Synthesis of LG186
Compounds referred below are shown in the schematic in Figure S1D. Compounds were dissolved in DMSO and stored at −20°C.
To a solution of cyclooctanone (10 mmol) in ethanol (10 mL) were added sulfur (320 mg, 10 mmol), ethyl cyanoacetate (1.07 mL, 10 mmol) and morpholine (875 µL, 40 mmol). The reaction mixture was stirred at 60°C for 5 h. Eight hundred and fifty-five milligrams of 1 was obtained (yield: 34%) after purification by chromatography using dichloromethane.
1H NMR (400 MHz, CDCl3)δ: 1.28 (m, 5H, CH3 + CH2), 1.39 (m, 2H, CH2), 1.50 (m, 2H, CH2), 1.56 (m, 2H, CH2), 2.54 (m, 2H, CH2), 2.75 (m, 2H, CH2), 4.21 (q, 2H, J = 7.5 Hz, CH2), 5.84 (brs, 2H, NH2). 13C NMR (100 MHz, CDCl3)δ: 14.5 (CH3), 24.6 (CH2), 25.0 (CH2), 25.9 (CH2), 26.1 (CH2), 29.2 (CH2), 31.5 (CH2), 58.8 (CH2), 105.6 (C), 119.5 (C), 134.3 (C), 160.8 (C), 164.2 (C). ES-MS m/z 254.1 (MH+).
Compound 1 was heated at 150°C in 5 mL formamide for 5 h. Upon cooling overnight, the product crystallized as slightly brownish crystals. The resulting crystals were collected and washed with a mixture of cold ethanol/water (1/1) to give the corresponding thienopyrimidone ring 2 in quantitative yield.
1H NMR (400 MHz, DMSO) δ: 1.27 (m, 2H, CH2), 1.42 (m, 2H, CH2), 1.62 (m, 4H, 2 CH2), 2.87 (m, 2H, CH2), 3.06 (m, 2H, CH2), 8.01 (s, 1H, CH), 12.28 (brs, 1H, NH). 13C NMR (100 MHz, DMSO) δ: 24.4 (CH2), 25.3 (CH2), 25.4 (CH2), 26.0 (CH2), 29.9 (CH2), 31.5 (CH2), 133.7 (C), 135.0 (C), 144.6 (C), 147.8 (C), 150.0 (CH), 157.7(C). ES-MS m/z 235.1 (MH+).
Six hundred and fifty milligrams of 2 was dissolved in hot DMF (dimethylformamide) and ice-cooled prior to the addition of 2 equivalent of POCl3. Upon stirring overnight, the product precipitated out. The white powder was collected and washed with cold water. Further addition of cold water into the mother liquor gave additional precipitate which is used straight away in the next step.
1H NMR (400 MHz, CDCl3)δ: 1.25 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.70 (m, 4H, 2 CH2), 2.92 (m, 2H, CH2), 3.12 (m, 2H, CH2), 8.67 (s, 1H, CH). 13C NMR (100 MHz, CDCl3)δ: 25.0 (CH2), 25.4 (CH2), 26.3 (CH2), 28.2 (CH2), 30.3 (CH2), 31.6 (CH2), 128.6 (C), 129.6 (C), 142.7 (C), 151.3 (CH), 156.4 (C), 158.7 (C). ES-MS (electrospray mass spectrometry) m/z 252.1 (MH+, 35Cl), 254.1 (MH+, 37Cl).
To a solution of chloride dissolved in methanol was added 10 equivalent of hydrazine monohydrate. The mixture was stirred for 2 h and water was added. The resulting precipitate was filtered off and washed with cold water to afford 255 mg of 4, with 37% yield starting from 2.
1H NMR (400 MHz, CDCl3)δ: 1.27 (m, 2H, CH2), 1.44 (m, 2H, CH2), 1.65 (m, 4H, 2 CH2), 2.50 (brs, 2H, NH2), 2.83 (m, 4H, 2 CH2), 6.54 (brs, 1H, NH), 8.41 (s, 1H, CH). 13C NMR (100 MHz, CDCl3)δ: 25.3 (CH2), 26.0 (CH2), 26.1 (CH2), 27.7 (CH2), 30.1 (CH2), 31.6 (CH2), 115.7 (C), 127.7 (C), 137.3 (C), 152.3 (CH), 158.7 (C), 164.8 (C). ES-MS m/z 249.0 (MH+), 271.0 (MNa+).
To a solution of 250 mg of 4 in methanol was added 1.2 equivalent of vanillin. The mixture was stirred for 2 h, diluted with water and extracted with dichloromethane. The organic layer was dried with MgSO4, filtered off and concentrated in vacuo. Crystallization from diethyl ether gave 100 mg of yellow crystals (yield: 26%).
1H NMR (400 MHz, DMSO) δ: 1.27 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.62 (m, 2H, CH2), 1.68 (m, 2H, CH2), 2.85 (m, 2H, CH2), 3.19 (m, 2H, CH2), 3.88 (s, 3H, CH3), 6.84 (d, 1H, J = 10. 7 Hz, CH), 7.60 (d, 1H, J = 10.7 Hz, CH), 7.79 (s, 1H, CH), 8.30 (s, 1H, CH), 9.45 (brs, 1H, OH) 11.70 (brs, 1H, NH). 13C NMR (100 MHz, DMSO) δ: 24.8 (CH2), 25.8 (CH2), 26.6 (CH2), 29.7 (CH2), 31.7 (CH2), 51.8 (CH3), 111.0 (CH), 115.4 (CH), 118.8 (C), 122.2 (CH), 126.8 (C), 126.9 (C), 133.5 (C), 135.1 (C), 143.6 (CH), 144.5 (C), 147.8 (C), 148.7 (C), 153.4 (CH). ES-MS m/z383.1 (MH+). HRMS (high resolution mass spectrometry) 393.1536, found 383.1530. Anal. (C20H22N4O2S.0.2MeOH) C, H, N.
Homology models of the Sec7 domains of the three human GEFs of interest [GBF1 (GenBank accession number NP_004184), BIG1 (NP_006412) and BIG2 (NP_006411)] were constructed based on the Arf1-GDP/BFA/ARNO [PDB entry 1RQ8, (10)] and Arf1-GDP/BFA/Gea1p [PDB entry 1RE0, (26)] complexes. Pairwise alignment of GBF1 and Gea1p gives a sequence identity of 43% and alignment of BIG1, BIG2 and ARNO gives a sequence identity of 48%. Hence, GBF1 was built on the template 1RE0, and BIG1 and BIG2 were built on the template 1RQ8.
For each model, the template structure was altered to the target sequence, loop insertions or deletions (in-dels) were rebuilt as follows. The long loop insertion in the GEA1 sequence between helices 7 and 8 (which is not resolved in the crystal structure) was rebuilt using the corresponding loop from ARNO and altered to the GBF1 sequence. Other loop in-dels were constructed using the loop-building function in InsightII. The only in-dels in the BIG1 and BIG2 models is a two-residue insertion in this region (with respect to ARNO). Of particular interest is the three-residue insertion in GBF1, with respect to the other GEFs (guanine nucleotide exchange factors), between helices 8 and 9, which is located close to both ARF1- and the BFA-binding site. This insertion was modeled as an extra turn in helix 8 and a different turn conformation. The canine GBF1 has three residue changes to the GEF domain with respect to human GBF1. Two are surface residues remote from the ARF binding interface, the third is the change M832L which prevents BFA binding. The leucine side chain is required to adopt the χ1 t, χ2 g-conformation because of steric constraints imposed by the GEF structure.
The conformations of residue side chains in the complete models were adjusted by inspection to remove bad clashes. Hydrogen atoms were added consistent with pH 7 and the model complexes soaked with a 10 Å layer of water molecules. The models were relaxed by 2000 steps of conjugate-gradient energy minimization, constraining the backbones to their original positions with harmonic restraint potential. This potential was reduced from 1000 to 0.5 kcal/Å during the minimization. The geometric quality of the models was examined using PROCHECK and found to be of similar quality to the original templates in each case.
The initial models of GBF1, BIG1 and BIG2 included BFA and Arf1 which are already present in the template structures. Ligand Exo2 was docked into the BFA site of the GBF1 complex by superimposing the Exo2 phenol group over the hydroxyl group of BFA. This maintains a hydrogen bond between the ligand and side chain of Tyr 828. Rotatable torsions in Exo2 were manipulated to allow the rest of the ligand to occupy the rest of the BFA site and causing the rest of Exo2 to project toward the extra turn in helix 8. The complex with LG186 was built in the same fashion, likewise the corresponding complexes with canine GBF1. These complexes were energy minimized using the protocol above. Models of Exo2 and LG186 with BIG1 and BIG2 were built by superimposing the protein backbones onto the corresponding minimized GBF1 complexes and transferring the ligand to the BIG structures. These non-minimized models illustrate the overlap of Exo2 and LG186 ligands with the shorter helix 8–helix 9 turn of BIG1 and BIG2.
Activated Arf pull-down
Analysis of Arf activation was done essentially as described before (41). HeLa cells were grown in 10-cm dishes and treated with the indicated chemical (at 50 µm or DMSO only) in culture medium for 1 h. After two quick washes in ice-cold PBS, cells were scraped in Buffer [200 mm NaCl; 50 mm Tris pH 7.4; 10 mm MgCl2; 1% Triton X-100; 0.1% SDS; 0.5% sodium deoxycholate; 5% glycerol; Proteases inhibitors Cocktails V (Calbiochem), pH 7.4] supplemented with the appropriate chemical. Insoluble material was pelleted by centrifugation for 10 min with 10 000 ×g at 4°C, and supernatant (about 1 mg of protein) was incubated with 50 µg of purified GST or GST-GGA2-GAT prebound on Gluthatione Sepharose for 30 min at 4°C on a rotating platform. Beads were then washed thoroughly and bound proteins were eluted in SDS–PAGE sample buffer. Samples were immunoblotted with the anti-Arf antibody 1D9 (AbCam) which recognizes all human Arf isoforms (29). The plasmid encoding for GST-GGA2-GAT (residues 157–331 of GGA2) was kindly provided by Jennifer Hirst and has been described previously (42). For Figure 2E, gels were probed using an IRDye-680CW-conjugated anti-mouse antibody (Licor) and blots scanned and quantified using a Licor Odyssey infrared imaging system (Licor).
In vitro analysis of the inhibition of Arf–GEF function
We measured the inhibitory activity of the four compounds (BFA, Exo2, GCA and LG186) toward several human Arf–GEF constructs representing three Arf–GEF families: ARNOSec7, BIG1Sec7 and BIG1DCB-HUS-Sec7, Brag2Sec7-PH, all of which were purified to high homogeneity and are efficient Arf–GEFs toward Δ17Arf1 in exchange assays in solution [(31,43); Brag2Sec7-PH to be published elsewhere]. The concentration of the inhibitors has been chosen as the highest LG186 concentration (10 µm) with no apparent aggregation. LG186 aggregation was measured at 384 nm after 1-h incubation in buffer WB (50 mm Tris pH 8, 50 mm NaCl, 2 mm MgCl2, 2 mmβ-mercaptoethanol) containing 0.2–0.4% DMSO before and after centrifugation (15 min, 20 000 ×g). The exchange activity without and with the inhibitors has been measured by tryptophan fluorescence (λEx = 292 nm, λEm = 340 nm), which monitors the conformational changes of Arf1 as it is converted from the GDP to the GTP-bound conformation. kobs (second−1) were determined from single-exponential fit of the fluorescence change. Exchange reactions were performed at 30°C, with highly purified protein in WB using a Flexstation (Molecular Devices) equipped with an eight-channel pipettor. Reaction component concentrations: Δ17Arf11 µm (soluble N-terminal truncated form), GEF 0.1 µm, compound 10 µm, DMSO 0.5%; incubation for 30 min at room temperature then for 5 min at 30°C. Reactions were started with 100 µm GTP.
Immunofluorescence and live-cell imaging
For immunofluorescence cells grown on glass coverslips were fixed either with methanol for 4 min at −20°C or with 3.5% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100 in PBS for 5 min, blocked with PBS containing 3% BSA and probed with primary antibodies, as indicated and detailed below, and Alexa-Fluor™ conjugated secondary antibodies (Invitrogen) or Cy-dye conjugated secondary antibodies (Jackson Immunoresearch). The antibodies used are as follows: polyclonal rabbit anti-COPII (Sec31A) was as previously described (44); polyclonal anti-COPI (BSTR) (45); rabbit polyclonal anti-giantin (Covance); monoclonal mouse anti-GM130 (BD Transduction Laboratories); polyclonal sheep anti-TGN46 (AbD Serotec); mouse monoclonal anti-Golgin-97 (CDF4) (Invitrogen); mouse monoclonal anti-58K Golgi protein antibody (58K-9) (AbCam); Nuclei were counterstained using DAPI (4',6-diamidino-2-phenylindole) (Invitrogen) and coverslips were mounted in Mowiol.
A plasmid encoding for TGN46-GFP was kindly provided by Vas Ponnambalam (University of Leeds, UK). The transferrin receptor in fusion with GFP (TfnR-GFP) was kindly provided by Gary Banker (Oregon Health and Science University, Oregon, USA). The TfnR-GFP cassette was excised from a pJPA5-TfnR-GFP vector using EcoRI and XbaI restriction sites and cloned into a pLVX-Puro (Clontech) using the same sites. The resulting vector (pLVX-Puro-TfnR-GFP) was verified by sequencing and used in transient overexpression.
For FRAP experiments, HeLa cells were grown on live-cell dishes and a plasmid encoding for GFP-GBF1 [kindly provided by Elizabeth Sztul (28)] transfected using Fugene6 or Lipofectamine2000 according to the manufacturer's instructions. Twenty-four to Forty-eight hours after transfection, cells were treated with chemicals in complete culture medium for 1 h. The culture medium was then supplemented by 30 mm HEPES pH 7.4 and the cells were live imaged in a 37°C heated chamber using a Perkin Elmer spinning disk confocal microscope (Ultraview ERS) with Photokinesis add-on controlled by Volocity software. Images were recorded at a rate of 1 frame per second, cells were subjected to 10 pre-bleach frames, and the recovery after bleaching was followed for 1 min. Data are presented as mean ± SD from 5 to 21 different regions of interest, from at least 3 independent transfections.
Molecular cloning and site-directed mutagenesis
To obtain the full-length human BIG1 in fusion with eGFP, a pCMV-HA-BIG1 construct (described in 46) was used as a template for PCR. A two-step procedure was used to amplify an N-terminal and a C-terminal half of BIG1 with the following primers: plus strand 5′-GGGGCTCGAGCATGTATGAGGGGAAG-3′ (XhoI, restriction sites are underlined and bold nucleotides indicates mutations introducing a single SalI restriction site) and minus 5′-CCTGAAAAGTCATGTTGGTCGACATATGCATACATGAC-3′ (SalI) for BIG1-N half; and plus strand 5′-GTCATGTATGCATATGTCGACCAACATGACTTT TCAGG-3′ (SalI) and minus 5′-GGGGGGATCCTCATTGCTTGTTTATT CCAAG-3′ (BamHI) for BIG1-C half. PCR products were cloned into pGEM-T (Promega) and BIG1-N half was cloned into pEGFP-C2 (Clontech) using XhoI and SalI sites. Then the BIG1-C half was subsequently cloned into the obtained vector using SalI and BamHI sites.
The obtained construct yields to a full-length BIG1 in fusion with an N-terminal GFP and was fully sequenced to ensure the success of the cloning. The obtained sequence corresponds to the deposited GenBank Accession number NM_006421 (BIG1).
To obtain the mutant GBF1 deleted for the NAP extra loop (residues 846–848 according to human GBF1, accession number: NP_004184), a GFP-tagged version kindly provided by E. Sztul (28) was used as a template for site-directed mutagenesis using the following sense primer: 5′-CAATGTTCGTAAACAGATGACCCT(G/C)GAGGAGTTTC GCAAAAATCTG-3′ in combination with its antisense primer. Nucleotide in brackets indicates the base substitution G2553C [according to the human open reading frame (ORF) for GBF1] producing a XhoI restriction site for simple screening of the colonies. Position of the deletion is indicated by a minus sign in brackets. The resulting construct was fully sequenced to ensure the fidelity of the PCR.