (Figure 1A). To analyze the efficiency of the inhibitors we used highly purified small GTPases and GEFs (Figures 1B and 1C) and measured their inhibition by fluorescence kinetics, which reports accurately and quantitatively on nucleotide exchange inhibition (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, Legionella pneumophila and Rickettsia prowazekii RalF. We used different Arf and ArfGEF constructs for the solution and membrane experiments, since membrane-binding elements are inhibitory in Arf and in several ArfGEFs. For experiments carried out in solution, we used the isolated Sec7 domain of the ArfGEFs and Arf constructs lacking their myristoylated N-terminal helix (Δ17Arf1 and Δ13Arf6 hereafter), which is autoinhibitory in solution and requires direct interaction with membranes for inhibition release (19)(22). For experiments carried out on membranes, we used lipidated full-length Arf1, which carries a myristate lipid in N-terminus. Membrane-binding elements in ArfGEFs 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


9

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

ARNO (50) and the capping domain of RalF proteins (42) are autoinhibitory, while the PH domains of BRAG2 (39, 40), EFA6 (41) and the DCD-HUS domains of BIG1 (22) are not. The concentrations of GEFs were selected to support efficiencies at which inhibitory effects could be accurately measured. Arf1 was used as reference in all experiments, except in the case of EFA6, which activates Arf1 only in the presence of membranes (41), in which case Δ13Arf6 was used for the solution assays. Inhibitors were used at fixed concentrations where significant inhibition of at least one ArfGEF could be measured. All inhibitors were assayed for non-specific effects in two control experiments. Firstly, effects outside the Arf/ArfGEF targets were assayed on the small GTPase Rac1 and its specific GEF Dock5 as described in (48). None exhibited a significant effect on the Dock5-mediated Rac1 activation (Figure S2A). Secondly, non-specific effects on liposomes were monitored by using dynamic light scattering (DLS), which indicated that none of the compounds induced aggregation or disruption of liposomes (Figure S2B).


10

N

N N

OH

Page 11 of 30

HO

HO

OH

O

HO

O

O

NN

OH

Biochemistry O O O

O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O

O O

O

O NH

NH

S S

N

O

N

O O

NH

S

O2N

BFA O

A

SecinH3

NH

BFA'

O SecinH3' HN

M-COPA

BFA

H H OH

HO HO

O Cl N N Cl N O

O

HO OH H H

HO

O

O M-COPA

O

O O

NH

NAV-2729 NAV"2729' NAV-2729

M-COPA M-COPA

O

O HO HO

H

H

Cl H

N O NAV-2729 N HN

NH

HN O

O

O

NH

N

M-COPA

in#solu(on# OH

H

390#

BRAG2Sec7#

HO

H

50#

Sec7#

527#

Sec7# HN

Sec7# O NH LpRalF

####1#

O BFA

####6#

RpRalFSec7# BIGSec7#

NH

N

691#

Dock5DHR2## 1212# Δ17Arf1##

18#

Δ13Arf6##

14#

Rac1#

C"

1#

727#

ON

Sec7# 2

N

O

191#

Sec7#

199#

Sec7#

889#

DHR2## # Arf1# # Arf6# ## Rac# #

1642#

O2N

390#

Sec7#

PH#

811#

50#

Sec7#

PH#

399#

527#

Sec7#

PH#

1024#

LpRalF#

1#

Sec7#

Cap#

374#

RpRalF#

1#

Sec7#

Cap#

462#

Sec7# # Arf1# #

888#

DCB#

BIG1DHSec7# 2#

HUS#

1

myrArf1##

175# 192#

ArfGEFs, , c7

,

, c7

, c7

H,

, PH

,

7 7 , PH 7P , e , 1, 2S Sec Sec7 Sec7 FSe lFSe Sec Sec Sec7 F, l a g2 O 6 al f1 Arf O G r 6 a 1 A N G A R R a N A R yr A 17 m BR AR BI EF Lp Rp Br AR EF Lp kDa, Δ

,

lF Ra Rp kDa, 75,

45,

250, 150, 100, 75,

35,

37,

25,

66,

25, 18,

181#

181#

Arfs,

kDa,

on#liposomes#

O N 594#N N BRAG2Sec7PH# HN Sec7PH# O O ARNO 256# N N SecinH3 EFA6Sec7PH# N

Sec7#

Cl

O N

EFA6Sec7#

ARNOSec7#

O2N O2N

N

NH

NAV-2729 Cl

O

O

NAV-2729

O

O

H HO B"HO H

S

N

O2N

N N N N HN O

NH

S

M-COPA

Cl

H

NH

S

O

M"COPA'

O

NAV-2729 NN N N N

SecinH3

25, 20, 15, 10,

c Se DH

7,

G BI

50, 37,

20, 15,

Figure 1. Small molecules and proteins used in this study. (A) Chemical structures of the compounds. (B) Small GTPases and GEF constructs used for in vitro nucleotide exchange assay in solution (left) and in the presence of membrane (right). Numbers refer to the first and last residue of each construct. (C) SDS-PAGE analysis of the recombinant proteins. The associated


11

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

Figure S2 shows control inhibition assays carried out with the related small GTPase Rac1 and its GEF Dock5 and analysis by DLS of liposome integrity in the presence of the inhibitors. 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 dissociation (21, 26). Its efficiency towards eukaryotic ArfGEFs has been quantified in solution assays, showing that it inhibits specifically large Golgi ArfGEFs such as BIG1, while PH domain-containing ArfGEFs are resistant (for example, (27)). We extended this analysis to the Sec7 domains of Legionella and Rickettsia RalF (Figures 2A and S3A). We observed that Rickettsia RalF is inhibited by BFA, whereas Legionella RalF is resistant, which is consistent with the presence of Tyr residue in the active site of the former and a Phe residue in the latter. Next, we determined the efficiency of BFA towards human and bacterial ArfGEFs in the presence of liposomes (Figures 2B and 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

myr

Arf1 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 effect due to auto-inhibition since 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


12

Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

uncovers an overlooked inhibition of Legionella RalF that could not be detected with the Sec7 domain alone in solution.

Figure 2. Analysis of the inhibitory efficiency of BFA (50 µM) 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. Right panel: A representative nucleotide kinetics curve. (B) Sensitivity of human and bacterial ArfGEFs to BFA with liposomes. All assays were carried out with myrArf1. The right panel shows a representative nucleotide kinetics curve. kobs 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 in this Figure. Inhibition of human and bacterial ArfGEFs by SecinH3 in solution and on membranes. SecinH3 inhibits cytohesins in vitro (29) and has been widely used as a proxy for the involvement of cytohesins in cellular processes (13, 36, 52). Currently, its specificity towards


13

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

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 efficiency on human and bacterial ArfGEFs in solution and with liposomes. SecinH3 appeared insoluble at concentrations as low as 15 µM, as judged by a noisy autofluorescence signal of the compound alone that stabilized after prolonged stirring (not shown); therefore, all experiments were performed at 5 µM, a concentration close to its theoretical solubility where autofluorescence was no longer observed. At this concentration, SecinH3 had a moderate inhibitory effect on ARNO in solution, which was slightly higher with liposomes (Figures 3A and S4A). No inhibition was observed for other human and bacterial GEFs, whether measurements were performed in solution or in the presence of membranes (Figures 3B and S4B). Because ARNO activates all Arf isoforms including Arf1 and Arf6 (46), we determined the efficiency of SecinH3 at blocking activation of Arf6. We observed that SecinH3 inhibited Δ13Arf6 with a slightly higher efficiency than Δ17Arf1 (Figure 3A). This analysis confirms that SecinH3 inhibits cytohesins specifically and this effect does not depend on which Arf GTPase is the substrate, with the caveat that the maximal inhibitory effect that could be measured (30% of inhibition of ARNO towards Δ13Arf6 in solution or myrArf1 on membranes) was limited by its poor solubility.


14

Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. Inhibition analysis of SecinH3 (5 µM) in solution and on membranes. (A) Sensitivity of human and bacterial ArfGEFs to SecinH3 in solution. All experiments were carried out with Δ17Arf1, except for the EFA6Sec7 experiment where Δ13Arf6 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 carried out with myrArf1. The right panel shows a representative nucleotide kinetics curve. kobs values are expressed as in Figure 2. The associated Figure S4 shows representative nucleotide kinetics curves for the experiments shown in this Figure. 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 profile 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 towards purified Arf GTPases and ArfGEFs has not been reported. A concentration of 16 µM provided efficient inhibition of a related Golgi ArfGEF, BIG1, in the solution GEF assay. M-COPA inhibited all


15

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

Sec7 domains in solution at this concentration, with efficiencies ranging from 20% to 60% inhibition (Figures 4A and S5A). All ArfGEF constructs containing a membrane-binding domain were also inhibited in the presence of liposomes, with slightly higher efficiencies (30-80% inhibition) (Figure 4B and S5B). There was no clear correlation between the efficiency of inhibition in solution and with liposomes. For example, the effect of M-COPA was highest for BIG1 in solution and for LpRalF with liposomes, and lowest 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 profile is different from that of BFA, despite their similar patterns of inhibition of cancer cell growth and dispersal of the Golgi (33, 54). Activity of M-COPA towards GBF1 could not be assessed directly, since 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.

Figure 4. Inhibition analysis of M-COPA (15 µM) in solution and on membranes. (A) Sensitivity of human and bacterial ArfGEFs to M-COPA in solution. All experiments were carried out with Δ17Arf1, except for the EFA6Sec7 experiment where Δ13Arf6 was used. The


16

Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

right panel shows a representative nucleotide kinetics curve. (B) Sensitivity of human and bacterial ArfGEFs to M-COPA with liposomes. All assays were carried out with

myr

Arf1. 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 in this Figure. 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). NAV2729 was synthesized as shown in Figure S1. We analyzed the inhibitory profile of NAV-2729 at a concentration of 25 µM, which was reported to result in almost total inhibition of spontaneous and GEF-stimulated Arf6 activation in vitro (12). In the conditions used in our assays, NAV2729 inhibited spontaneous nucleotide exchange of Δ13Arf6 by about 15% (Figures 5A and S6A). To determine the efficiency of NAV-2729 on GEF-stimulated nucleotide exchange, we first used BRAG2Sec7PH, which is highly active towards Δ13Arf6 in solution (39, 40). NAV-2729 inhibited the activation of Δ13Arf6 by BRAG2Sec7PH by 25% (Figure 5A and S6A). Δ17Arf1 has no measurable spontaneous nucleotide exchange. Alternatively, GDP/GTP exchange in Δ17Arf1 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 effect on the spontaneous activation of Δ17Arf1. Surprisingly, activation of Δ17Arf1 by BRAG2Sec7PH was inhibited by NAV-2729, and the efficiency was markedly higher than for Arf6 (50%). In a dose-response experiment, nucleotide exchange rates were reduced by 50% by 10 µM NAV-2729 for Δ17Arf1 while 50% inhibition was not achieved even at 25 µM NAV-2729 for Δ13Arf6 (Figure 5B). It should be noted that


17

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

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 specificity of NAV-2729 in solution towards the Sec7 domains of human ArfGEFs using Δ17Arf1 as a substrate (except for EFA6 where the substrate was Δ13Arf6) with a concentration of 25 µM at which strong inhibition was observed (Figure 5C and S6B). Potent inhibition was seen for the Sec7 domain of BRAG2 (60%), and a moderate inhibition of about 20% was seen for all other Sec7 domains evaluated. This is consistent with the observation that knockdown of BRAG2, but not ARNO, phenocopied the reduced proliferation of uveal melanoma cells resulting from treatment with NAV-2729 (12). Finally, we sought to analyze the effect of NAV-2729 in the presence of liposomes. No matter which GEF was used, we observed complex kinetics that precluded further investigation. This effect was not due to liposome aggregation or disruption (Figure S1), 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 efficiency for BRAG2 and for Arf1.


18

Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5. Figure 5. Inhibition analysis of NAV-2729. (A) Effect of NAV-2729 on spontaneous and BRAG2Sec7PH-stimulated activation of Δ17Arf1 and Δ13Arf6. (B) Dose-response of NAV2729 towards the activation of Δ17Arf1 and Δ13Arf6 by BRAG2Sec7PH. (C) Sensitivity of human and bacterial Sec7 domains to NAV-2729 (25 µM) in solution. The right panel shows a representative nucleotide kinetics curve. kobs values are expressed as in Figure 2. The associated Figure S6 shows representative nucleotide kinetics curves for the experiments shown in this Figure.

DISCUSSION In this study, we determined the efficiency of four unrelated chemical compounds at inhibiting six ArfGEFs of human and bacterial origin, based on fluorescence kinetics assays with highly


19

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

purified proteins. Our analysis confirms that all inhibitors are active at inhibiting directly at least one ArfGEF, and it reveals that each of them has a unique inhibitory profile despite the similarity of the Arf and ArfGEF targets included in this study (Tables 1 and S1). Notably, family-wide characterization of SecinH3 confirms that it targets only cytohesins, although its inhibitory efficiency is rather low whether the Sec7 domain in solution or the full-length protein with liposomes were used. Our analysis also identifies M-COPA as a potent pan-ArfGEF, and shows that NAV-2729 inhibits all ArfGEFs with a higher efficiency towards BRAG2. We note that the concentration at which we observed inhibition of ArfGEFs by M-COPA is higher than those that resulted in growth defects or dispersal of the Golgi, which were in the 0.1-1 µM range (33),(54). A possible explanation is that M-COPA is more efficient on GBF1 than on any of the ArfGEFs included in this analysis. Alternatively, our observation that M-COPA is a potent inhibitor of other ArfGEFs raises the possibility that M-COPA also affects the activity of ArfGEFs other than GBF1, each with a moderate efficiency but with effects that add up to yield bio-activity. The observation that NAV-2729 also inhibits several ArfGEFS, with BRAG2 being the most affected, suggests that it may also function by small and cumulative effects on several ArfGEFs to achieve bio-activity. All inhibitors were efficient at inhibiting the activation of Arf1, which was used as a reference in most experiments. Inhibitory effects were also measured on Arf6 for all inhibitors, except for BFA. Notably, our family-wide analysis showed that NAV-2729 is a dual Arf1/Arf6 inhibitor and is more effective towards Arf1 than Arf6; this is in contrast to a previous report that found that it targets only Arf6 (12). Based on these observations, it is likely that the other Arf family members (Arf3, Arf4 and Arf5) not assayed in this study, are also inhibited by these small molecules with similar ArfGEF specificity profiles.


20

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Because membranes are integral components of the activation of Arf GTPases by their GEFs and productive interaction between Arf GTPases and GEFs takes place on membranes (22), we also compared the efficiencies of the inhibitors using lipidated Arf GTPases and ArfGEF constructs comprising a membrane-binding domain (Tables 1 and S1). Surprisingly, membranes had in general a moderate effect on the efficiency and selectivity of inhibition. A notable exception was Legionella RalF. The Sec7 domain of Legionella 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 only be detected in the presence of membranes (42), was strongly inhibited by BFA. Based on the presence of aromatic residues in the inhibitory capping domain that mimic equivalent residues of Arf, a plausible model is that BFA binds at the interface between the Sec7 and capping domains to stabilize RalF in an autoinhibited conformation. Thus, inhibition of LpRalF by BFA would be an intra-molecular variation of the interfacial mechanism whereby BFA inhibits Golgi ArfGEFs by trapping an abortive Arf/Sec7 complex (21). Together, our analysis indicates that inhibitors that target the Sec7 domain can in general be assayed for specificity by using a simple setup with soluble versions of the small GTPases and the isolated Sec7 domain of their GEFs. However, the inhibition by BFA of full-length LpRalF, but not its Sec7 domain alone, underlines the potential of small molecules to target the exchange mechanism as a whole by incorporating elements provided by appended regulatory domains. Caveats in the interpretation of the efficiencies and specificities of inhibitors can lead to misleading results (55). First, most inhibitors except BFA are poorly soluble as predicted from their theoretical solubilities, which can lead to misinterpretation if used at the concentrations above full solubility. Direct measurement of the actual solubility is difficult, and caution is


21

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

required in order to avoid solubility issues. This was exemplified in our study of SecinH3, which exhibited a bizarre autofluorescence signal. This signal stabilized upon prolonged stirring, pointing to a solubility issue that was resolved by lowering its concentration from 15 to 5 µM, which is close to its predicted solubility. At this concentration, specific interference with cytohesins but no other ArfGEFs was observed, consistent with previous studies (29), although this was at the cost of a maximal inhibition, which did not exceed 30%. Another approach would be to monitor the inhibitory response at increasing inhibitor concentrations; a discontinuity with the appearance of a plateau of inhibition can suggest that the solubility limit has been reached. Finally, analysis of the kinetics using initial velocities or single points can misestimate the inhibitory effects for inhibitors that have intrinsic fluorescence or absorbance at the wavelengths used to measure the kinetics. Modelling the kinetics over the entire duration of the assay (ideally to the plateau) alleviates such issues. With these technical precautions in mind, it is desirable to measure the efficiencies and specificities of small molecules using purified proteins as an important initial step towards validating them as tools for cellular or chemical biology. Mounting evidence indicates that Arf GTPases and their GEFs are pivotal regulatory nodes in major physiological functions such as insulin signaling (29, 36), vascular stability (13) or lipolysis (11) and in pathologies such as cancers (10-12) and infections (14-16). Small molecule inhibitors are therefore highly needed to interrogate the biology of these pathways (34), and their accurate biochemical characterization is an important step towards the identification of which target(s) they engage to yield bioactivity (56). Our family-wide inhibitory profiles of ArfGEF inhibitors will provide increased accuracy for future analysis of molecular pathways involved in diseases and help attain therapeutic goals. Ultimately, this knowledge will feed the development


22

Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

of drugs that modulate Arf and ArfGEF interactions and of other biotechnology applications (57, 58).

Table 1. Family-wide inhibition profiles of ArfGEF inhibitors. BFA SecinH3 M-COPA NAV-2729 sol. mb. sol. mb. sol. mb. sol. BRAG2 0* + 0 0 ++ ++ +++ ARNO 0* + + ++ ++ +++ + EFA6 0* 0 0 0 ++ + ++ BIG1 +++* +++ 0 0 +++ +++ + LpRalF 0 +++ + + ++ +++ + RpRalF +++ +++ 0 0 +++ +++ + Percentages of inhibition were evaluated from kobs determined without and with the inhibitor as indicated. Sol: in solution, mb: in the presence of 100 µM liposomes. 0: 40% inhibition; *: determined in (27). The percentages of inhibition can be found in the associated Table S1.

SUPPORTING INFORMATION The supporting information listed below is available free of charge. Chemical synthesis of NAV-2729; analysis of non-specific effects; representative kinetics curves for the experiments with BFA, SecinH3, M-COPA and NAV-2729; table of percentages of inhibition. (PDF) SMILES molecular formula strings. (CSV)


23

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

AUTHOR INFORMATION Corresponding Authors * [email protected] * [email protected] Present Addresses π

Present Affiliation: Pharmaceuticals and Medical Devices Agency, Shin-Kasumigaseki

Building. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was supported by grants from the Fondation pour la Recherche Médicale, the Institut National du Cancer and the CNRS to JC and from the Institut National du Cancer to RR.

ACKNOWLEDGMENT We are grateful to Lionel Duarte, Yann Ferrandez, Alexandra Joubert and Lurlène Akendengue (ENS Paris-Saclay) for protein purifications. We thank TOCRIS Bioscience for performing quality controls on SecinH3.

ABBREVIATIONS


24

Page 25 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

BFA, Brefeldin A; DCB-HUS, dimerization and cyclophilin binding-homology upstream of sec7 domain; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; FRET, Föster resonance energy tranfer; GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GTP, guanosine triphosphate; IC50, half-maximal inhibitory concentration; LpRalF, Legionella pneumophila RalF protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PH, Pleckstrin homology; PI(4,5)P2, phosphatidylinositol 4,5 bisphosphate; PS, phosphatidylserine; RNA, ribonucleic acid; RpRalF, Rickettsia prowazekii RalF protein; TGN, trans-golgi network; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Takai, Y., Sasaki, T., and Matozaki, T. (2001) Small GTP-binding proteins, Physiol Rev 81, 153-208. Wittinghofer, A., and Vetter, I. R. (2011) Structure-function relationships of the G domain, a canonical switch motif, Annu Rev Biochem 80, 943-971. Cherfils, J., and Zeghouf, M. (2013) Regulation of small GTPases by GEFs, GAPs, and GDIs, Physiol Rev 93, 269-309. 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, 842-857. Aktories, K. (2011) Bacterial protein toxins that modify host regulatory GTPases, Nat Rev Microbiol 9, 487-498. Arkin, M. R., Tang, Y., and Wells, J. A. (2014) Small-molecule inhibitors of proteinprotein interactions: progressing toward the reality, Chem Biol 21, 1102-1114. Cherfils, J., and Zeghouf, M. (2011) Chronicles of the GTPase switch, Nature chemical biology 7, 493-495. D'Souza-Schorey, C., and Chavrier, P. (2006) ARF proteins: roles in membrane traffic and beyond, Nat Rev Mol Cell Biol 7, 347-358. Donaldson, J. G., and Jackson, C. L. (2011) ARF family G proteins and their regulators: roles in membrane transport, development and disease, Nat Rev Mol Cell Biol 12, 362375. 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, 85-92.


25

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11. 12.

13.

14. 15. 16.

17. 18. 19. 20. 21. 22. 23. 24. 25.

Page 26 of 30

Singh, S. R., Zeng, X., Zhao, J., Liu, Y., Hou, G., Liu, H., and Hou, S. X. (2016) The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila, Nature 538, 109-113. 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, 889-904. 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, 252-255. 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. 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, 107-111. Wesolowski, J., Weber, M. M., Nawrotek, A., Dooley, C. A., Calderon, M., 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. Cox, R., Mason-Gamer, R. J., Jackson, C. L., and Segev, N. (2004) Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers, Mol Biol Cell 15, 1487-1505. Casanova, J. E. (2007) Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors, Traffic 8, 1476-1485. 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, 1035-1041. Goldberg, J. (1998) Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching, Cell 95, 237-248. Renault, L., Guibert, B., and Cherfils, J. (2003) Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor, Nature 426, 525-530. Nawrotek, A., Zeghouf, M., and Cherfils, J. (2016) Allosteric regulation of Arf GTPases and their GEFs at the membrane interface, Small GTPases 7, 283-296. 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. 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. 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, 275-285.


26

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

26. 27. 28.

29. 30. 31.

32.

33.

34. 35. 36. 37. 38.

39.

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, 1403-1411. Zeeh, J. C., Zeghouf, M., Grauffel, C., Guibert, B., Martin, E., Dejaegere, A., and Cherfils, J. (2006) Dual specificity of the interfacial inhibitor brefeldin a for arf proteins and sec7 domains, The Journal of biological chemistry 281, 11805-11814. 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, Proceedings of the National Academy of Sciences of the United States of America 104, 10370-10375. 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, 941-944. 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, Nature chemical biology 5, 157-165. 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. 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, 596-601. 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, The Journal of biological chemistry 287, 3885-3897. Mishev, K., Dejonghe, W., and Russinova, E. (2013) Small molecules for dissecting endomembrane trafficking: a cross-systems view, Chem Biol 20, 475-486. Jackson, C. L. (2000) Brefeldin A revealing the fundamental principles governing membrane dynamics and protein transport, Subcell Biochem 34, 233-272. Fuss, B., Becker, T., Zinke, I., and Hoch, M. (2006) The cytohesin Steppke is essential for insulin signalling in Drosophila, Nature 444, 945-948. Schlienger, S., Ramirez, R. A., and Claing, A. (2015) ARF1 regulates adhesion of MDAMB-231 invasive breast cancer cells through formation of focal adhesions, Cell Signal 27, 403-415. 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, 3895-3903. 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.


27

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40. 41.

42.

43. 44. 45.

46. 47. 48.

49.

50.

51.

Page 28 of 30

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, The Journal of biological chemistry 287, 24273-24283. 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, Proceedings of the National Academy of Sciences of the United States of America 111, 12378-12383. 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. 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, 41-56. Franco, M., Chardin, P., Chabre, M., and Paris, S. (1995) Myristoylation of ADPribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels, The Journal of biological chemistry 270, 1337-1341. 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, 36513659. 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, 1259-1272. 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, 98-104. 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. 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, The Journal of biological chemistry 282, 28834-28842. 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-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors, Mol Cell 28, 569-583. Franco, M., Peters, P. J., Boretto, J., van Donselaar, E., Neri, A., D'Souza-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.


28

Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

52. 53. 54. 55.

56. 57. 58.

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. 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, Journal of medicinal chemistry 56, 150-159. 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, 530-544. 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., O'Hagan, 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, Nature chemical biology 11, 536-541. Schurmann, M., Janning, P., Ziegler, S., and Waldmann, H. (2016) Small-Molecule Target Engagement in Cells, Cell Chem Biol 23, 435-441. Zhu, Y., Xiao, T., Lei, S., Zhou, F., and Wang, M. W. (2015) Application of chemical biology in target identification and drug discovery, Arch Pharm Res 38, 1642-1650. Milroy, L. G., Grossmann, T. N., Hennig, S., Brunsveld, L., and Ottmann, C. (2014) Modulators of protein-protein interactions, Chem Rev 114, 4695-4748.


29

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 30

Family-wide analysis of the inhibition of Arf guanine nucleotide exchange factors with small molecules: evidence for unique inhibitory profiles. Sarah Benabdi, François Peurois, Agata Nawrotek, Jahnavi Chikireddy, Tatiana Cañeque, Takao Yamori, Isamu Shiina, Yoshimi Ohashi, Shingo Dan, Raphaël Rodriguez, Jacqueline Cherfils, and Mahel Zeghouf.

For Table of Content use only


30

Family-wide analysis of the inhibition of Arf guanine nucleotide

Recommend Documents