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`of the nine mutants exhibited an editing defect
`based on their sensitivity to the structurally re-
`lated noncognate amino acid norvaline (fig. S1).
`These results suggest that the editing pocket of
`Cdc60p is the binding site for AN2690.
`To delineate its mode of action, we inves-
`tigated the effect of AN2690 on the ability of
`LeuRS to hydrolyze mischarged tRNALeu. Ad-
`dition of AN2690 to the posttransfer editing
`assay inhibited the hydrolysis of Ile-tRNALeu in
`a dose-dependent manner (Fig. 1B). In addition,
`we found that AN2690 inhibited tRNA amino-
`acylation (fig. S2A), and, as would be expected
`for a LeuRS inhibitor, it blocked protein syn-
`thesis in vivo (fig. S2B). Initial aminoacylation
`experiments also revealed that AN2690 required
`the presence of tRNA for effective inhibition of
`aminoacylation activity. Kinetic analysis of
`aminoacylation inhibition showed that AN2690
`acted as a noncompetitive inhibitor with respect
`to both adenosine triphosphate (ATP) and leu-
`cine (fig. S3, A and B). Analysis of the noncom-
`petitive nature of AN2690 revealed that
`the
`inhibition constant (Ki) decreased on increas-
`ing AN2690’s incubation time with tRNA and
`Cdc60p, before initiating the aminoacylation
`reaction with ATP. When enzyme and tRNA
`were incubated with AN2690 for 2 min, the Ki
`was 31.4 ± 2.8 (SEM) mM, whereas after a 20-
`min incubation the Ki decreased to 1.85 ± 0.1
`mM (fig. S3). To better understand this
`process, we measured inhibition of amino-
`acylation as a function of incubation time and
`AN2690 concentration (Fig. 2A). We found a
`direct linear relationship between the observed
`rates of inactivation (kobs) and AN2690 concen-
`trations, with no apparent plateau even at the
`highest concentration tested (fig. S4). From
`these data, we deduced a rate of inactivation of
`−1
`the enzyme (kinactivation) of 0.66 ± 0.10 min
`
`An Antifungal Agent Inhibits an
`Aminoacyl-tRNA Synthetase by
`Trapping tRNA in the Editing Site
`Fernando L. Rock,1* Weimin Mao,1* Anya Yaremchuk,2,3 Mikhail Tukalo,2,3
`Thibaut Crépin,2 Huchen Zhou,1,4 Yong-Kang Zhang,1 Vincent Hernandez,1
`Tsutomu Akama,1 Stephen J. Baker,1 Jacob J. Plattner,1 Lucy Shapiro,5
`Susan A. Martinis,6 Stephen J. Benkovic,7 Stephen Cusack,2 M. R. K. Alley1†
`
`Aminoacyl–transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino
`acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial
`drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-
`benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast
`cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNALeu-AN2690 adduct in the editing
`site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2′-
`and 3′-oxygen atoms of tRNA’s 3′-terminal adenosine. The trapping of enzyme-bound tRNALeu in
`the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNALeu and
`consequentially blocking protein synthesis. This result establishes the editing site as a bona fide
`target for aminoacyl-tRNA synthetase inhibitors.
`
`induced AN2690-resistant mutants in the yeast
`Saccharomyces cerevisiae (7). These genetically
`dominant mutants were 32- to 512-fold more re-
`sistant to AN2690 than the parental S. cerevisiae
`strain (table S2), and their resistance mutations
`were found to lie in the CDC60 gene, which en-
`codes the cytoplasmic LeuRS (Cdc60p). Further-
`more, all AN2690-resistant mutations mapped
`to the editing domain (Fig. 1A and table S2)
`and all but two, Cys326 → Arg326 (C326R) and
`Cys326 → Phe326 (C326F) (8), to the two highly
`conserved regions that form the editing active
`site of LeuRS (9). Four mutations lie in the
`threonine-rich region, a locus known in bacterial
`LeuRS homologs to be involved in binding and
`hydrolyzing mischarged tRNAs (9–12). Seven
`
`Aminoacyl-tRNA synthetases (AARSs)
`
`perform a pivotal role in translating the
`genetic code by catalyzing the attach-
`ment of the correct amino acid to its cognate
`tRNA (1). The aminoacylation reaction occurs
`in two steps: the formation of an enzyme-bound
`aminoacyl-adenylate, followed by transfer of this
`activated amino acid to either the 2′- or 3′-hydroxy
`group on the 3′-terminal adenosine of tRNA. The
`accuracy of the tRNA aminoacylation reaction is
`critical to ensuring the fidelity of the genetic code
`(2). To achieve this accuracy, many AARS en-
`zymes possess a proofreading (editing) mechanism
`that hydrolyzes tRNAs aminoacylated with the in-
`correct amino acid (3). Leucyl-tRNA synthetase
`(LeuRS) is a proofreading AARS, which pos-
`sesses distinct synthetic (aminoacylation) and edit-
`ing active sites separated by more than 30 Å (4, 5).
`We show that 5-fluoro-1,3-dihydro-1-hydroxy-
`2,1-benzoxaborole (AN2690) inhibits LeuRS by
`trapping tRNALeu in the editing active site.
`AN2690 is a member of a new class of
`broad-spectrum antifungals (table S1), the ben-
`zoxaboroles, which have an unusual chemical
`attribute: a boron atom (6). We isolated sponta-
`neous and ethyl-methanesulfonate (EMS)–
`
`1Anacor Pharmaceuticals, Incorporated, 1060 East Meadow
`Circle, Palo Alto, CA 94303, USA. 2European Molecular Biol-
`ogy Laboratory, Grenoble Outstation 6 rue Jules Horowitz,
`BP181, 38042 Grenoble Cedex 9, France. 3Institute of Mo-
`lecular Biology and Genetics, National Academy of Science
`(NAS) of Ukraine, 252627 Kiev, 3143, Ukraine. 4School of
`Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan
`Road, Shanghai 200240, China. 5Department of Develop-
`mental Biology, Beckman Center, Stanford University
`School of Medicine, Stanford, CA 94305, USA. 6Depart-
`ment of Biochemistry, University of Illinois, Urbana,
`IL
`61801–3732, USA. 7Department of Chemistry, Pennsylva-
`nia State University, University Park, PA 16802, USA.
`*These authors contributed equally to this work.
`†To whom correspondence should be addressed. E-mail:
`dalley@anacor.com
`
`Fig. 1. (A) S. cerevisiae AN2690 resistance mutations in the editing active site of Cdc60p (8). Align-
`ment of the conserved regions of the LeuRS editing domains from S. cerevisiae (Sce) from
`CAA97865, Pyrococcus horikoshii (Pho) from O58698, Escherichia coli (Eco) from AAC73743, and T.
`thermophilus (Tth) from BAD69984. The amino acid substitutions that confer resistance in S.
`cerevisiae to AN2690 are in black (table S2). (B) AN2690 inhibits posttransfer editing. Deacylation
`of total brewer’s yeast tRNA mischarged with isoleucine, no enzyme control (circles), enzyme control
`(squares), Cdc60p treated with 6 mM AN2690 (triangles), and enzyme treated with 66 mM AN2690
`(asterisks). All reactions were performed in triplicate, and the mean values were plotted. The SD for
`each point was less than 4%.
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`sistent with AN2690 being characterized as a
`slow-tight-binding inhibitor.
`Equilibrium dialysis demonstrated that
`adenosine-containing ribonucleotides were essen-
`tial for measurable binding of AN2690 to
`Cdc60p (fig. S6A). To determine more precisely
`the mode of action of AN2690, we obtained a
`3.5 Å crystallographic structure of Thermus
`thermophilus LeuRS complexed with tRNALeu
`and AN2690 (Fig. 3A). Significant positive
`difference density found in the editing site (fig.
`S7) was interpreted as a tRNA-AN2690 adduct
`with the boron from the oxaborole ring bound to
`the 2′,3′-hydroxy groups on the 3′-terminal
`adenosine (A76). A 1.85 Å resolution structure
`of an adenosine monophosphate (AMP)–
`AN2690 adduct bound in the editing site of
`LeuRS (Fig. 3B) confirmed this tetrahedral
`spiroborate structure, whose configuration is
`stabilized by two hydrogen bonds to the
`conserved threonine-rich peptide and a water
`molecule (Fig. 3C). These results show that AMP
`the 3′-terminal
`can act as a surrogate for
`adenosine of tRNALeu, as in our direct binding
`assay, and that AN2690 occupies the noncognate
`amino acid–binding pocket in the editing site.
`The adenosine and the 2′-hydroxy group of
`the AMP-AN2690 adduct can be exactly over-
`laid on the analogous groups of a posttransfer
`editing substrate analog, 2′-(L-norvalyl) amino-
`2′-deoxyadenosine (Nva2aa), bound in the edit-
`ing site (9). However, the planar benzoxaborole
`only partially overlaps with the noncognate amino
`acid (Fig. 3D). In particular, AN2690 has no
`equivalent of the amino acid amino group, and
`the absence of any associated strong interactions
`with the enzyme probably explains why the
`compound has very low affinity for the editing
`site in the absence of AMP or tRNA.
`Because benzoxaboroles can bind to the cis-
`diols of sugars (13, 14), we tested the require-
`ment for adenosine’s 2′- and 3′-hydroxy groups
`in AN2690 binding to Cdc60p and found that
`both hydroxy groups were required (fig. S6C).
`This implies that AN2690 can only form an
`
`REPORTS
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`(fig. S5). Measurement of the rate of recovery
`demonstrated that AN2690’s inactivation of
`Cdc60p was reversible, albeit with a very slow
`
`
`−4reactivation rate of 1.64 × 10−4 ± 0.15 × 10
`
`
`min−1 (Fig. 2B), which corresponds to a half-life
`of 424 min. These kinetic properties are con-
`
`Fig. 2. (A) The rate of Cdc60p inactivation by AN2690. The percentage of inhibition from different
`concentrations [3.3 mM (squares), 5 mM (triangles), 6.6 mM (asterisk), 9.9 mM (open diamonds), and
`16.5 mM (circles)] of AN2690 were plotted versus the time of incubation. All reactions were performed
`in triplicate, and the mean values were plotted. (B) The recovery of Cdc60p activity from AN2690 inhibi-
`tion. All reactions were performed in triplicate, and the mean values were plotted. The data were fitted to a
`first-order exponential decay.
`
`Fig. 3. AN2690 forms an adduct with the terminal adenosine (A76) of tRNALeu in the editing active site
`of LeuRS. (A) Overall structure of the complex of T. thermophilus LeuRS with tRNALeu and AN2690,
`showing the adenosine-AN2690 adduct (ball-and-stick model, ringed in red) in the editing site and
`leucine (space-filling model) in the synthetic site. The editing domain is cyan; the catalytic domain, yellow;
`Zn-1 domain, purple; the leucyl-specific insertion domain, black; the anticodon-binding domain, red; the
`C-terminal domain, gold; zinc atoms, gray spheres; and tRNA, blue tube. (B) Unbiased difference map
`(1.85 Å resolution) for the AMP-AN2690 adduct in the editing site. (C) Diagram showing water molecules
`(dark blue spheres) and hydrogen bonds (green dotted lines) between editing site residues of LeuRS and
`the AMP-AN2690 adduct (orange). Amino acid residues that are mutated in the S. cerevisiae AN2690-
`resistant mutants are labeled and colored in purple (table S2). The atoms are colored accordingly: boron,
`mauve; fluorine, green; oxygen, red; nitrogen, light blue; carbon, yellow; and phosphate, purple. (D)
`Superposition of bound posttransfer editing substrate analog (Nva2AA, brown) (9) and the AMP-AN2690
`adduct (orange) obtained after superposing the Ca positions of the editing domain of each complex.
`
`Fig. 4. Boron and the oxaborole ring are re-
`quired for inhibition of aminoacylation. All reac-
`tions were performed in triplicate, and the mean
`values were used to determine a median inhib-
`itory concentration (IC50) with Prism 4 (17).
`
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`adduct with nonaminoacylated tRNA: The free
`3′ end of the bound tRNA must sample the
`editing site before aminoacylation for AN2690
`to inhibit LeuRS (fig. S8). This is the
`configuration observed in the T. thermophilus
`LeuRS-tRNALeu crystal structure (15).
`The requirement for boron and the oxaborole
`ring in aminoacylation inhibition was tested
`by using various AN2690 analogs (Fig. 4). Ex-
`panding the oxaborole ring to form a ben-
`zoxaborin, compound A, adversely affected
`its ability to inhibit the aminoacylation reac-
`tion (Fig. 4). Because the ring-opened boronic
`acids are known also to bind to tRNA (16),
`we tested compounds B, C, and D. Each was
`found to be inactive, further demonstrating the
`importance of the oxaborole ring (Fig. 4).
`Lastly, the compounds E and F, where boron is
`replaced with a sp2 or sp3 carbon, were inactive,
`showing that boron cannot simply be replaced
`by carbon (Fig. 4).
`This work demonstrates that AN2690 is a
`highly specific inhibitor of LeuRS and that the
`boron atom in the oxaborole ring is essential for
`AN2690’s unique mechanism of inhibition. With
`appropriate compounds, the oxaborole tRNA
`
`trapping (OBORT) mechanism could be used
`to inhibit other AARS enzymes that perform
`posttransfer editing. Therefore, the incorpora-
`tion of a boron atom into rationally designed
`enzyme inhibitors represents a promising ap-
`proach to the discovery of new classes of
`therapeutic agents.
`
`References and Notes
`1. M. Ibba, D. Soll, Annu. Rev. Biochem. 69, 617 (2000).
`2. T. L. Hendrickson, P. Schimmel, in Transfer RNA-Dependent
`Amino Acid Discrimination by Aminoacyl-tRNA Synthetase,
`J. P. D. Lapointe, L. Brakier-Gingras, Eds., Translation
`mechanisms (Landes Bioscience/Eurekah.com, Austin, TX,
`2003), pp. 34–64.
`3. P. Schimmel, E. Schmidt, Trends Biochem. Sci. 20,
`1 (1995).
`4. S. Cusack, A. Yaremchuk, M. Tukalo, EMBO J. 19, 2351
`(2000).
`5. R. Fukunaga, S. Yokoyama, J. Mol. Biol. 346, 57
`(2005).
`6. S. J. Baker et al., J. Med. Chem. 49, 4447 (2006).
`7. Materials and methods are available on Science Online.
`8. Single-letter abbreviations for the amino acid residues
`are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe;
`G, Gly; H, His; I, Ile; L, Leu; M, Met; N, Asn; P, Pro;
`Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
`9. T. L. Lincecum Jr. et al., Mol. Cell 11, 951 (2003).
`10. Y. Zhai, S. A. Martinis, Biochemistry 44, 15437
`(2005).
`
`11. M. G. Xu, J. Li, X. Du, E. D. Wang, Biochem. Biophys. Res.
`Commun. 318, 11 (2004).
`12. R. S. Mursinna, T. L. Lincecum Jr., S. A. Martinis,
`Biochemistry 40, 5376 (2001).
`13. M. Dowlut, D. G. Hall, J. Am. Chem. Soc. 128, 4226
`(2006).
`14. A. Ricardo et al., J. Org. Chem. 71, 9503 (2006).
`15. M. Tukalo, A. Yaremchuk, R. Fukunaga, S. Yokoyama,
`S. Cusack, Nat. Struct. Mol. Biol. 12, 923 (2005).
`16. T. F. McCutchan, P. T. Gilham, D. Soll, Nucleic Acids Res.
`2, 853 (1975).
`17. GraphPad, GraphPad Software, Incorporated.
`18. We thank M. Kully and J. Khan at NAEJA Pharmaceuticals,
`Incorporated, for their contribution to the minimal
`inhibitory concentrations in table S1. Part of this work
`was funded by an NIH grant awarded
`to J.J.P. (R01 DE16835). The coordinates for LeuRSTT:
`tRNA:AN2690 and LeuRSTT:AMP:AN2690 have been
`deposited in the Protein Data Bank as 2V0G and 2V0C.
`S.J.B. and L.S. are co-founders and members of the
`board of Anacor Pharmaceuticals, Incorporated. S.C.
`and S.A.M. have received honoraria and/or consulting
`fees from Anacor Pharmaceuticals, Incorporated.
`Supporting Online Material
`www.sciencemag.org/cgi/content/full/316/5832/1759/DC1
`Materials and Methods
`Figs. S1 to S9
`Tables S1 to S3
`References
`6 March 2007; accepted 23 May 2007
`10.1126/science.1142189
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`An Antifungal Agent Inhibits an Aminoacyl-tRNA Synthetase
`by Trapping tRNA in the Editing Site
`Fernando L. Rock, Weimin Mao, Anya Yaremchuk, Mikhail
`Tukalo, Thibaut Crépin, Huchen Zhou, Yong-Kang Zhang, Vincent
`Hernandez, Tsutomu Akama, Stephen J. Baker, Jacob J. Plattner,
`Lucy Shapiro, Susan A. Martinis, Stephen J. Benkovic, Stephen
`Cusack and M. R. K. Alley (June 22, 2007)
`316
` (5832), 1759-1761. [doi: 10.1126/science.1142189]
`Science 
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