article: Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products.
  Macrocyclic natural products are a rich source of biologically active compounds, and identification of their targets can open up new opportunities for therapeutic intervention (Driggers et al. , 2008; Villar et al. , 2014). Cyclic peptide natural products, especially those containing one or more N-methyl (or N-alkyl) amino acids, comprise a privileged class of macrocycles due to their modular structure, relative ease of synthesis, intrinsic cell permeability, and constrained three-dimensional architectures (Bockus et al. , 2013; Giordanetto and Kihlberg, 2014; Hewitt et al. , 2015). Examples of cyclic peptides with well characterized cellular targets include cyclosporin (Liu et al. , 1991), trapoxin and romidepsin (Taunton et al. , 1996; Nakajima et al. , 1998), and cotransin/CAM741 and decatransin (Besemer et al. , 2005; Garrison et al. , 2005; Junne et al. , 2015). These macrocyclic compounds target conserved eukaryotic proteins that play essential roles in calcium-mediated signaling (cyclophilin/calcineurin), epigenetic regulation (histone deacetylases), and secretory protein biogenesis (Sec61 translocon), respectively. In addition to serving as drugs and potential leads for cancer and immune diseases, these cyclic peptides have proven to be useful as chemical probes, revealing new cellular roles for their target proteins, as well as mechanistic insights into the biochemical processes they orchestrate. Ternatin (1, Figure 1a) is an N-methylated cyclic heptapeptide that inhibits adipogenesis at low nanomolar concentrations (EC50 20 nM) and becomes cytotoxic at ten-fold higher concentrations (Shimokawa et al. , 2008a). Previous work established an important role for leucine-4, as substitution with alanine (ternatin-4-Ala, 2) abolished biological activity (Shimokawa et al. , 2008b). Motivating the current study, ternatin' s molecular target and mechanism of action were completely unknown. While searching for cyclic peptides that, like ternatin, incorporate a β-hydroxy leucine, we found a patent application that describes partially elucidated structures of five related cyclic heptapeptides isolated from an Aspergillus fungus in Malaysia (Blunt et al. , 2010). All five compounds were reported to be cytotoxic to cancer cells in the picomolar to low nanomolar range (IC50 0. 1–24 nM vs HCT116, MCF7, and P388 cells). One of the more potent congeners, termed ‘A3’, is shown in Figure 1a. Although only 4 out of 11 stereocenters in A3 were assigned, we were able to map the amino acid sequence, stereochemistry, and N-methylation pattern directly onto the structure of ternatin. The apparent structural similarity to ternatin was not mentioned in the patent application. 10. 7554/eLife. 10222. 003Figure 1. New ternatin variants inspired by Aspergillus-derived cyclic peptides. (a) Design of ternatin variants 3 and 4, based on the partially elucidated structure of A3. (b) Effect of cyclic peptides 1–4 (three-fold dilutions) on HCT116 cell proliferation over 72 hr. (c) Compounds 1 and 4 were tested against a panel of 21 cancer cell lines. Shown is a scatter plot comparing IC50 values of 1 and 4 for each cell line (Spearman correlation, 0. 84, p < 0. 0001). DOI: http: //dx. doi. org/10. 7554/eLife. 10222. 00310. 7554/eLife. 10222. 004Figure 1—source data 1. Antiproliferative activity of 1 and 4 against cancer cell lines. DOI: http: //dx. doi. org/10. 7554/eLife. 10222. 00410. 7554/eLife. 10222. 005Figure 1—figure supplement 1. Epimers 4 and 4b have similar antiproliferative activity. (a) Chemical structure of 4b, containing (2S, 4S) -dehydro-homoleucine. (b) Antiproliferative activity of 4 and epimer 4b toward HCT116 colorectal cancer cells (4b IC50 7. 4 ± 1. 3 nM). DOI: http: //dx. doi. org/10. 7554/eLife. 10222. 005 Based on the potential similarity between A3 and ternatin structures, we hypothesized that replacement of N-Me-Ala-6 and Leu-4 in ternatin with two A3-specific residues – pipecolic acid and dehydro-homoleucine, respectively – would be sufficient to confer increased cytotoxic potency. These considerations led to the design of cyclic peptides 3 and 4 as potential ‘north/south’ hybrids of ternatin and A3 (Figure 1a). Compound 3 differs from ternatin only at position 6, whereas 4 additionally has dehydro-homoleucine at position 4. Here, we demonstrate that ternatin and its structural variants inhibit cellular protein synthesis, with compound 4 showing the greatest potency. We further identify the elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a direct target of ternatin-related cyclic peptides. This family of N-methylated cyclic heptapeptides represents a new structural class of macrocyclic elongation factor-1A inhibitors.
  In this study, we sought to identify the target of the cytotoxic natural product, ternatin, previously characterized as a potent adipogenesis inhibitor. Inspired by the partially elucidated structure and cytotoxic activity of the cyclic heptapeptide natural product A3, we designed compounds 3 and 4, which are up to 500-fold more potent than ternatin. Ternatins inhibit protein synthesis with potencies that correlate with their ability to block cell proliferation, and photo-ternatin 5 identified eEF1A as a plausible target. Surprisingly, photo-ternatin crosslinked to purified eEF1A only in the context of its ternary complex with GTP and aminoacylated tRNA. To our knowledge, specific binding of a small molecule to the eEF1A ternary complex has not been reported previously. Whether this binding event blocks translation elongation by a mechanism analogous to the antibiotic kirromycin, as suggested by the genetic dominance of ternatin sensitivity, is an exciting question that awaits future biochemical and structural studies. Also unexpected was our discovery that two structurally unrelated natural products, didemnin and ansatrienin, compete with 5 for binding to the eEF1A ternary complex. Combined with the recent finding that nannocystin A targets domain III of eEF1A (Krastel et al. , 2015), our study suggests that four natural products with highly diverse chemical structures have evolved independently to target a shared interaction surface near Ala399 on eEF1A. A didemnin variant, plitidepsin, is currently in advanced clinical trials for multiple myeloma and T-cell lymphoma (PharmaMar Pipeline). Hence, drugging this surface on eEF1A may have clinical utility. Of the four structurally divergent natural products now identified as eEF1A inhibitors, ternatins may be particularly attractive as leads for future medicinal chemistry efforts, due to their relative structural simplicity and accessibility by total synthesis.
  HCT116 cells (ATCC, Manassas, VA) were maintained in McCoy’s 5A media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Axenia Biologix, Dixon, CA), 100 units/mL penicillin, and 100 ug/mL streptomycin (Gibco). Mutant HCT116 cells were obtained from Dominic Hoepfner (Krastel et al. , 2015). HEK293T cells (ATCC) and HeLa cells (ATCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. All other cell lines were obtained from the Cancer Cell Line Encyclopedia collection and were maintained as described (Barretina et al. , 2012). All cell lines were tested for mycoplasma (Lonza, CH) before use and were SNP-typed to verify that their identity matched that of the originally procured sample. None of the tested lines are present in the list of cross-contaminated/misidentified cell lines (http: //iclac. org/wp-content/uploads/Cross-Contaminations-v7_2. pdf). All cells were cultured at 37°C in a 5% CO2 atmosphere. Growing cells were briefly trypsinized and repeatedly pipetted to produce a homogenous cell suspension. 2,500 cells were seeded in 100 μL complete growth media per well in 96-well clear-bottom plates. After allowing cells to adhere overnight, cells were treated with 25 μL/well 5X drug stocks (0. 1% DMSO final) and incubated for 72 hr. AlamarBlue (Life Technologies, Grand Island, NY) was used to asses cell viability per the manufacturer’s instructions. Briefly, 12. 5 μL alamarBlue reagent was added to each well, and plates were incubated at 37°C. Fluorescence intensity was measured hourly to determine the linear range for each assay (Ex 545 nm, Em 590 nm, Spectra Max M5, Molecular Devices, Sunnyvale, CA). Proliferation curves were generated by first normalizing fluorescence intensity in each well to the DMSO-treated plate average. Normalized fluorescence intensity was plotted in GraphPad Prism (GraphPad, La Jolla, CA), and IC50 values calculated from nonlinear regression curves. The reported IC50 values represent the average of at least three independent determinations (±SEM). Proliferation assays with the 21-cell line panel (Figure 1c) and EEF1A1 mutant and parental HCT116 cells (Krastel et al. , 2015) were performed using an ultra-high throughput screening system (GNF Systems, San Diego, CA). For the 21-cell line panel, cells were harvested and suspended at a concentration of 50,000 cells/mL in the appropriate medium. Cells (250 cells/well, 5 μL/well) were then dispensed into Greiner (Monroe, NC) white, solid-bottom, TC-treated, 1536-well assay plates (Griener # 789173-A) and incubated for 10 hr at 37°C (95% humidity, 5% CO2). Compounds (15 nL/well, 16-point two-fold dilution series) and controls (15 nL/well, MG-132 and DMSO) were then added to the assay plates (n = 4) using an Echo acoustic liquid dispenser (Labcyte, Sunnyvale, CA). Assay plates were incubated 3 days at 37°C (95% humidity, 5% CO2) before addition of CellTiter Glo (4 μL/well, Promega, Madison, WI). Assay plates were incubated for 15 min at room temperature and then luminescence was measured using a ViewLux uHTS Microplate Imager (PerkinElmer, Waltham, MA). Reported IC50 values were calculated as described (Barretina et al. , 2012). Experiments with EEF1A1 mutant and parental HCT116 cells were conducted in the same manner as described above, except compounds were applied by 22-point two-fold dilution series (30 μM–0. 1 nM). WT and A399V eEF1A coding sequences were inserted by Gibson assembly (Gibson et al. 2009) into a pHR lentiviral vector containing an eEF1A promoter and P2A-mCherry sequence (gift of A. Weiss, UCSF). Lentiviral particles were generated as described (http: //www. broadinstitute. org/rnai/public/resources/protocols). Briefly, HEK293T packaging cells in 6-well plates were transfected with eEF1A-P2A-mCherry expression plasmid, 2nd-generation packaging plasmid pCMV-dR8. 91, and envelope plasmid pMD2. G using TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI) according to the manufacturer’s instructions. After 24 hr, media was replaced with viral harvest media (complete growth media +12 mg/mL BSA). Virus-containing media was collected after 24 and 48 hr, pooled, and used for infections without further processing. Parental and EEF1A1 A399V homozygous mutant HCT116 cells at 70% confluence in 6-cm dishes were treated with polybrene (8 μg/mL, AmericanBio, Natick, MA) and virus (0. 5 mL). After 24 hr, cells were trypsinized and passaged in complete growth media. Trypsinized cells (8. 6 × 105) were washed with PBS, resuspended in 1 mL PBS, and treated with CFSE (1 mL, 5 μM in PBS, Life Technologies). After 5 min, FBS (2 mL) was added, and cells were washed once with complete growth media. CFSE-labeled cells were plated (8 × 104 cells/well) in 6-well dishes and treated with 4 or DMSO. After 72 hr, plates were centrifuged (500 ×g, 5 min), media aspirated, and cells trypsinzied. Trypsinized cells were washed with FACS buffer (PBS –Mg/Ca + 2% FBS + 2 mM EDTA) and resuspended in the same buffer (0. 4 mL). Samples were analyzed by flow cytometry (BD LSRFortessa, BD, Franklin Lakes, NJ), with data analysis using FlowJo software (Tree Star, Ashland, OR). mCherry expression gates were defined to span a ten-fold expression range with at least 500 events for each data point. HCT116 cells at 80% confluence in 12-well plates were incubated with compounds for 5 hr at 37°C. The cell culture media was replaced with methionine- and cysteine-free DMEM with the appropriate amount of compound. 35S-methionine (PerkinElmer EasyTag, 40 μCi/well) was added and cells were incubated for 1 hr. Subsequently, media was aspirated and cells were washed twice with ice-cold phosphate-buffered saline (PBS). For samples analyzed by SDS-PAGE/autoradiography (Figure 2a), cells were lysed in buffer (PBS, 0. 5% Triton X-100, Roche complete protease inhibitor cocktail, Roche, Indianapolis, IN) and protein content was normalized by Bradford assay. Dried gels were exposed to a storage phosphor screen (Amersham Biosciences, UK) overnight and scanned with a Typhoon imager (GE Life Sciences, Pittsburgh, PA). Bulk incorporation measurements (Figure 2b) were made by treating cells with cold 10% trichloroacetic acid (TCA, 0. 5 mL) for 10 min. Precipitated monolayers were washed with 10% TCA (2 × 0. 5 mL) and solubilized in 0. 2 N NaOH (0. 5 mL). Aliquots (100 μL) were diluted into liquid scintillation fluid and radioactivity was quantified by scintillation counting. HeLa cells grown to 80% confluence in 10-cm dishes were treated with DMSO or 4 (5 μM) for 20 min (Figure 2c, left panel), or were first treated with DMSO, 4 (5 μM), or cycloheximide (CHX, 100 μg/mL, Sigma, St. Louis, MO) for 15 min, followed by harringtonine (2 μg/mL, LKT Laboratories, St. Paul, MN) for 20 min. Following treatment, dishes were placed on ice, media aspirated, and cells were washed once with ice-cold PBS. Cells were scraped into 400 μL lysis buffer (20 mM Tris-HCl pH 7. 5,150 mM NaCl, 5 mM MgCl2,1 mM DTT, 1% Triton X-100,100 μg/mL CHX). Lysates were incubated for 10 min on ice, then passed through a 26-G needle 5 times. Samples were clarified by centrifugation at 14,000 rpm for 10 min at 4°C. Lysates were applied to 10–50% sucrose gradients (prepared in 20 mM Tris-HCl pH 7. 5,150 mM NaCl, 5 mM MgCl2,1 mM DTT, 100 μg/mL CHX), followed by centrifugation at 35,000 rpm for 3 hr in a SW41 rotor at 4°C. Ribosome/polysome profiles were obtained by flowing the gradient top to bottom through an in-line spectrometer (254 nm, EM-1 Econo UV Monitor, BioRad, Hercules, CA). Lysates were prepared from HEK293T cells by washing a monolayer of cells once with ice-cold PBS and briefly freezing at –80°C. Lysis buffer (25 mM HEPES-KOH pH 7. 3,100 mM KOAc, 5 mM MgOAc2,1 mM TCEP, 1 mM PMSF, Roche EDTA-free complete protease inhibitor) was added, and cells were detached with a cell scraper. Crude lysates were clarified by centrifugation (15 min at 14,000 rpm), protein was quantified by Bradford assay, and the protein concentration was brought to 1. 5 mg/mL with lysis buffer. Lysates were processed for photo-labeling as described below. For samples treated with RNase A, RNase A (Roche) was added to lysates (10 mg/mL final concentration) and incubated for 20 min at RT. Photo-labeling experiments were preformed essentially as described (MacKinnon and Taunton, 2009; MacKinnon et al. , 2007). Reactions (100 μL) were treated with DMSO (1 μL) or competitor in DMSO as 100X stocks and incubated for 10 min at RT, then treated with photo-probe 5 and incubated for 20 min. Reactions were transferred to 96-well plates and irradiated with long-wave UV light (λmax = 355 nm, 1000 W, 90 s). To each reaction (34 μL) was added a click chemistry master mix (8. 3 μL) made from the following stock solutions: 1. 7 mM TBTA in 1: 4 DMSO/tBuOH [2. 5 μL]; 5 mM TAMRA-N3 [0. 5 μL]; 50 mM TCEP [0. 8 μL, freshly prepared]; 10% SDS [4. 5 μL]. Then, CuSO4 (0. 8 μL, 50 mM stock), was added and reactions were incubated for 1 hr at RT. Reactions were quenched with 5X Laemmli sample buffer, and separated by SDS-PAGE. Gels were scanned for TAMRA fluorescence (532 nm excitation laser, 568 nm emission measured with LPG filter) on a Typhoon FLA 9000 scanner (GE Life Sciences). HEK293T cells were transfected overnight with Flag-eEF1A DNA (pQCXIP-Flag-eEF1A1, provided by Orna Elroy-Stein [Sivan et al. , 2011]) in 6-well dishes using Lipofectamine 2000 (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. Flag-eEF1A1 A399V was produced by PCR amplification of the plasmid with the following primers: 5’-CTTGAAGTCTGGTGATGTTGCCATTGTTGATATGGTTCC-3’ 5’-GGAACCATATCAACAATGGCAACATCACCAGACTTCAAG-3’ Lysates were prepared, treated with 5 ± competitor, and photolyzed as described above. After photolysis, samples were immunoprecipitated overnight at 4°C with anti-Flag magnetic beads (Sigma). Beads were washed four times with buffer (50 mM Hepes pH 7. 5,150 mM NaCl, 0. 5% Triton X-100), then eluted (5 min, 100°C) in 1% SDS, 25 mM Hepes pH 7. 5. Eluates were subjected to click chemistry with TAMRA-N3 and processed for in-gel fluorescence scanning as described above. Gels were transferred to nitrocellulose by the semi-dry method, blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE), incubated with anti-EF1A (Millipore, DE, cat. 05–235) or anti-Flag (Sigma, cat. F3165), and detected with goat anti-mouse light chain-specific secondary antibody conjugated to 680 nm near-IR dye (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots were scanned on an Odyssey infrared imager (LI-COR Biosciences). Native eEF1A was purified from rabbit reticulocytes as described (Carvalho et al. , 1984). Bulk rabbit reticulocyte tRNA was purified as described (Merrick, 1979). tRNA was charged with phenylalanine using a crude reticulocyte tRNA synthetase preparation (Merrick, 1979) as described (Walker and Fredrick, 2008). Reactions (100 μL) were assembled by adding the following components in order: 50 μL 2X buffer (100 mM HEPES pH 7. 3,250 mM KOAc, 10 mM MgCl2), TCEP (1 μL, 100 mM), GTP or GDP (1 μL, 10 mM), ± Phe-tRNA (2 μL, ~0. 1 mM), water to 97 μL, and eEF1A to 1 μM. Samples were incubated for 20 min at RT before processing for photo-labeling as described above. Ansatrienin B was purchased from Enzo Life Sciences (Farmingdale, NY) and prepared as a 10 mM stock in DMSO. Didemnin B was obtained from the NCI/DTP chemical repository and was prepared as a 10 mM stock in DMSO.
  Materials purchased from commercial vendors were of reagent grade and used without further purification. (R) -2,5-Dihydro-3,6-dimethoxy-2-isopropylpyrazine (Schöllkopf auxiliary) was purchased from Green Chempharm Inc (Bardonia, NY). N-Boc amino acids were purchased from Chem-Impex International (Wood Dale, IL). All other reagents were purchased from Sigma-Aldrich unless otherwise noted. (S) -Boc-photo-leucine was prepared from Boc-4,5-dehydro-Leu-OH (Chem-Impex Intl.) as described (MacKinnon et al. , 2007). (2R, 3R) -ethyl 2-amino-3-hydroxy-4-methylpentanoate (Shimokawa et al. , 2007) (11, Figure 8), tripeptide 17 (Figure 8) (Shimokawa et al. , 2009), and iodides 6a and 6b (Nakamura and Mori, 2000; Smith et al. , 1996) were prepared as described. Water and air sensitive reactions were conducted in flame-dried glassware under an inert argon atmosphere. Dry solvents were prepared on a Glass Contour (Laguna Beach, CA) system, which dispenses solvents through either alumina or activated molecular sieve columns. 1H and 13C NMR spectra were obtained on a Varian (Palo Alto, CA) Inova 400 MHz spectrometer, a Bruker (Billerica, MA) Avance DRX500 spectrometer equipped with a QCI-Cryoprobe, or a Bruker AvanceIII 600 spectrometer, and referenced to the residual solvent peak. Spectral data are reported as follows: chemical shift (in ppm), integration, multiplicity (br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; or as a combination of these), and coupling constant (s) in Hertz. LC-MS analysis was performed on a Waters (Milford, MA) Acquity LCT UPLC equipped with a TUV detector (monitored at 215 nm) and a Waters Acquity UPLC 1. 7 μm C-18 column, eluting with a water/MeCN (+0. 1% formic acid) gradient at 0. 6 mL/min. Purification by reverse-phase HPLC was performed on either a Varian ProStar 210 purification system with a UV-Vis detector (model 345) monitoring at 215 nm, or a Waters binary gradient purification system (model 2545) with a photodiode array detector (model 2998). Two dehydro-homolecuine diastereomers were prepared according to the scheme in Figure 7. 10. 7554/eLife. 10222. 015Figure 7. Synthesis of (2S, 4R) - and (2S, 4S) -methyl 2-amino-4-methylhex-5-enoate (dehydro-homoleucine). Reagents and conditions: (a) nBuLi, THF, –78°C; (b) TBAF, THF, 0°C; (c) Dess-Martin periodinane, CH2Cl2; (d) MePPh3Br, nBuLi, THF, 0°C; (e) TFA, H2O/MeCN. DOI: http: //dx. doi. org/10. 7554/eLife. 10222. 015 Ternatin (1) and ternatin-4-Ala (2) were synthesized as described, and all spectral data were in accordance with those reported (Shimokawa et al. , 2008b). Cyclic peptides 3,4, 4b, and 5, were synthesized by the same route as ternatin (Figure 8). 10. 7554/eLife. 10222. 016Figure 8. Synthesis of ternatin variants. Reagents and conditions: (a) HATU, DIPEA, DCM/DMF; (b) 2 M HCl, MeOH, 30°C; (c) 1 N LiOH, H2O/THF; (d) HATU, DIPEA, DMF. DOI: http: //dx. doi. org/10. 7554/eLife. 10222. 016
summary: Many plants, fungi, and bacteria have evolved to produce small molecules that have powerful effects on the cells of other living organisms, and can even kill them. These naturally produced compounds are often used as starting points for developing new drugs. One such class of compounds are the cyclic peptides, which can be relatively easily produced in the laboratory and are able to penetrate cells. Some cyclic peptides have also proved to be useful for treating cancer and immune diseases, so researchers are keen to identify others that have similar effects. One promising prospect, called ternatin, is produced by several species of fungi. In high doses, ternatin can kill mammalian cells, but it was not clear how it does so. To learn more, Carelli et al. searched a chemical database for cyclic peptides related to ternatin and identified several similar compounds that were reported to kill cancer cells. Inspired by the structures of these cyclic peptides, Carelli et al. synthesized modified versions of ternatin. One of these was 500 times more potent than ternatin, which means a much lower dose of the compound is still able to kill cancer cells. Further experiments showed that ternatin blocks the production of new proteins in cells. Specifically, ternatin binds to a complex that includes a protein called elongation factor-1A (eEF1A). Mutations in a particular region of eEF1A prevent ternatin from killing cells, suggesting a potential binding site for ternatin. The next challenge is to dissect the mechanism by which compounds binding to this site on eEF1A block protein synthesis and kill cells. A related challenge is to understand why certain cancer cells are hypersensitive to ternatin and other eEF1A inhibitors, while other cancer cells are relatively resistant. These questions are relevant to the development of eEF1A inhibitors as cancer treatments.
keywords: biochemistry and chemical biology
  cell biology
title: Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex