From the Departments of Basic Animal Science Research
and § Medicinal Chemistry, Merck Research Laboratories,
Rahway, New Jersey 07065
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ABSTRACT |
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Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.
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INTRODUCTION |
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The elongation phase of translation in fungi requires the soluble
elongation factors EF1, EF2, and EF3. EF1
and EF2 are members of
the GTPase superfamily of proteins and are characterized by common
structural motifs and their ability to alternate between conformational
states in response to binding GDP or GTP. These proteins are required
for translation in all eukaryotes, while EF3 is unique to fungi and
essential for fungal protein synthesis (1). EF2 catalyzes the
translocation of the ribosome along messenger RNA, presumably by
stimulating a gross rearrangement of the ribosome that results in
peptidyl-tRNA transfer and the movement of mRNA by one codon. The
protein sequence of EF2 has been highly conserved throughout evolution,
with Saccharomyces cerevisiae EF2 sharing 66% identity and
85% homology to human EF2. Despite this high degree of similarity, a
class of tetracyclic diterpene glycoside natural products, the
sordarins, has now been identified as selective inhibitors of EF2
function in fungal protein synthesis. Sordarin, produced by species of
the fungal genus Sordaria, was described as an antifungal
agent in 1970 (2, 3), but the mode of action of this family has not
been examined until now. In this report, we show that sordarins
specifically bind to the S. cerevisiae EF2-ribosome complex
and block protein synthesis by inhibiting the release of EF2 from the
post-translocational ribosome. Our observations show that it is
possible to inhibit fungal EF2 specifically, which may provide an
opportunity for developing antifungal agents with a unique and
selective mechanism of action.
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EXPERIMENTAL PROCEDURES |
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Sordarin was isolated essentially as described for Sordaria arenosa (2). Reticulocyte and wheat germ lysates were obtained from Promega.
Assays-- IC50 values were determined from growth inhibition assays in which cells were inoculated at 2 × 105 cells/ml in YPAD medium (4) containing sordarin serially diluted 2-fold from 0.2-100 µg/ml, followed by incubation at 29 °C for approximately 16 h. S30 extracts and translation factors EF1, EF2, and EF3 were prepared from S. cerevisiae cells harvested in mid-to-late logarithmic phase (A600 ~2). Translation assays using S30 extracts were performed at 22 °C essentially as described in Ref. 5 with poly(U) (Sigma) at 160 µg/ml as message and [3H]phenylalanine as precursor during the linear period of incorporation. Translation factors were purified essentially as described by Skogerson (6) except for the substitution of a Mono Q cartridge (Pharmacia Biotech Inc.) for DEAE Sephadex. Yeast EF2 was nearly homogeneous by SDS-gel electrophoresis and silver staining and had a final specific activity in the diphtheria catalyzed ADP-ribosylation assay (6) of 2.5 pmol/µg. Rat liver EF1 and EF2 were prepared as described in (7) and the wheat germ proteins as in Ref. 8. Ribosomes were prepared from all three sources by sedimentation three times through 0.5 M KCl, 20% sucrose, 10 mM MgCl2 cushions and concentrations estimated using the figure of 18.6 pmol/A260 unit. In vitro translation assays with purified ribosomes and translation factors were performed as described for S. cerevisiae (7) except that [3H]Phe-tRNA was prepared as precursor at a specific activity of 2000 dpm/pmol.
[3H]L-793,422 (Fig. 1) was prepared at 20 mCi/mg (8000 mCi/mmol) and a concentration of 0.004 mg/ml by the Drug Metabolism Department at Merck Research Laboratories. Each binding assay contained 2 µg of yeast S-30, 25 µM GTPYeast Strains and Plasmids--
The sordarin-resistant strains
sR1 and sR2 were generated by selecting spontaneous mutants of the
parental strain YPH98 (MATa ade2 leu2 lys2 trp1
ura3) (9) on SC medium (4) containing 5 µg/ml sordarin. The
haploid S. cerevisiae strain YEFD12h/ pURA3-EFT1 (MATa ade2 lys2 ura3 his3 leu2 trp1 eft11:HIS3
eft2
:TRP1), deleted for the genomic copies of EFT1
and EFT2, and harboring the plasmid pURA3-EFT1 for
viability, has been described (10). Strain YEFD12h/YCpEFT2 was
generated by transforming YEFD12h/ pURA3-EFT1 to leucine prototrophy
with plasmid YCpEFT2, followed by eviction of the plasmid pURA3-EFT1.
Strains deleted for either EFT1 or EFT2 were
constructed by mating YEFD12h/pURA3-EFT1 with the strain YPH54
(MAT
ade2 his3 lys2 trp1 ura3) (9) to obtain spores with
the genotype EFT1/eft2
:TRP1 or EFT2/eft1
1:HIS3. The
EFT2 yeast expression plasmid YCpEFT2 was constructed by
subcloning a 5-kilobase pair BamHI-PstI fragment
of DNA that includes the native promoter and the entire EF2 coding
region into plasmid Ycplac111 (11).
Molecular Mapping of EF2
Mutations--
Plasmid-dependent sordarin-resistant
mutants were spontaneously generated from the
eft1/eft2
deletion strain YEFD12h harboring an
episomal copy of EFT1 (pEFT1URA) or EFT2
(YCpEFT2). 1 × 107 and 1 × 108
yeast cells were plated on SC medium containing 2% agar and sordarin at 5 µg/ml and incubated at 29 °C until colonies appeared (3-7 days). Plasmid DNA was recovered and transformed into the
Escherichia coli strain DH5
(Life Technologies, Inc.) by
established methods (4, 12). Plasmid DNA was prepared from E. coli using Qiaprep spin columns (Qiagen) and transformed into the
yeast strains YEFD12h/pEFT1URA or YEFD12h/ YCpEFT2 to demonstrate
plasmid-dependent resistance to sordarin. Plasmids that
conferred resistance were sequenced with an ABI Prism 373 DNA sequencer
according to the manufacturer's recommendations (Applied Biosystems).
Sequences were analyzed using Sequencher DNA analysis software (Gene
Codes Corp.). The mutations in the sR1 and sR2 strains were identified
by sequencing the PCR products. Based on the locations of the plasmid
mutations, specific regions of approximately 0.4 kilobase pairs were
amplified from genomic DNA using Klentaq (CLONTECH)
according to the manufacturer's instructions. Following initial
identification of a mutation, five additional independent PCR reactions
were performed and the products sequenced to rule out the possibility
of errors due to misincorporation.
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RESULTS AND DISCUSSION |
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Sordarin as a Selective Inhibitor of Fungal Protein
Synthesis--
Exposure of intact S. cerevisiae to low
concentrations of sordarin rapidly inhibits the incorporation of
[3H]threonine into proteins while having no effect on
incorporation of uracil into RNA or adenine into
DNA.2 The IC50
for this effect (6 µg/ml) corresponds closely to the IC50
values for growth inhibition of wild-type strains in YPAD medium. In
contrast, sordarin has no effect on HeLa protein synthesis (leucine
incorporation) or cell proliferation at concentrations of 100
µg/ml.2 In vitro translation experiments using
S30 extracts of S. cerevisiae and a poly(U) message revealed
that sordarin inhibits phenylalanine incorporation (IC50
30 ng/ml) while having no effect on similar systems from rabbit
reticulocyte or wheat germ at levels of up to 100 µg/ml.2
Based on these findings it appears that sordarin is a specific inhibitor of protein synthesis in fungi while having no effect on other
eukaryotic systems, a specificity within eukaryotes that is without
precedent for known translation inhibitors.
Sordarin Binding Assay--
Hydrolytic cleavage of sordarin
to sordarose and sordaricin abolishes activity both for inhibition of
whole cell growth and for in vitro translation. However
activity is restored by replacement of the sugar with a short chain
alkyl ether. L-793,422 (Fig.
1A), a sordaricin derivative
with an isobutyl ether side chain, is 1000-fold more active in growth
inhibition assays than sordarin (IC50 of 6 ng/ml) with the
wild-type strain. A tritiated analog (2,3-ditrito-2-methyl propyl
ether) of specific radioactivity high enough for a sensitive binding
assay was prepared. With the addition of both Mg2+ and GTP,
or the nonhydrolyzable analog GTPS or GDP, to desalted S30 extracts,
binding of [3H]L-793,422 was associated with a complex
excluded by filtration through Sephadex G-75. Binding of
[3H]L-793,422 is displaced by cold sordarin with an
IC50 of 3 ng/ml. A Scatchard plot of binding indicated a
single binding site with a Kd of <3 nM.
S30 extracts of wild-type yeast bound a maximum of 80 pmol/mg of
protein.
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Fungal Specificity Conferred by EF2-- The ability of EF2 and ribosomes purified from rat liver and from wheat germ to bind [3H]-L793,422 in the presence of GTP was examined. In contrast to S. cerevisiae EF2, neither rat liver nor wheat germ EF2 shows any binding when tested with ribosomes from rat liver, wheat germ, or yeast. Nor does binding occur using S. cerevisiae ribosomes and either higher eukaryotic EF2. However, substantial binding is conferred by S. cerevisiae EF2 upon addition of ribosomes from either rat liver or wheat germ (Fig. 1A). Thus EF2 is the determinant of the observed fungal specificity of sordarin. Furthermore, fungal specificity in polymerization can be demonstrated in reconstituted translation assays dependent on added purified EF2. Polyphenylalanine formation from [3H]Phe-tRNA as precursor by rat liver ribosomes plus rat liver EF1 is insensitive to sordarin when rat liver EF2 is used, but sensitive with yeast EF2. Incorporation by yeast ribosomes plus yeast EF1 and EF3 is sensitive with yeast EF2, reconstituting the sensitivity of the unfractionated system, but becomes insensitive when rat liver EF2 is used (Fig. 1B).
Stabilization of the EF2-Ribosome Complex-- During the translocation cycle, GTP is bound by the EF2-ribosome complex, followed by an extremely rapid hydrolysis to EF2-GDP, and a conformational change that releases EF2 for the next round of translocation (13). Fusidic acid is a universal EFG and EF2 inhibitor that inhibits translocation by stabilizing the EF2-GDP-ribosome complex (14). Fusidic acid stabilization was demonstrated for the S. cerevisiae complex by measuring cold excess GTP exchange with prebound ring-labeled [3H]GTP to EF2 plus ribosomes. In similar experiments sordarin increases the half-life of the GDP-EF2-ribosome complex (Fig. 2), from less than 0.5 min to approximately 6 min (t1/2 for fusidic acid ~10 min). Sordarin analogs that show little or no effect in vivo fail to cause stabilization in vitro. Fusidic acid levels up to 5 mM fail to compete with the binding of [3H]L-793,422 to the S. cerevisiae EF2-ribosome complex. Thus the universal binding site for fusidic acid appears not to overlap the fungal specific sordarin binding site.
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Genetic Linkage of Sordarin Resistance--
Genetic confirmation
that fungal EF2 confers specificity to sordarin was demonstrated by
analysis of mutant yeast strains resistant to sordarin. Spontaneous
mutants resistant to sordarin were selected on SC medium containing 5 µg/ml sordarin, and these appeared at a frequency of approximately
1 × 108. Progeny from crosses of the
sordarin-resistant strains sR1 and sR2 with strain YEFT2Dh
(eft2
:TRP1) that is deleted for the genomic copy of the
EFT2 gene were analyzed. Dissection of 20 tetrads from each
cross showed that sordarin resistance was never associated with
tryptophan prototrophy, confirming genetic linkage, and that a mutant
allele of eft2 is responsible for sordarin resistance in
both sR1 and sR2 strains.
Identification of Mutations That Confer Sordarin
Resistance--
To facilitate determining which substitutions can
result in sordarin resistance, mutants conferring
plasmid-dependent resistance were spontaneously generated
by plating cells on solid medium containing sordarin, using
eft1/eft2
deletion strains that harbor an episomal
copy of EFT1 or EFT2. Fifteen mutant strains were chosen that range in resistance from having an IC50
10 µg/ml sordarin, to an IC50
100 µg/ml (Table
I). Plasmids conferring sordarin
resistance were recovered, clonally purified, and sequenced. A single
base change causing an amino acid substitution was identified in each
clone, with the exception of pSR7, which has a three base pair
deletion, resulting in the loss of G790 (Table I). Based on the
locations of the mutations in the episomal copies of the EFT1 and EFT2 genes, we used PCR to amplify
specific regions of EFT2 from genomic DNA prepared from the
original sR1 and sR2 mutants. Single base changes that result in amino
acid substitutions in each mutant were identified. The amino acid
changes conferring resistance to sordarin are clustered into three
regions of the EF2 protein (Fig. 3).
Amino acid sequence alignment of EF2 with its prokaryotic counterpart
EFG demonstrates that the EF2 substitutions are located in regions with
homology to domains I, III, and IV in EFG. These domains of EFG are
thought to interact with one another during GTP hydrolysis and
translocation (15-17). The sequence alignment also revealed that
mutations in EF2 conferring resistance to sordarin are located in
proximity to mutations in EFG that give rise to fusidic acid resistance
(18, 19) (Fig. 3). Since fusidic acid does not permeate yeast cells,
translational sensitivity to fusidic acid was determined in S30
extracts prepared from each sordarin-resistant mutant. Many mutations
that confer resistance to sordarin also confer resistance to fusidic
acid (Table I), although levels of resistance to the two drugs are not
closely correlated. The partial cross resistance of mutations and the proximity of mutations that confer sordarin and fusidic acid resistance in the alignment of EF2 with EFG support the biochemical evidence of
mechanistic similarity of the two drugs. One or more of the EF2
residues altered in the resistant mutants may be located in the
sordarin binding site, but there is not sufficient evidence, without
cross-linking or other biochemical approaches, and a three-dimensional structure for EF2, to identify the binding site.
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ACKNOWLEDGEMENTS |
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We thank Guy Harris, Wendy Clapp-Shapiro, Gabe Dezeny, Anne Dombrowski, Francisca Vicente, and Angela Basilio of Natural Product Drug Discovery for fermentation and isolation of sordarin; Stuart Hayden and Allen Jones of Drug Metabolism for preparation of labeled L-793,422; Karen Carniol of Wesleyan University for technical assistance as a Merck summer intern; Michael Amendola and Ralph Mosley of Molecular Design and Diversity for modeling EF2 upon EFG; and Michael Metzger of Human Genetics for DNA sequencing. We particularly thank James Bodley of the University of Minnesota for yeast strains and helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 732-594-6799; Fax: 732-594-1399; E-mail: jennifer_nielsen_kahn{at}merck.com.
1
The abbreviations used are: GTPS, guanosine
5
-3-O-(thio)triphosphate; PCR, polymerase chain
reaction.
2 J. Nielsen, unpublished results.
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REFERENCES |
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