CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2051 - 1/7
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`

`
`
`
`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
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`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
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`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
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`CONTENTS continued >$
`
`www.sciencemag.org
`
`SCIENCE VOL316
`
`22 JUNE 2007
`
`1659
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`
`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.
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`SCIENCE VOL 316
`22 JUNE 2007
`1661
`
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`
`CONTENTS continued >>
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2051 - 4/7
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`
` 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
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`
`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’|5-01776 ANACOR EX. 2051 - 6/7
`22 JUNE 2007 VOL 316
`SCIENCE www.sciencemag.org
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2051 - 6/7
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`

`
`
`
`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 5-01776 ANACOR EX. 2051 - 7/7
`www.sciencemag.org
`SCIENCE VOL 316
`22 JUNE 2007
`
`1761
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2051 - 7/7