Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

The reaction mixture was cooled to room temperature, and then filtered through a pad of Celite using Ethyl Acetate (50 mL) as the eluent and the filtrate concentrated. The residue was purified by column chromatography (silica, EtOAc-hexanes, 1: 4) gave desired compound (1.4 g, 91%) as a white solid. To a solution of this (1.3 g, 5.1 mmol) in AcOH (20 mL) was added HNO3 (5 mL) at 0 o C. The reaction mixture was stirred at the same temperature for 12 h at which point TLC (silica, 1:3 EtOAc-hexanes) showed the reaction was complete. The reaction mixture was diluted with Ice-Water (60 mL), and then filtered, washed with Ice-Water (20 mL). The precipitated product was recrystallized from ethanol to yield product (1.1g, 75%) as a yellow solid. The yellow solid (1.1 g, 4.3 mol) was slowly added to TFA (15 mL) at 0 o C for 1 h, and then the result mixture was stirred at room temperature overnight at which point TLC (silica, 1:3 EtOAc-hexanes) showed the reaction was complete. The reaction mixture was diluted with Ice-Water (60 mL), and then filtered, washed with Ice-Water (20 mL). The precipitated product was purified by column chromatography (silica, EtOAc-hexanes, 1:4 → 1:1) gave compound S3 (590 mg, 69%) as a white solid. 1
The reaction mixture was stirred at room temperature 0.5 h. At this point, TLC (silica, 3:1 EtOAchexanes) showed the reaction was complete. The reaction mixture was quenched with saturated NaHCO3 (10 mL), and the aqueous layer was extracted with Ethyl Acetate (20 mL), washed with brine (10 mL).
The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (silica, EtOAc) gave desired compound S11 (152 mg, 92%) as a yellow oil. 1
The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (silica, EtOAc-hexanes, 1: 1) gave desired compound S12 (150 mg, 81%) as a yellow oil. 1

Syntheses of Negative Controls 8 and 9
Compound S18.
The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (silica, EtOAc-hexanes, 1: 1) gave desired compound S22 (150 mg, 81%) as a yellow oil. 1

General Methods
All reagents were purchased from commercial sources listed below and were used without further was determined. GraphPad Prism 7 software was used to make a plot of % viability versus sample concentration and to calculate the concentration at which a compound exhibited 50% cytotoxicity (IC50).

Activity Based Protein Profiling
MDA-MB-231 cells were seeded in five groups of 6 cm petri dishes with 3 ml DMEM high glucose culture and incubated at 37 º C and 5% CO2 until 90% confluency. Each group was treated with fresh medium containing 20 µM competitor or an equal volume of DMSO vehicle for 30 min at 37 º C and 5% The mixed solution was added into the previously prepared protein solution containing 0.25 mg protein.
The total volume was adjusted to 100 µl with the lysis buffer 1 to make a final protein concentration of 2.5 mg/ml, and the click reaction was performed at room temperature for 1.5 h. Proteins in 15 µl of the reactant were fractionated by an SDS-PAGE gel. The protein gel was scanned with a typhoon scanner (Typhoon™ FLA 9500) at 532 nm excitation with 700 mV gain setting and then stained with Coomassie blue.

Biotin Affinity Pulldown
Live Cell-based Pulldown According to the experimental needs, MDA-MB-231 cells were seeded into 3-7 groups of 15-cm tissueculture dishes and incubated at 37 º C and 5% CO2 until 90% confluency. Each group was treated with fresh medium containing a competitor or an equal volume of DMSO vehicle for 30 min at 37 º C and 5% CO2, followed by addition of a probe or an equal volume of DMSO. After 1 h incubation at 37 º C and 5% CO2, the cells were harvested by scraping and washed with cold PBS for 3 times and lysed with the lysis buffer 2 (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 10 mM NaF, 10 mM β-glycerophosphate, and 1 mM Na3VO4). The supernatant of each group was collected after centrifugation at 4 º C and 21,100 g for 10 min, and the protein concentration was quantified with the BCA kit. After adjusting protein concentration with lysis buffer 2, each protein solution with equal amount of total protein in the same volume was incubated with 20 µl of washed streptavidin bead at 4 º C for 2 h with S21 rotation. After incubation, the beads were washed with washing buffer (50 mM Tris, pH 7.4, 150mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 10 mM NaF, 10 mM β-glycerophosphate, and 1 mM Na3VO4) for 3 times and boiled at 95º C for 5 min in 2x sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 2% β-mercaptoethanol and 0.02% bromophenol blue). After centrifugation at 21,100 g and 4 º C for 1 min, the cleared eluted proteins were divided into two parts.

Delayed/Reverse Competition in Live Cell-based Pulldown
The procedure is almost identical to that of the regular pulldown except for the switched order of addition for the biotin probe and ipomoeassin F.

Mass Spectrometry (MS) Analysis and MS Data Processing
In the pulldown, the bound proteins were eluted via boiling the high capacity streptavidin agarose beads in 1x sample buffer. The eluted proteins were fractionated with a 12% SDS-PAGE and the fractionated proteins were visualized by blue silver staining. 8  identifications were accepted if they could achieve an FDR of <1.0% and contained at least 2 identified peptides. Total spectral counts were exported from Scaffold and used to represent relative protein abundance from different samples based on the positive, linear correlation between spectral count and the abundance of a protein in complex samples. [11][12][13] Contaminant proteins such as keratins were discarded.
For reliable representation of protein abundance, proteins identified with <4 spectra in the probe 7enriched sample were discarded. 12

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MDA-MB-231 cells were seeded on fibronectin-coated coverslips at 70% confluency in a 48-well plate and cultured at 37 º C and 5% CO2. After 24 h, culture medium was replaced with fresh medium containing a 20 µM competitor or an equal volume of DMSO vehicle for 30 min at 37 º C and 5% CO2, followed by addition of 0.2 µM (final concentration) fluorescent probe 12. After 1 h incubation at 37 º C and 5% CO2, the coverslips were mounted onto a glass slide with a mounting medium with or without 0.5 µg/ml DAPI.
The fluorescent images were acquired with a confocal microscope (Leica TCS SP5) using 405 nm excitation and 562 nm excitation. For the ER co-localization studies, 1 uM ER-Tracker™ Blue-White DPX was added to the medium after 1 h incubation with probe 12. After 5 min incubation, the coverslips were mounted onto a glass slide and the images were acquired as described above.

Photo-Affinity Labeling.
CT7 photo-affinity labeling and click chemistry were done as previously described. 14 Briefly, SRMs (sheep rough microsomes, isolated as described in reference 15 ) containing 100 nM Sec61 were treated with 10 µM ipomoeassin F/mycolactone or 1% DMSO for 30 min at 0°C, followed by incubation with 100 nM CT7 for 10 min RT. Samples were then photolyzed for 10 min and crosslinked proteins were detected by click chemistry, SDS-PAGE, and in-gel fluorescent scanning (Typhoon Trio, GE Healthcare).

Statistical Analysis
Quantitative analysis of Ii membrane insertion was performed using AIDA v.5.0 (Raytest Isotopenmeβgeräte) whereby the ratio of the signal intensity for the 2Gly/0Gly forms was used as a readout to estimate efficiency of integration into the ER membrane. This value was then expressed relative to the matched DMSO control in order to derive the mean relative ER insertion from three independent experiments (n=3). Statistical significance of the effectiveness of Ipom-F analogues (RM one-way ANOVA) was determined using Tukey's multiple comparisons test. IC50 value estimates were determined using non-linear regression to fit data to a curve of variable slope (four parameters) using the least squares fitting method in which the bottom and top plateaus of the curve were defined as 0% and 100% respectively. The differences between the estimated IC50 values for the three compounds analysed were statistically significant (****) according to the extra-sum-of-squares F test. Statistical analyses and IC50 estimates were performed using Prism 7.0d (GraphPad) with statistical significance given as n.s., nonsignificant and ****, P < 0.0001.

SH-SY5Y GLuc Secretion and Cell Viability Assays.
SH-SY5Y human neuroblastoma cells stably expressing a constitutively secreted variant of Gaussia luciferase (GLuc-'No Tag') were previously described. 19 Cells were cultured in DMEM (4.5 g/L D- Luminescence was measured using a ViewLux microplate imager equipped with clear filters.
For cell viability assays, cells were cultured, plated, and treated with test compounds as outlined for the secretion assays. 48 h after treatment, viability was assessed using a CellTiter-Glo assay by adding 3

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µL of reagent to each well, incubating 15 min at room temperature, and reading luminescence using a ViewLux microplate imager.

Metabolic Labelling Assays in HCT116 Cells
HCT116 cells were counted using an automated cell counter (BioRad TC20) immediately prior to each experiment. 3 ml of HCT116 cells at a density of 0.166x10 6 cells/mL were seeded to 6-well plates (0.5x10 6 cells/well) and then incubated at 37 º C and 5% CO2 for 24 h. The compounds were dissolved in DMSO to make drug stocks (10 mM). The stock solutions were diluted with DMEM high glucose culture medium lacking methionine and cysteine to make a fresh working solution immediately prior to each test. Cells were washed twice and 1ml of fresh DMEM lacking methionine and cysteine was added to each well.
Subsequently, the cells were treated with 250 µl of compound solutions in a total volume of 1,250 µl/well.
The plate was then incubated at 37 º C and 5% CO2 for 30 min before 100 µCi 35 S-labelled methionine/cysteine was added (Expre 35 s 35 s protein labelling mix). Plates were incubated for a further 30 min, then media collected and cells harvested by scraping after four washes in ice-cold PBS. Total protein was acquired by RIPA extraction from the cell pellet, cytoplasmic fraction was acquired by partial permeabilization of the outer cell membranes with 0.15% Digitonin followed by centrifugation, secreted proteins were acquired by TCA precipitation of the media.

Pulse-Chase Analysis of Protein Secretion in HepG2 Cells
The secretion assay was performed using a pulse-chase approach as described previously. 20

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of precipitated proteins in the media and clarified lysates were analyzed by SDS-PAGE and phosphorimaging (FLA-3000; Fuji).

T Cell Assays
Jurkat E6.1 (ATCC TIB-152TM) T cells were cultured in RPMI GlutamaxTM (Life Technologies), supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen) and penicillin/streptomycin (100 U/ml, 100 µg/ml). Cells were treated with Ipomoeassin F or mycolactone for 24 h, then pelleted and stained with FITC-conjugated anti-human CD62L (clone DREG-56 (BioLegend #304804) at 500 ng/ml) during 40 min at 4°C. Fluorescence signals were acquired on an Accuri CD (BD) and analyzed with the FlowJo X software. Immunosuppressive activity on T cells was measured through the inhibition of IL-2 production by Jurkat T cells incubated with mycolactone for 1 h prior to 24 h of activation with phytohemagglutinin-ionomycin, as described. 21 In both assays, blanks corresponded to cells treated with the vehicle volume of the highest concentration of sample tested (DMSO for IpoF; EtOH for mycolactone).

Washout assay
The reversibility of the inhibition of secreted protein production by ipomoeassin F was determined by following the production of TNF in RAW264.7 cells as described before. 22 Briefly, RAW264.7 cells were maintained at 37 ⁰C and 5% CO2 in high glucose DMEM (Merck), supplemented with 10% heatinactivated FBS (GIBCO). Cells were plated into 24 well plates at a concentration of 1 x 10 5 /ml 24 hour prior to assay, then incubated for 1 hour in the presence or absence of 125 ng/ml mycolactone, 250 nM Ipomoeassin F or DMSO carrier. Cells were either stimulated directly with 100 ng/ml TLR-grade LPS (Enzo) or washed 3 times with medium and incubated 24 hour, then stimulated with LPS. In each case, cells were incubated with LPS for 4 hour then the supernatants were removed. The concentration of TNF in supernatants was quantified by ELISA (eBioscience).

Figures:
Cell Cytotoxicity Assay A. B.

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In Vitro Membrane Insertion Assays of a Model Type II Protein.