Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex
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
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 .
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 .