`
`
`
`
`
`COVER
`
`DEPARTMENTS
`
`
`
`Volume 316, Issue 5832
`
`A female Aedes aegypti mosquito attempts
`to take flight after a blood meal. The complete
`sequencing of this disease vector is reported
`on page 1718, with an accompanying
`Perspective on page 1703.
`Photo: James Gathany/CDC
`
`Science Online
`1663
`This Week in Science
`1665
`Editors’ Choice
`1670
`1672 Contact 5Ci€nC€
`1675 R3"d°m 5amPl€5
`1677 Newsmakers
`1769
`Science Careers
`
`
`
`
`
`
`
`EDITORIAL
`
`1669
`
`Fixing the Drug Laws
`by Donald Kennedy
`
`NEWS OF THE WEEK
`
`Elephants Take Center Ring at CITES
`Corals: Suffering From Whiplash
`
`No lifeline for Proposed Breast Cancer
`Prevention Trial
`
`Osaka University Researchers Reject Demand
`to Retract Science Paper
`SCIENCESCOPE
`
`Gene-Synthesis Companies ]oin Forces '
`to Self-Regulate
`U.S. National Medals: For Men Only?
`Science Editor-in-Chief to Retire
`
`NEWS FOCUS
`The Last of the Leviathans
`On Life Support
`
`(an the Bald Eagle Still Soar After It is Delisted?
`
`Population Geneticists Move Beyond the Single Gene
`
`Eongress Splits Over Plan to Consolidate
`Intelligence Research
`
`1678
`
`1679
`
`168-1
`
`1681
`
`1682
`
`1683
`
`1683
`
`1684
`
`1 689
`
`1 690
`
`1 693
`
`
`
`LETTERS
`
`The Utility of Standardized Tests
`M. Lerdau and C. Avery; B. Brown; 1. L. Sherley
`Response N. R. Kuncel and S. A. Hezlett
`CORRECTIONS AND CLARIFICATIONS
`
`BOOKS ETAL.
`
`Theoretical Ecology Principles and Applications
`R. M. May and A. R. McLean, Eds.,
`reviewed by S. A. Levin
`
`Ecology and Evolution of Flowers
`L. D. Harder and S. C. H. Barrett, Eds.,
`reviewed by B. 3. Simpson
`
`POLICY FORUM
`
`Taking Science Out of the Box—Foresight Recast
`D. A. King and S. M. Thomas
`
`PERSPECTIVES
`
`A Breakthrough for Global Public Health
`D." D. Chadee et al. >> Research Article p. 1718
`
`Resolving an Elusive Structure
`R. L. Penn >> Report p. 1726
`
`Is There Glue in Cuprate Superconductors?
`P. W. Anderson
`
`Making Energy Count
`F. F. Crim >> Report p. 1723
`
`Reassessing Carbon Sinks
`D. E Baker >> Reports pp. 1732 and 1735
`
`NuclearActin as Choreographer of Cell Morphology
`and Transcription
`]. I. Wu and G. R. Crabtree » Report p. 1749
`
`Birth Order and Intelligence
`F. ]. Sulloway >> Brevia p. 1717
`
`1 694
`
`1 698
`
`1 699
`
`1700
`
`1701
`
`1 703
`
`1 704
`
`1705
`
`1707
`
`1708
`
`1710
`
`1711
`
`www.sciencemag.org
`
`SCIENCE VOL316
`
`22 JUNE 2007
`
`CFAD V. Anacor, |PR2015-01780 ANACOR EX. 2051 - 2/7
`1657
`
`CONTENTS continued >>
`
`
`
`CFAD v. Anacor, IPR2015-01780 ANACOR EX. 2051 - 2/7
`
`
`
`CONTENTS
`
`. Cience
`
`IENCE EXPRESS
`
`.sciencexpress.org
`
`UCAT|ON'FORUM:_Empowering Green Chemists in Ethiopia
`Asfaw, P. Licence, I Engida, M. Poliakoff
`10.1126/science.1144439
`
`LICY FORUM: Willingness to Donate Frozen Embryos for
`m Cell Research
`D. Lyerly and R. R. Faden
`
`10.1126/science.1145067
`
`LL BIOLOGY
`
`I uin 2 Inhibitors Rescue on-Synuclein—Mediated Toxicity
`Models of Parkinson's Disease
`F. Outeiro et al.
`- inhibitor of a microtubule deacetylase can rescue dopamine-containing cells
`I: Drosophila from the toxicity of a protein aggregate associated with Parkinson's
`sease.
`
`10.1126/science.1143780
`
`
`
`ECHNICAL COMMENT ABSTRACTS
`EUROSCIENCE
`
`mment on "Tequila, a Neurotrypsin Ortholog,
`egulates Long-Term Memory Formation in
`rosophila"
`Sonderegger and L. Patthy
`1 text at wwmsciencemag. org/cgi/content/full/316/5332/1698b
`
`1 698
`
`sponse to Comment on "Tequila, a Neurotrypsin
`rtholog, Regulates Long-Term Memory Formation
`' Drosophila"
`*Preat et al.
`
`OCEAN SCIENCE
`
`Free-Drifting Icebergs: Hot Spots of Chemical and Biological Enrichment
`in the Weddell Sea
`K. L. Smith Jr. et al.
`Trace elements and iron released from free-drifting Antarctic icebergs stimulate
`local productivity that enhances carbon sequestration in the Southern Ocean.
`10.1126/science.1142834
`
`PHYSICS
`
`Single-Atom Single-Photon Quantum Interface
`72 Wilk, S. C. Webster, A. Kuhn, G. Rempe
`A sequence of laser pulses targeted on a single atom trapped in a cavity can
`generate a source of entangled photon pairs.
`
`10.1126/science.1143835
`
`BREVIA
`PSYCHOLOGY
`
`Explaining the Relation Between Birth Order
`and Intelligence
`P. Kristensen and T. Bjerkedal
`The tendency for first-born children to have higher I05 can be
`explained by social interaction within the family rather than by
`biological effects in utero. >> Perspective p. 1711
`
`1717
`
`RESEARCH ARTICLE
`GENETICS
`.
`
`Genome Sequence of Aedes aegypti, a Major
`Arbovirus Vector
`V. Nene et al.
`The genome of the mosquito that carries dengue and yellow fever
`consists of almost 50 percent transposable elements and over 15,000
`protein-coding genes. >> Perspective p. 1703; Report p. 1738
`
`1718
`
`REPORTS
`CHEMISTRY
`
`1723
`
`Do Vibrational Excitations of CHD3 Preferentially
`Promote Reactivity Toward the Chlorine Atom?
`S. Yan, Y.-72 Wu, B. Zhang, X.-I-'. Yue, K. Liu
`Precisely controlled molecular collision experiments unexpectedly
`reveal that translational energy can promote reactivity as effectively
`as vibrational energy. >> Perspective p. 1707
`GEOCHEMISTRY
`
`1726
`
`The Structure of Ferrihydrite, a Nanocrystalline
`Material
`F. M. Michel et al.
`Analysis of x-ray scattering data reveals the crystal structure
`of ferrihydrite, a ubiquitous nanometer-sized iron phase,
`and shows that it is a single compound, not a mixture.
`>> Perspective p. 1704
`
`
`
`I
`
`text at wwmsciencemag.org/cgr7conten1/full/316/5332/1698c
`EVIEW
`3- OLOGY
`
`i[
`
`rent Problems'in the Management of
`rine Fisheries
`
`I R. Beddington, D. ]. Agnew, C. W. Clark
`
`1713
`
`1704 &
`1 726
`
`CFAD V. Anacor, |PR2015-01780 ANACOR EX. 2051 - 3/7
`
`CONTENTS continued >$
`
`www.sciencemag.org
`
`SCIENCE VOL316
`
`22 JUNE 2007
`
`1659
`
`CFAD v. Anacor, IPR2015-01780 ANACOR EX. 2051 - 3/7
`
`
`
`
`
`
`
`
`
`
`
`CONTENTS-
`
`1749
`
`Nuclear Actin Regulates Dynamic Subcellular
`Localization and Activity of the SRF Cofactor MAL
`M. K. Vartiainen; S; Guettler, B. Larijani, R. Treisman
`in cultured cells, serum regulates the interaction of nuclear actin
`with a transcriptional coactivator, facilitating its nuclear transport
`and thus stimulating gene expression. >>- Perspective p. 1710
`CELL BIOLOGY
`
`1753
`
`Quantitative Morphological Signatures Define
`Local Signaling Networks Regulating Cell Morphology
`C. Bakal, J. Aach, G. Church, N. Perrimon
`A large genetic screen and automated characterization of cell
`morphology allowed mapping of the pathways controlling
`cell adhesion and membrane protrusion and tension.
`VIROLOGY
`
`1756
`
`Restriction of an Extinct Retrovirus by the Human
`TRlM5oc Antiviral Protein
`“
`A
`S. M. Kaiser, H. S.Ma1i/<, M. Emerman
`Resistance that humans acquired 4 million years ago to a
`now extinct virus that infected chimpanzees and gorillas
`may have left us more sensitive to HIV infection today.
`MOLECULAR BIOLOGY
`
`An Antifungal Agent Inhibits an Aminoacyl-tRNA
`Synthetase by Trapping tRNA in the Editing Site
`I-'. L. Rock et al.
`A boron-containing antifungal drug forms an adduct with oxygen
`atoms in the tRNA, inhibiting attachment of the amino acid to the
`tRNA and blocking protein synthesis.
`
`1759
`
`
`
`
`CELL BIOLOGY
`
`
`
`
`
`
`
`
`
`
`
`
`1729
`
`1732
`
`
`
`
`
` EEPORTS CONTINUED...
`OPHYSICS
`
`
`ormation of (Mg,Fe)SiO3 Post-Perovskite
`D” Anisotropy
`
`I Merkel et al.
`
`!- formation of a deep mantle mineral is accommodated
`
`dominately by slip along certain planes in the crystal,
`
`laining seismic signatures at the core mantle boundary.
`
`
`5 IMATE CHANGE
`ak Northern and Strong Tropical Land Carbon
`
`
`take from Vertical Profiles of Atmospheric C02
`
`‘ B. Stephens et al.
`
`ospheric models that account for the vertical distribution of
`
`in the atmosphere imply that Northern Hemisphere ecosystems
`up less carbon than previously thought.
`, Perspective p. 1708
`
`CEAN SCIENCE
`
`
`turation of the Southern Ocean C02 Sink Due to
`
`cent Climate Change‘
`
`. Le 0ue'ré et al.
`eamount of C02 taken up by the Southern Ocean, a major
`nk, has decreased since 1981, despite the continued increase
`t
`atmospheric CO2 levels.
`> Perspective p. 1708
`
`1735
`
`E OLUTION
`Evolutionary Dynamics of immune-Related Genes
`ind Pathways in Disease-Vector Mosquitoes
`.M. Waterhouse et al.
`
`1738
`
`mparison among the genomes of Drosophila and two mosquito
`' ecies reveals that stages of the insect innate immune response
`olved via distinct mechanisms and rates.
`Research Article p. 1718
`COLOGY
`
`1743
`
`ulling Prey Promotes Predator Recovery—
`
`glternative States in a Whole-Lake Experiment
`ii". Persson et al.
`‘
`collapsed freshwater fishery was shown to recover when a prey
`ecies was culled, causing it to go into reproductive compensation
`nd produce prey of an edible size for the predator.
`COLOGY
`
`1746
`
`fluence of Phylogeny on Fungal Community
`sembly and Ecosystem Functioning
`.Maherali and]. N. Klironomos
`Communities of soil fungi contain a larger number of species
`§nd are more productive when the founding species are more
`distantly related.
`IcIe
`
`‘
`
`‘
`
`SCIENCE (ISSN 0036-8015) is published rreeirly on Friday, except the last noel in December, by the American Association
`tortheMvancement ogscience, 1200 NewVorlr Avenue, NW, Washington, DC 20005. Periodicals Mail postage (publication No.
`484460) paid at Washington, DC. and additional mailing offices. Copyright O 2007 by the American Association for the Advancement
`of Science. The title SCIENCE is a registered trademark of the AAAS. Domestic individual membership and subscription (51 issues): 5142
`(574 allocated to subscription). Domestic institutional subscription (51 issues): 5710; Foreign postage extra: Mexico, Caribbean (surface
`I
`mail) 555; other countries (air assist delivery) 585. First class, airmail, student, and emeritus rates on request. Canadian rates with GST
`;DVA N CI N G S C I E N C E . S E RV I N G S 0 CI E T Y
`available upon request, GST #1254 88122. Publications Mail Agreement Number 1069624. Printed in the U.S.A.
`thange ofaddress:Allow 4 weeks, giving old and new addresses and 8—digit account number. Postmaster. Send change ofaddress toMAS, P.0. Box 96178, Washington, DC 20090-6178. Single-copy sales:
`510.00 current issue, $15.00 back Issue prepaid includes surface postage; bulk rates on request. Authorization to photocopy material for internal or personal use under circumstances not falling within the
`Mr use provisions of the Copyright Act is granted by AAAS to libraries and other users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that 518.00 per article is
`beid directlyto CCC, 222 Rosewood Drive, Danvers, MA01923. The identification code forScienceis 0036-8075.Scienceis indexed in the Reader's Guide to PeriodicalLiteratureand in several specialized indexes.
`If
`”
`*
`
`CFAD V. Anacor, |PR2015-01780 ANACOR EX. 2051 - 4/7
`www.sciencemag.org
`SCIENCE VOL 316
`22 JUNE 2007
`1661
`
`.
`
`CONTENTS continued >>
`
`CFAD v. Anacor, IPR2015-01780 ANACOR EX. 2051 - 4/7
`
`
`
` This material may be protected by Copyright law [Title 17 u.s. Code)
`
`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,“ Yong-Kang Zhang,1 Vincent Hernandez,1
`Tsutomu Akama,1 Stephen ]. Baker,‘ Jacob J. Plattner,1 Lucy Shapiro,5
`Susan A. Martinis,‘ Stephen ]. Benkovic,7 Stephen Cusack,2 M. R. K. Alleylt
`
`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 tRNALe“-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 tRNA”“ in
`the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyt—tRNAL°” and
`consequentially blocking protein synthesis. This result establishes the editing site as a bona fide
`target for aminoacyl-tRNA Synthetase inhibitors.
`
`minoacyl-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 anrinoacylation 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 arninoacylated with the in-
`correct amino acid (3). Leucyl-tRNA synthetase
`(LeuRS)
`is a proofieading AARS, which pos-
`sesses distinct synthetic (aminoacylation) and edit-
`ing active sites separated by more than 30 A (4, 5).
`We show that 5-fluoro-1,3-dihydro-1-hydroxy-
`2,1-benzoxaborole (AN2690) inhibits LeuRS by
`tapping tRNAL°“ in the editing active site.
`AN2690 is a member of a new class of
`
`lbroad-spectrum antifiingals (table S1), the ben-
`toxaboroles, which have an unusual chemical
`attribute: a boron atom (6). We isolated sponta-
`neous and ethyl-methanesulfonate (EMS)-
`
`Incorporated, 1060 East Meadow
`‘Anacor Pharmaceuticals,
`(ircle, Palo Alto, CA 94303, USA. 2European Molecular Biol-
`ogy Laboratory, Grenoble Outstation 6 rue ]ules Horowitz,
`IPIB1, 38042 Grenoble Cedex 9, France. slnstitute of Mo-
`hcular Biology and Genetics, National Academy of Science
`MAS) of Ukraine, 252627 Kiev, 3143, Ukraine. ‘School 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. °Depart-
`;ment of Biochemistry, University of
`Illinois, Urbana,
`IL
`31801-3732, USA. 7Department of Chemistry, Pennsylva-
`fftia State University, University Park, PA 16802, USA.
`"lhese authors contributed equally to this work.
`-‘Ho whom correspondence should be addressed. E—mail:
`,ilalley@anacor.com
`
`
`
`REPORTS
`
`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 tRNAL”“. Ad-
`dition of AN2690 to the posttransfer editing
`assay inhibited the hydrolysis of Ile-tRNAL°“ in
`a dose-dependent manner (Fig. 1B). In addition,
`we found that AN2690 inhibited tRNA arnino-
`
`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 etfective 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 i 2.8 (SEM) uM, whereas after a 20-
`min incubation the K decreased to 1.85 3: 0.1
`uM (fig. S3). To better understand this
`process, we measured inhibition of arnino-
`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
`the enzyme (kinactivm-ion) of 0.66 i 0.10 min
`
`
`
`
`
`No Enzyme
`
`66 ptM AN2690
`
`6 uM AN2690
`
`0 uM AN2690
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`Time (minutes)
`
`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 th6 CDC60 gene, which en-
`codes the cytoplasmic I_euRS (Cdc60p). Further-
`more, all AN2690-resistant mutations mapped
`to the editing domain (Fig. 1A and table S2)
`and all but two, Cys326 —» Argm (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 (942). Seven
`
`"Wag $ “me
`
`QTCC
`See 3t4TLRP
`VT
`Pho 229'I‘LRP
`FMGCT
`Eco 24-yrlfizs
`Tth
`247T
`P -oar
`“Threonine—Rich"
`Region
`
`__ 80
`g
`;' 60
`3
`
`40
`
`<Z
`
`0
`
`mnagtm 5I
`rcvp NSPDD
`g 20
`P
`APE-‘D
`P
`P
`
`see
`Pho
`Eco
`Tth
`
`40
`31
`331
`3
`
`G
`G
`GA
`G
`
`GTG
`Region
`
`Catalytic
`Region
`
`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 058698, 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 52). (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 pM AN2690 (triangles), and enzyme treated with 66 M AN2690
`(asterisks). All reactions were performed in triplicate, and the mean values were plotted. The SD for
`each point was less than 4%.
`
`www.sciencemag.org
`
`SCIENCE
`
`CFAD V. Anacor, |PR20’|5-01780 ANACOR EX. 2051 - 5/7
`VOL 316
`22 JUNE 2007
`1759
`
`CFAD v. Anacor, IPR2015-01780 ANACOR EX. 2051 - 5/7
`
`
`
`REPORTS
`
`reactivation rate of 1.64 X 10-4 i 0.15 X 10"4
`(fig. S5). Measurement of the rate of recovery
`demonstrated that AN2690’s inactivation of min‘! (Fig. 2B), which corresponds to a half-life
`Cdc60p was reversible, albeit with a very slow
`of 424 min. These kinetic properties are con-
`
`A
`
`1 .5 txM AN2690
`
`E
`
` 8AminoacytationInhibition(Va) 03
`
`
`
`
`
`8
`
`
`
`
`
`AminoacylationInhibition(°/o)o888
`
`15
`10
`Time (minutes)
`
`20
`
`25
`
`250
`
`500
`
`0
`
`750 1000 1250 1500
`Time (minutes)
`
`sistent with AN2690 being characterized as w
`slow-tight-binding inhibitor.
`T
`Equilibrium dialysis demonstrated I=Ii
`adenosine-containing ribonucleotides were es '-:4.
`tial
`for measurable binding of AN2690 :1
`Cdc60p (fig. S6A). To determine more prec‘ - ’
`the mode of action of AN2690, we obtained ,
`3.5 A crystallographic structure of Therm
`thermophilus LeuRS complexed with tRNA
`and AN2690 (Fig. 3A). Significant positivé
`difference density found in the editing site (fig
`S7) was interpreted as a tRNA-AN2690 add i.
`with the boron fiom the oxaborole ring boundb
`the 2',3’-hydroxy groups on the 3’-terminal,‘
`adenosine (A76). A 1.85 A resolution structure
`of an adenosine monophosphate (AMP)J
`AN2690 adduct bound in the editing site I:
`LeuRS (Fig. 3B) confirmed this tetrahedrd
`spiroborate structure, whose configuration it’
`stabilized by two hydrogen bonds to tilt:
`conserved threonine-rich peptide and a wate"
`molecule (Fig. 3C). These results show that AME
`can act as a surrogate for the 3’-terminal?
`adenosine of tRNAL°“, as in our direct binding
`assay, and that AN2690 occupies the noncognatoi
`amino acid—binding pocket in the editing site:
`The adenosine and the 2’-hydroxy group
`the AMP-AN2690 adduct can be exactly oven-
`laid on the analogous groups of a posttransfe=
`editing substrate analog, 2’-(L-norvalyl) amino?
`2'-deoxyadenosine (Nva2aa), bound in the edit.
`ing site (9). However, the planar benzoxabomls i
`only partially overlaps with the nonoognate
`acid (Fig. 3D). In particular, AN2690 has In
`equivalent of the amino acid amino group, all:
`the absence of any associated strong inter-actiom’,
`with the enzyme probably explains why tin
`compound has very low aflinity for the editing‘
`site in the absence of AMP or tRNA.
`.f
`
`Because benzoxaboroles can bind to the
`diols of sugars (13, 14), we tested the require-
`ment for adenosine’s 2'- and 3'-hydroxy groupt
`in AN2690 binding to Cdc60p and found thl.
`both hydroxy groups were required (fig. S6C)‘{
`This implies that AN2690 can only form at
`1..-l
`
`Corr\;'>r>und iC~—,o(.UM) Corrrrgiolmci
`
`'05:)‘
`
`a’\
`o
`9”
`
`_
`
`D
`
`E
`
`QH
`B.
`.0 W
`0
`
`0
`
`9H
`F
`OH
`"gr: >100 F >100
`OH
`
`£j\8oH >100
`
`T
`
`T
`i.
`
`ul
`
`l
`
`vi
`
`‘Fig. 4. Boron and the oxaborole ring are «]
`quired for inhibition of aminoacylation. All reacy ’
`tions were performed in triplicate, and the meat
`values were used to determine a median inh
`itory concentration (lC5o) with Prism 4 (17).
`
`(A) The rate of Cdc60p inactivation by AN2690. The percentage of inhibition from different
`Fig. 2.
`concentrations [3.3 uM (squares), 5 uM (triangles), 6.6 pM (asterisk), 9.9 uM (open diamonds), and
`16.5 M (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.
`
`AN2690-AMP
`
`Fig. 3. AN2690 forms an adduct with the terminal adenosine (A76) of tRNAL°“ in the editing active site
`of LeuRS.
`(A) Overall structure of the complex of T.
`thermophilus LeuRS with tRNAL°“ 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. (3) Unbiased difference map
`(1.85 A 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 52). 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 Co positions of the editing domain of each complex.
`
`CFAD V. Anacor, |PR20’|5-01780 ANACOR EX. 2051 - 6/7
`22 JUNE 2007 VOL 316
`SCIENCE www.sciencemag.org
`
`CFAD v. Anacor, IPR2015-01780 ANACOR EX. 2051 - 6/7
`
`
`
`
`
`REPORTS
`
`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-tRNAL"“ 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 hen-
`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 spz 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. Biachem. 69, 617 (2000).
`2. T. L. Hendrickson, P. Schimmel, in Transfer RNA-Dependent
`Amino Acid Discrimination by Aminoacyl-tRNA Synthetase,
`]. P. D. Lapointe, L. Brakier-Gingras, Eds., Translation
`mechanisms (Landes Bioscience/Eurekah.com, Austin, TX,
`2003), pp. 34-54.
`3. P. Schimmel, E. Schmidt, Trends Biochem. Sci. 20,
`1 (1995).
`4. S. Cusack, A. Yaremchuk, M. Tukalo, EMBO ]. 19, 2351
`(2000).
`5. R. Fukunaga, S. Yokoyama, ]. Mol. Biol. 346, 57
`(2005).
`6. S. ]. Baker et al.,]. 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;
`6, Gly; H, His;
`l, Ile; L, Leu; M, Met; N, Asn; P, Pro;
`0, Gln; R, Arg; 5, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
`9. T. L. Lincecum Jr. et al., Mal. Cell 11, 951 (2003).
`10. Y. Zhai, S. A. Martinis, Biochemistry 44, 15437
`(2005).
`
`t-‘I-‘o-i->-Ir--I-I9°.".°‘P‘?E”!"
`
`11. M. G. Xu, ]. Li, X. Du, E. D. Wang, Biochem. Biophys. Res.
`Cammun. 318, 11 (2004).
`R. S. Mursinna, T. L. Lincecum Jr., 5. A. Martinis,
`Biochemistry 40, 5376 (2001).
`M. Dowlut, D. G. Hall, J. Am. Chem. Soc. 128, 4226
`(2006).
`A. Ricardo et al., ]. Org. Chem. 71, 9503 (2006).
`M. Tukalo, A. Yaremchuk, R. Fukunaga, S. Yokoyama,
`S. Cusack, Nat. Struct. Mal. Biol. 12, 923 (2005).
`T. F. Mccutchan, P. T. Gilham, D. Soll, Nucleic Acids Res.
`2, 853 (1975).
`GraphPad, GraphPad Software, Incorporated.
`We thank M. Kully and ]. Khan at NAE]A Pharmaceuticals,
`Incorporated, for their contribution to the minimal
`inhibitory concentrations in table 51. Part of this work
`was funded by an NIH grant awarded
`to ].].P. (R01 DE16835). The coordinates for LeuRSTT:
`tRNA:ANZ690 and LeuRSTT:AMP:AN269O have been
`deposited in the Protein Data Bank as ZVOG and 2VOC.
`S.].B. and LS. 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
`wwwsciencemag.org/cgi/content/fulU316/5832/1759/DC1
`Materials and Methods
`Figs. 51 to 59
`Tables 51 to 53
`References
`
`6 March 2007; accepted 23 May 2007
`10.1126/science.1142189
`
`CFAD V. Anacor, |PR201 5-01780 ANACOR EX. 2051 - 7/7
`www.sciencemag.org
`SCIENCE VOL 316
`22 JUNE 2007
`
`1761
`
`CFAD v. Anacor, IPR2015-01780 ANACOR EX. 2051 - 7/7