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`ILMN EXHIBIT 1017
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`Page 1 of 5
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`ILMN EXHIBIT 1017
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`NATURE nseonrs ‘ -i
`
`'
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`I"
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`4
`'
`
`
`
`‘I 7*__NoVOI'l‘I_ber £931 '
`Vol. 354 Issue no. 8348
`4 Astronauts ‘aboard the space shuttle
`have described '.the curious phenomenon
`of ‘shuttle
`glow’ fl‘0l‘I‘l around the Shl.lItIE'S
`tail-end. which faces Into the oribital wind
`as the shuttle circles the Earth. The cover
`shows a simulated shuttle glow caused by
`nitric oxide release from the shuttle’:
`payload bay, part -or an experiment
`to
`find an explanation for the phenomenon.
`Page 48.
`
`K...T|-iISWEEK...'l'HISWEEK...
`
`US high-energy physics faces
`‘peace dividend‘ I Soviet scientisls:e_n'i_i';lr"
`
`JESS] in jeopardy? I Two approtrchesztrrl’-"”'__k__I.
`g _.
`Japanese patent disputes I A Swiss A
`transition I The Ukraine will shut Chernob
`A cry for help front Yugoslavia
`—j‘,__'
`‘V -
`3
`
`2 .. -CORRESPONDENCE .
`
`No ‘plus’ from AIDS I Species intro-dution_s._
`I Astronomical costs.
`
`I
`
`3';
`
`
`
`
`
`
`
`COMMENTARY
`
`Quenching the wild wells of Kuwait
`R L Garvvin & H W Kendall
`
`NEWS AND VIEWS
`
`Metamorphosis of B British laboratory
`John Maddox
`
`Molecular neurobiology: NMDA receptors cloned
`at last
`
`Mark L Mayer
`Cosmology: Quark soup, do not boil
`M Fulcugita & C J Hogan
`Developmental psychology: Face to face with babies
`P E Bryant
`I
`I
`Gamma-ray bursts: Astronomy versus astrophysics
`David Lindley
`Fluid dynami: Forging the missing link
`John D S Jones
`
`Viral proteases: Molecular metamorphosis
`Dagmar Ringo & Gregory A Pctsko
`Evolution: Sex, slime and selfish genes
`Laurence D Hurst
`
`Daedalus: Arresting waste
`
`SCIENTIFIC CORRESPONDENCE
`
`Hazard from volcanic ash A J Prata, IJ Barton,
`R W Johnson. K Karno «Sc 1 Kingwell I Camouflaged
`DNA B E Griffin
`Out on a limb S A Newman; Reply — J M W Slack I‘
`Lead correction D Beliingcr
`-
`
`._
`
`BOOK REVIEWS
`
`t L
`t" '
`
`A History of Geology by G Gohnrr Peter J Smith
`Wanderers in Space: Exploration and Discovery in '
`the Solar System by K R Lang & CA Whitney
`ft
`Clark R Chapman
`The Biology of the Naked Mole-Rat eds P WShei_rgia_z§
`Jla.-vi": & A Alexander Brian Bertram I In '
`_
`Trefiicking of Proteins eds CJ Steer & J A Hana
`John Armstrong
`The Soiid Earth: An Introduction to Global:
`
`_.
`
`'"
`n .
`
`1.‘;
`
`'
`
`‘
`
`by C M R Fowler Leigh Royden'
`
`305.
`
`
`
`whale conservation
`Genetic
`sequences
`flanking
`simple sequence length poly-
`rnorphisrns (SSLPSJ
`In whales
`seem to have been conserved to
`a remarkably high degree In
`whales of all kinds. This could
`reflect
`unprecedentediy
`slow
`evolution.
`or
`that modern
`whales are a product of a rel-
`atively recent. explosive specia-
`tlon event. This finding will en-
`able researchers to use stan-
`dard probes on many — if not
`all — whale species. Page 63.
`
`Viral core
`The crystallographic structure of
`the core protein of Sindbis virus
`reveals
`a
`polypeptide
`fold
`homologous
`to
`that
`of
`chymotrypsin-like
`serine
`pro-
`teases. This explains why its
`proteolytic activity is blocked af-
`ter autocleavage from the spike
`protein. and predicts a virion
`structure with T=4 symmetry.
`This
`research has interesting
`ramifications for the design of
`antiviral dmgs active against
`togavlruses.
`some
`of which
`cause serious diseases such as
`encephalitis and arthritis. Pages
`3? and 22.
`
`Hot trick
`Ultraviolet light from the flash of
`the explosion that generated
`supernova SN198'i'A is
`now
`travelling through the circum-
`stellar material surrounding the
`supernova.
`creating the
`so-
`called Napoleon's hat emission
`nebula. A new model to explain
`the nebulae structure makes
`predictions for the development
`of the emission structure over
`the next few months. Page 43.
`
`Building a library
`On pages 82 and 84 two related
`new approaches are described
`for constructing libraries of pep-
`tides
`including
`all
`possible
`sequences. which can be used
`to screen for ligands to recep-
`tors. new antimicrobial agents
`and other bloactive peptides.
`
`Guide to Authors
`Page 87.
`
`
`
`.i
`
`teaming
`:
`is‘ an important ex-
`neurotransmitter In the
`-. us system that acts
`« r of different classes
`r. one of which. the
`_ ptor,
`is thought
`to
`key role In processes
`learning and mem-
`ge 31. Moriyoshi er
`the cloning of a rat
`.: product functions
`DA receptor, while on
`mar er at. describe a
`clone
`encoding
`a
`binding protein which
`lit a subunit of an
`or complex. News
`~ page 16. On page-66
`ing and expression of a
`'
`transporter from rat
`reported.
`
`r graphite
`-
`- of carbon structure
`of
`needle-like
`microtubules. made up
`.: tubes of between 2
`graphite sheets,
`is de-
`on page 56. These
`iare up to a few tens of
`= s In diameter. sug-
`that
`carbon-based
`engineering
`at
`a
`er than that of the
`is a possibility.
`
`_
`
`II
`
`fled
`'3 Commentary de-
`the various proposais
`_ scientists earlier this
`contributed to putting
`hundreds of
`fires in
`_oil wells. The
`fires
`' under control by mid-
`. much earlier
`than
`lestimates. Page 11.
`
`drift
`assessing future world
`s an understanding of
`Antarctic ice sheet will
`climate change. New
`from a group of sites
`“Land points to a re-
`Increase in snow
`:
`tlon rates to the high-
`.
`‘ for 170 years. Intensi-
`"lo activity since 1958
`lble cause. Page 58.
`
`'
`
`-
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`—
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`Page 2 of 5
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`tluulustvrockin Deocrubenbyh-i.Icmiilan Magaa'nest.td{4£.altlc Easustreet.
`on'l‘lunsiia .e
`irlhlnedwee
`fl-(B36 is
`payment). US and Canadian orders to: Nature. Sohsniptinu Dept. PO Box I733. Riverinn. . U
`I35
`~ h}. I1
`. Dlhcrordcn tn-
`.
`..
`.-of“
`nu-
` , Hutu RG21 2XS. UK. Somndciur postage paid It New York. NY IDJIZ. and Idditiflnal Illailingoffiioes. AtlIhD1‘lnr_.luI! to
`__
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`__
`_
`unorqaecifl: clients. kn-mind by Nauru to libraries and other: registered with iii: oopyn 1Clcarenoc Centre (OOC}Tran1.acboItlll Reporting Sorrel.‘ provided the bnseleevi $1-00
`Street.
`Nnrurz: 1 SLIII + $0.10. U5l"ofllIfl_U:t'fiIIIl.ddrradr_an,[cslo: Nature. 65
`. MA 01970. USA. tdentifrcauon code
`I
`_
`‘
`_
`_
`NY llIl12.. Published in Japan by Nature Japan KJL. Shin-Mitsukc Bldg. 36 Icltigtya Tlmolrhi. Shlnjllkla-ku. Tokyo ICE. lull) D 191
`Murmurs I-Id.
`- *fi;:Lwuwwmmc,zrcmgm
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`Page 2 of 5
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`
`‘L-"'ETTE*_'_?l5.'iS.’£"TE) ‘nnruinr
`
`.
`
`raam M. 51170 1 ill. 1225-1236 l1991l.
`sol. M. Clark. M. w.. vijayraymavan u. a. Abelson. J. Mole: gen. Genet 2:4, 72.30 (1990:.
`arll. K. L.
`-in Sotague. Jr. G. F. Moles‘. cell. Biol. 9. 2832-2694 (19693.
`. R. Ohtsubc. M., seieiguaui. M. a. Nishimutn. 7. Marco. coil. aioi. a. 2o2r—2o32 (1986)
`tsmo. M. et at Genes Dev. 1. 585-593 E1967].
`taubo. M. Okazdsi. H. & Nishlmoto. T. .l GM. Biol 109. 1389-1397 £1989).
`‘schoff. F.R.MaieI_l3.11I:. G. &PorIntirIfl.H. Hot: nelrr. load 56:. USA. II. 8617-8621. l1990l.
`.
`10. Blamafl. F. R. at Ponatingl. it Pi-oi: natn. Acae Sci LISA. inn me pressl.
`11. DrLva5.Gt T.. Bill. 8.. Coutauas. E. RI£l"i.M. G &D‘Eustai:nio. P. Miler‘. cell BN1‘. 1|). 1793-1795
`{.1990}.
`12. Valencia. A. Chardin. P., l'l"IttlI'Iql'3o‘leI‘. A. 5: Serluer. C. Elocliernlsrry SD. -56217454-B H991}.
`1:3. crecrm. 1-3. at al. Science am. 866-565 (1990).
`14. Woilman. A. .3. Macaro. I. G. science 2&8. 8?-69 E1990).
`1.5 West. M, Kong H..F a. Kalnata. ‘I’. -5% Lett. 299. 245-243 (19901.
`15. Boothe, H. R.. Sanders. D. A. a Mncnrrnid-. F. nature 943. 117/127 (19911
`1?. John. J., Frech. M. t. Wdtlngttoler. A. J hid. chem ass. t:lr92—11.799 E1983].
`ta hlatsumoto. r. at. Beach. 0. can OI. 347-360 (1991!
`19. Nishirnoto. T.. Ellen. E & Basilica. C. call 15. 47548.3 (19731.
`20 Chemo, P. Cancer Cells 5. 117-126 (1991).
`ACKNOWLEDEIVENTS. we that-It Ft. ti Himes for discussions, J. Kratscnmer for tetzhriirat aasmmoe.
`T, Nishimoto for a gilt oi ariu-RGC1-mtbotty. and A. Wirlinghofer for in gill cl Ha-ms protein and
`for discussions This work was amporled by the Dautscho Forsdiungsgemainsohaft
`
`A new type of synthetic
`peptide library for
`identifying ligand-binding
`activity
`
`Ill 5. Lam. Sydney E. Salmon, Evan M. I-Iarah.
`Victor J. l'lruby"', Wlealaw M. Kazmleraltlt
`It ttlchartl J. Knapp?
`Arizona Cancer Center and Department of Internal Medicine.
`College of Medicine. Tucson. Arizona 85?24. USA
`’ Department of Chemistry. Faculty of Science. University of Arizona.
`Tucson. Arizona. USA
`1' Selectide Corporation. 10900 N. Stallarcl Place. Tucson.
`Arizona 85737. USA
`
`OUR aim was to improve techniques for drug development by
`facilitating the identification of small molecules that bind with
`high aflinityto acceptor molecules (for example, cell-surface recep-
`tors, enzymes, antibodies) and so to mimic or block their interaction
`with the natural ligand”. Previously such small molecules have
`been characterized individually on a serial basis. The systematic
`synthesis and screening of peptide libraries of defined structure
`represents a new approach. For relatively small libraries, predeter-
`mined sequence variations" on solid-phase supports have been
`used“. and large librari have been produced using a lane-
`teriophage vector into which random ollgodeoxynucleotide sequen-
`ces have been introdaced"', but than techniques have severe
`limitations. Here we investigate an alternative approach to syn-
`thesis and screening of peptide libraries. Our simple methodology
`greatly enhances the production and rapid evaluation of random
`libraries of millions of peptides so that acceptor-binding ligands
`of high aflinity can be rapidly identified and sequenced. on the
`basis of a ‘one-bead, one-peptide’ approach.
`Our method involves creating a large peptide library consist-
`ing of‘ millions of beads, with each bead containing a single
`peptide and with the complete collection representing the uni-
`verse of possible random peptides in roughly equimolar propor-
`tion. II is clearly not enough to use a random mixture ofactivatcd
`amino acids in a peptide synthesis protocol, because the widely
`different coupling rates of dificrent amino acids will lead to
`unequal representation and because each head will contain a
`mixture of difiercnt peptides. Our solution was to use a ‘split
`synthesis‘ approach. The first cycle consisted of distributing a
`pool of resin beads into separate reaction vessels each with a
`single amino acid, allowing the coupling reactions to go to
`completion, and then repooling the beads. This cycle was
`repeated several times to extend the peptide chain (Fig. la). In
`this fashion, each bead should contain only a single peptide
`species.
`
`We then developed a rapid approach for screening the lib‘
`to find beads containing peptides able to bind to any parti as
`acceptor molecule. Acceptor molecules were coupled t
`enzyme (alkaline phosphatase) or to fluorescein and add ‘
`soluble form to the peptide-bead library. Typically. a few -'
`were intensely stained and were visible to the naked eye
`easily seen with a low-power dissecting microscope (
`diameter of l00-200 um) against a background of colon if
`nonreactive beads (Fig. 2). With the aid oftiny forceps cou
`to a mlcromanipulator, the intensely staining beads could
`removed for analysis (Fig. lb). We washed each head with :-
`guanidin: hydrochloride to remove the acceptor complex, F
`then determined the peptide sequence contained on the -
`by placing it on a glass filter which was inserted into a mi‘
`microscqucnccr (model 477A, Applied Biosystems}. A lib" -
`containing several million beads could be screened in I_
`Petri dishes in an afternoon. Afterwards. it could be w
`with SM guanidine hydrochloride and subsequently reused I
`screening new acceptors.
`Our sequencing studies established that the peptide ..-.
`on any given single bead is sufiicicnt for unambiguous seque .
`analysis as each head sequenced contained 50-200 pmo|_
`peptide (the lower limit ofscnsitivity ofthc instrument is in 3
`range oi'5 pmoll. Furthermore. as measured by preview anal
`for several dozen individual beads from this library, the coup 5
`reactions in the split synthesis procedure were virtually com - _
`as most individual beads displayed peptides that were over "
`pure (range 97—l00%}. At least three pcntapeptide beads
`sequenced daily using the microsequcncer.
`We applied the process to produce a library of millions‘-
`peritapcptidcs and screen it against two well-studied acct: I
`molecules. Using the split synthesis approach with 19 «IE.
`vessels, we synthesized pentapeptide libraries incorporating
`the natural amino acids except for cysteine (for simplicity
`eliminate disulphide crosslinkingl. The random incorpora
`of 19 amino acids into pentapeptides can produce a total of"
`to 2,476,099 (195) individual peptides of dillcring sequence
`any one sequence represented on at least one solid-phase —;
`bead (of course. the number of beads of any given scquc
`will follow a Poisson distribution. and many multiples of {
`minimal number of beads are required to assure that
`-
`maximum theoretical number of possible peptide entities-
`approximated in the library}. Libraries were readily synthe-5'
`in a few days.
`We studied a monoclonal antibody against ,3-endorphin
`high aflinity (Ki: 115 nMi for the epitope sequence YGG
`(single-letter amino—acid code}. A total of six reactive b =
`were retrieved from about 2 million beads screened from <
`pentapeptidc library. One peptide ligand sequence retrie -
`YGGFQ. had an afiinity (#1.: 15.0 nM) nearly identical to _5
`native epitope. Two of the other peptide ligands retrieved .5
`Kis of less than 3'.’ nM (Table 1). These aflinitics were m"
`than 50-fold better than those obtained for the identical mo
`clonal antibody by Cwirla et at, who used a phage lib =:
`method’.
`
`Native ligand
`Peptide ligand
`
`:1."
`TABLE 1 Affinity of anti.,3—endorphin ligands: comparison of the
`ligand and peptide ligands identified from a large pentapeptide iibr .-
`Sequence
`K. lnMl
`YGGFL
`1?.5e3.2
`15.0 s 1.7
`32.9 x 2.0
`36.9 s 7.7
`726 = 134
`19cc 2 303
`3730 : 1500 .
`
`YBCFQ
`YGGFA
`‘EGG-"F
`YGGLS
`YGALQ
`YGGMQ
`
`I
`
`Affinity constants for the peptide ligands determined with com «-
`radioliganct binding assays using trltiated YGGFL as the standard.
`.
`NATLRE - VOL354 ' YNOVEMBER
`
`
`
`Page 3 of 5
`
`

`
`
`
`TABLE 2 Aminoacid sequences of individual pentapeptide beads that
`interacted with streptavldln
`
`HPQFV
`HPQGP
`HQPAG
`
`LHPQF
`Fl-PQG
`GI-PQN
`TWQN
`QHPQG
`IHPQG
`GFPQG
`
`WN-PM -
`WTI-IPM
`_Vll_>l\iiA
`Ml-PMA (21
`
`_
`
`-
`
`-
`
`-i
`_
`‘r
`
`‘
`
`'
`
`MYI-PQ
`RE!-PQ
`iQi-FQ
`Gilt-IPQ
`TVHPQ
`IGHPQ
`Wlvl-IPQ
`'
`GAHPQ
`‘."-.__
`PLHPQ
`AIHPQ
`-1.
`._ ..
`AAHPQ
`TPl-lPQ(2l
`
`
`
`All sequences listed above were found on single beads eiteapt
`_
`E
`and NI-IPMA for which two beads were obtained. The first
`list the sequences found with I-PQ located in the amino terminus_'. contra‘!
`region or carboaty terminus respectively. The fourth column iisti; "
`sequences retrieved.
`.
`
`investigators have attempted to develop peptt
`Other
`libraries for similar purposes. For example. Geysen and so .
`leagues’ synthesized peptides or known amino-acid sequcn'_ .
`on plastic pegs in 96-well plates. This approach permitted tligi
`synthesis of several thousand peptides‘. A related techniqué'.'7
`using complex instrumentation, photochemistry. and computer-'
`ized inventory control reported by Fodor at at.‘ perrnittcd syn;
`thesis of known arrays of at least 1,024 peptides on an individual""
`microscope slide. Finally. Smith and colleagues pioneered the
`concept of using a recombinant bacteriophage incorporating
`random nucleic acids to produce phage displaying millions of
`random peptides‘. This approach is innovative but faced with
`the inherent
`limitations of synthetic and selection biases of
`.
`biological systems. Various investigators have used these tech-
`niques to identify ligands forsevcral monoclonal antibodies and -.
`strcptavidin”. On the basis of comparisons with this published ;
`information, it is clear that our process is for simpler and more
`rapid than any of the alternative methods and unlike these"-'
`methods has identified peptide ligands with alfmities virtually
`identical
`to those of the native ligands. Additionally, our.
`approach has far greater potential for applying the richness of
`well-established peptide chemistry to synthesize libraries incor-
`porating D-amino acids or unnatural amino acids as well as-
`specific secondary structures including cyclic peptides. All is
`
`7
`
`. .' 3'09
`-I
`; A V
`A -0
`13-0
`V-C
`hrldollitfltliiill
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`AV -0
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`GAV
`car. -0
`cos-O
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`
`1. stained bead retrieved and washed
`2 Peptide microsauuenced from a single head
`
`.
`
`
`
`-
`
`in peptide library synthesis and screening. 3. Flow diagrarn of
`».
`—
`-- example of solid phase ‘split synthesis’ of tripeptides consisting
`(M. Elrcine (G3 and valine (Vi using standard solid-phase peptide
`methods with Front: or Bot: chemistry. After each coupling step.
`
`tom each of the three reaction vessels are combined {or
`n and then split again to the three vessels for the next coupling
`l
`' iter three such steps the 2'! possible peptide sequences (33) are
`-
`ted on separate heads. 1:. A single head binding an acceptor
`tagged with an enzyme is identified after being stained by enzymatic
`.__on a dye substrate. The stained beat: is physically removed from
`-
`- beads remaining in the library. washed free of the acceptor
`and subjected to rnlcrosequencing. Once identified. the reactive
`then synthesized in larger quantities for confirmatory binding and
`studies
`
`.
`
`so used the same pentapeptide bead library to find
`binding strcptavidin (chosen as a receptor-like target).
`ctive beads retrieved, 28 were sequenced and a triplet
`- sequence of HPQ was found in either the amino or
`fa -terminus or the central portion of 23 of the recovered
`prides (Table 2). The live other peptide ligands sequen-
`ed the triplet sequence HPM in the pentapeptide-
`ds containing LHPQF were synthesized, competitive
`studios established that the HPQ sequence was recogn-
`c same binding site on streptavidin as the native ligand
`
`_
`
`' - and high-power photomicrographs of a peptide ligand library
`in which a reactive {dark} bead stained with the alitaline phos-
`ion can be easily identified in a background of many thousands
`ttve (colourless) beads.
`
`.:.-. VOL 354 - 7 NOVEMBER 1991
`.L
`
`Page 4 of 5
`
`
`
`Page 4 of 5
`
`

`
`i Ll'-.*l'?liElri"§7’|'.‘@)- tin‘ ;
`
`libraries of longer or more diverse peptides should they be
`required for any given application.
`We have expanded the applications of our peptide library
`approach by modifying the synthesis procedure to incorporate
`cleavable linkers on each head. After exposure to the cleaving
`agent. such beads can then release a portion of their peptides
`into solution for biological assay while still retaining sufficient
`peptides on the beads for subsequent structure determination.
`The one-bead, one-peptide concept and its applications dis-
`cussed above demonstrate that this approach provides important
`new tools with which to search for specific ligands of potential
`diagnostic or therapeutic value. Such information should also
`enhance Fundamental understanding of interactions between
`ligands and acceptor molecules.
`El
`
`5°”"".°t.UiP
`
`rtnmwett 30 May. accepted 10 his-roar 1991,
`.l K. 246-262 (1990).
`1. Ruby. ti‘. J, AI.Coeltll. F. at Klrllllflli. W. Bfl:ID‘ieI'l't
`2.
`ieuoy. v. J. 6- Shamas D. cm. Ooh fltrJl'II:‘l 2. 599-605i1991I
`3. Susan. it M. Melveu. R. H. E BI‘llIil[S. 1. Pro; natrt Acct: so. use 81. 3998-4002 (19841.
`Fooor. 5. P. at at Science 251. T67-‘I73 l1991l.
`Pa-miey. S. F a. Srritui. G. F. Gene ‘#3. 305-31at198Bl.
`5“-,u_ 1].; gs-I-pm G_p_5¢g-_¢ga,g_3gfi.3g)(1gg3;i_
`il:1w9-gas.E..r=oters.E_L.Barrutt.n.w..socwec.w_J.Pm: rratn acau 5431. t.is.iio1.63rs-6332
`DMHJ LPag'm_LLcl&°'W'P_,_swmE”9_4m_w6u99°)‘
`iiiau. it D..Tre5eor.G.'i'i'.&.hoobs.1 ln Diet-niso'ymdB4'alagy arpepuau tee Melenl'ioler.ll.
`°°5""'“'°°'-*°"I-'~19‘-’='-
`AG€NO\I\lI.ED@t£l*ll‘S. We own 3‘. At-Ocelot. M Ross and ii. Hirschmarin tor valuable suggestions
`tInoE. Lridortoroornunlltonttnniariuscript I:.S.L. Isa Special Fellow or In! Letlcarriia Society at
`America. The peptide synthesis and screening process is referred to as the Seleeiitls Process and
`is the subject of patmt this went was supported tn; the National institutes at Health, the Arizona
`Disease Current Research Oorrirnissieri end the Solectioe Corporation.
`
`Generation and use of
`synthetic peptide
`I
`I
`I
`I
`combinatorial libraries
`f
`b
`'
`ch
`(1
`
`rug Iscovery
`.
`lllollrd Ac
`Sylvlo E. Blomlolo, Jon R. Appel, Colette T. Dooley
`& J In H. c
`u
`'1...“
`grrey Pines institute for Molecular Studies. 3550 General lttomics Court.
`" °'°g°' °“'”°""" 92121“ U5“
`screening of large nurn-
`EXISTING miethoils for the
`tiers of peptides are limited by their Inability to generate and
`screen the requisite number (mlliions) of Individual peptiiIes""
`and/or their inability to generate unmodified free peptides in
`quantities able to interact in solution""‘. We have circumvented
`these limitations by developing synthetic peptide combinatorial
`libraries composed of mixtures of free peptides in qllantitis which
`can be used directly in virtually all exlstllgastiay systems. The
`screening of these heterogeneous libraries. along with an Iterative
`selection and synthesis pt-oeem, permits the systematic identi-
`fication of optimal peptide ligands. Sllrlllg with 3 library com-
`posed of more than 34 million llexil-peptides. we present here the
`precise identification of an antigenic iletenninnnt recognized by a
`monoclonal antibody as well as the straightforward development
`of new potent antimicrobial peptides.
`library (SPCL}
`The initial synthetic peptide combinatorial
`prepared and used in this work consisted of six-residue peptide
`sequences with acetylated N terminals and atnidated C ter-
`minals. The first two positions in each peptide were individually
`and specifically defined, whereas the last four positions consisted
`of‘ equimolar mixtures of I8 of the 20 natural L-amino acids
`{for ease of synthesis. cysteine and tryptophan were omitted in
`34
`
`Page 5 of 5
`
`this initial library). Such libraries can be generally represe F
`by the sequence Ac-0,0,XXXX-NH, (where Ac repres_
`acetyll (see legend to Fig. 1).
`:_'_
`it
`Using a competitive enzyme«linked imrnunosorbent
`(ELISA), each of the 324 diflerent peptide mixtures ofthe S _
`(Ac-O,01XXXX-NH,) was assayed to determine its ability '.
`inhibit the interaction of a monoclonal antibody with a la"
`13-residue peptide (Ac-YPYDVPDYASLRS-NH,; single-le
`amino-acid code). Of the 324 peptide mixtures examined { "'7
`ll. Ac-DVXXXX—NH, caused the greatest inhibition of a
`body binding (Table 1}. Twenty new peptide mixtures were! ._
`synthesized in which the third position of the peptide rriixt
`Ac-DVXXXX-NH, was defined (Ac-DVOXXX-NI-I2.
`t
`-5
`tophan now included in the X positions). Each new pep
`mixture contained 6,859 (193) individual peptides {l37.l80 '
`total). The most effective inhibiting peptide mixture was
`_
`DVPXXX-NH; (50% inhibitory concentration, ]C5n:4l p.
`-
`Table 1b). The above iterative process. which reduces the ri
`ber of peptide sequences by 20-fold each time it is repeat
`was then carried out For the remaining three positions (Ta
`l,
`l'.'-E}. ll should be Tlolifid that On defining the
`positi
`{Ac-DVP[)0)(.NH2, Tame ml),
`the
`[C50 found for
`V
`DVPDYX-NH: {(3.38 p.M) was at least 3,500-fold lower! -'
`any of the other 19 peptide mixtures. Also, the peptide mixt
`Ac-DVPDXX-NH; and Ac—t)vt=xxx-NH, had 1c_.u val
`lower than all of‘ the peptide mixtures with the firth positi
`defined, with the exception of Ac-DVPDYX-NH_.. This cle :_
`
`TABLE 1
`
`Identification of the antigenic determinant recognized by
`clonal antibody 19310
`
`-
`
`_‘-
`
`ff“? ""*‘“'°
`a
`Ac~nvxxx=<-NH.
`C-
`-
`§°'3l.”§i‘§;“i'E.
`Ac-IDLXXXX-NH:
`tn)
`A -DVPXXX
`Agofixxxfi
`Ac.ovoxxx.NH,
`Ac-DVXXXX-Ill,
`AC-DVMXXX-NH,
`
`2
`
`-
`.
`Ac Dvcxxx NH
`M
`,qc_Dvppxx.m.|2
`Ac-DVPXXX-Ill,
`Ac-Dv'PAxx.Ni-I,
`
`('59
`
`Ac_DvPDAx_NH?
`
`Icy,
`‘W’
`250
`$23
`>-1.400
`41
`145
`215
`250
`451
`133$
`,
`5-1400
`
`4_4
`41
`>1,400
`
`3'23
`>1.400’
`
`Icm
`W’
`'
`o. r.
`|
`'-:
`"-
`
`
`
`I "em
`(
`e
`Ac-ovvnmvn.
`if;.‘§‘.}'£-‘.’.‘~}i$£5a”f.
`Ac-DVPDYC-NH,
`‘Ac-DVPDW-NH,
`Ac-DVPOYT~Nl-I,
`A -DVPDYG-N
`A:_DVp-DyE_N|i.::
`Ac-DVPDW-NH;
`Ac-DVPDYM-l'fl'lg
`Ac-DVPDYQ-NH,
`
`-
`.
`Ac DVPDYR NH,
`
`Ac.gvpo*m.NHa
`Ac-DVPDW-Ni-I,
`Ac-DVPDYP-NH,
`N=~DVi'D‘|'W-NH:
`N3-DVPDVD-NH:
`
`-=4»-'
`The lcms of the most effective inhibitory peptide mixtures Dblai
`each iterative step are illustrated for a. peptide mixtures from the -
`'-
`screening of the spec D. the third position defined (AC-DVOXXBC-NH-_.l: c..
`fourth position defined [Ac-DVPOXX-NH;l: at the fifth position defined
`DVPDOX-NHQJ: and e. the sixth position defined (Ac-DVPDYO-Ni-lg). The '_
`of the peptide mixture derived from the previous iterative step is in "
`for comparison. Peptide mixtures were assayed by competitive ELJSA
`Fig. 1). The concentration ol each peptide mixture reoessary to Irtiibit ‘i'
`ol the antibody binding to the control peptide on the plate was obtained
`serial dilutions of the peptide mixture. The Icgos were calculated using5
`software GRAPI-PAD (Isl, San Diego}. The four-step iterative screening —
`synthesis process totes approximately 4 weeks. This time frame will
`‘
`depending on the assay being used and the number of cases moved fa
`at each iterative step.
`
`NATLRE ' VOL 354 - T NOVI-JVBER :3-
`
`
`
`Page 5 of 5