West-Ward Pharm.
`Exhibit 1035
`Page 001
`
`

`

`AMERICAN
`ASSOCMTION FOR. THE
`AD\«'ANCEMi3N‘t' op
`SCIENCE
`
`Etiiim‘i:ti
`
`SciENcE ISSN 0036-8075
`
`18 JANUARY 199i
`VOLUME 25:
`NLJM nun 4.991
`
`This Week in Scierrte
`
`Teaching and Research
`
`Safety of Bovine Growth Hormone: D. S. KRONI-‘ELD; J. C. Jusrzevrcn .-mo
`C. G. Goren I Interpreting Cancer Tests: J. D. WII.SON; G. W. Giunntia I
`Kidney Transplantation: Overlooked Pioneer: G. B. ELION
`
`The Rush to Publish I Lessons from Physics
`Third Strike for Idaho Reactor
`
`CDC Ab-andons Pians For AIDS Survey
`Heaiy Nominated
`GAO and DOD Get Into a Cat Fight
`Science Literacy: The I:'.nemy Is Us I Sr:ience‘s Top 20 Greatest Hits
`
`New Light on Writing in the Americas
`Montagnier Pursues the Mycopiasma-AIDS Link
`Despite Reports of Its Death, the Big Bang Is Safe
`Giobai Temperature Hits Record Again
`Briefings: Radiation Research Shake—Up I Private Initiative on Fetal Research I
`U.K. Antes Up for Telescopes I George Mason to Set Up Think Tank
`
`Subsistence Economy of E! Parafso, an Early Peruvian Site: J‘. QUii.’l'E.R,
`B. Drum E., D. M. Ptii\RSAt.I., D. H. SANDWRISS, J. G. Iomss, E. 8. Wine
`Chemistry and Biology of the Immunophiiiris and Their Irnmuriosuppressive
`Ligands: S. I... SCI-iRE.IB1~‘.I't
`
`CCAAT-Enhancer Binding Protein: A Component of a Differentiation Switch:
`R. M. UMEK, A. D. FRIEDMAN, S. L. MCKNlGH'i'
`
`An Antimony Sulfide with a Two-Dimensional, intersecting System of Channels:
`I. B. P.-titlste
`
`SCIENCE [ISSN 0036-8075] is published weeirty on Friday. except the lea! week In December. by the American
`Aeeocleiien tor the Advancement of Science. 1333 I-I street. NW. Washington. Dc 20005. Second-class puslaga
`{publication No. 434-660] paid at Washington. DC. and additional mailing oiiices. Copyright 113 1990 by the American
`Association for the Advancement oi Science. The Iille SCIENCE is a registered trademark oi the MAS. Domestic
`lndivitlual membership and subscription (51 issues]-: $52. Domestic inslilutionai subscription [SI issues}: 3150. Foreign
`postage extra: Canada S46. other [surface mail} $46. air freight $90. First ciass. airmail. echcoi-year. and student rates
`on request. Canadian GST Number: Pending. Change at addreee: ailow 5 weeks. giving old and new addresses and
`I1-digit account number. Postmaster: Send change of address In Science. P,O. Box 1?23. Fiivorlnn. NJ IJIJD??_ single
`copy sales: $6_t:lrti per issue prepaid Includes suriaoa postage; Guide Io Biotechnology Products and Instruments. $20.
`Bulk tales on request. Authorization to photocopy maierim for internal or personal use under cireurnslances not falling
`within the fair use provisions oi the Copyright Act is granted by AME to libraries and other users registered with the
`Copyright Clearance Center [CEO] Transactional Flap-erling Service. provided Ihat the base fee or Si per copy plus
`$0.10 per page is -paid directly to (300. 2? Congress Street. Salem. Massachusetts more. The identification code for
`Science-is 0CI3B—BiIi?5f33 $1 + .10. Scienoeis indexed in lhe Reader's Guide to Periodical Liter.“-rtirre and in savarai
`specialized indexes.
`The American Association for the Advancement of Science was iounded in 1348 and incorporated in rent. its
`objectives are to iunher Ihe work oi‘ scienlisls. Io Iacilitara cooperation among them. Io Ioster scientific Iraedom and
`responsibility. In improrva Iha attentiveness of science in the promotion of human weriere. Io advance educallon In
`science. and to increase public understanding and appreciation ot the importance and promise of line methods of
`science in human progress.
`
`.‘r'('2[F.NC|‘C, VOL. 25]
`
`est-Ward Pharm.
`Exhibit 1035
`Page 002
`
`West-Ward Pharm.
`Exhibit 1035
`Page 002
`
`

`

`Lakes and ponds on the arctic tundra with ltigaltnit Mountain in the
`COVER
`background, North Slope, Alaska. These aquatic ecosystems are continuously
`releasing carbon dioxide to the atmosphere. Much of the carbon originates in
`terrestrial environments. and accounting for this release substantially lowers the
`estimate of the worldwide arctic sink for atmospheric carbon dioxide. See page 298.
`[Photograph by George W. Kling]
`
`Local Structure and Chemical Shifts for Six~Coordinated Silicon in High-Pressure
`Mantle Phases: I. F. S'I‘E.i1BINs AND M. KANZAKI
`Arctic Lakes and Streams as Gas Conduits to the Atmosphere: Implications for
`Tundra Carbon Budgets: G. W. KLING, G. W. Kn-PHUT. M. C. MILLER
`Putative Skeletal Neural Crest Cells in Early Late Ordovician Vertebrates from
`Colorado: M. M. SMIT1-1
`
`Altered Perception of Species-Specific Song by Female Birds After Lesions of a
`Forebrain Nucleus: E. A. Bitewowrrz
`
`The Efiect of Anti-Neoplastic Drugs on Murine Acquired Immunodeficiency
`Syndrome: C. SLMARD AND P. IOLICOEUR
`Evidence for Biased Gene Conversion in Concerted Evolution of Ribosomal DNA:
`D. M. HILLIS, C. Monrrz, C. A. PORTER, R. I. BAKER
`The Efiect of the Floor Plate on Pattern and Polarity in the Developing Central
`Nervous System: 8. Hm.-wo, S. Fuss, G. S. Sonar.
`Regulation of Interleukin—2 Gene Enhancer Activity by the T Cell Accessory
`Molecule CD28: 1. D. FRASE.1{, B. A. IRVING, G. R. CRARTREE, A. WEISS
`
`Microwave Sounding Units and Global Warming: B. L. GARY AND S. I. KEIHM;
`R. W. SPENCER AND I. R. CI-IRISTY I Lipid Flow in Locomoting Cells:
`M. S. B1u=.'rst:Hi-Lit; K. IACOBSON, 1. LEE, M. Gusrarsson, K.-E. MAGNUSSON I
`Bryozoan Morphological and Genetic Correspondence: What Does It Prove?:
`I. LEVINEON; I. B. C. }ACl(.‘-SUN arm A. H. CHE]-;‘1‘I-{AM
`
`Authors of Their Own Lives, reviewed by A. SICA I Australian Ecosystems,
`M. IJOWMAN I Thalamic Oscillations and Signaling mo Brainstem Control of
`Wakefiilness and Sleep, C. KOCH I Books Received
`
`Protein Immunoblotting Incubation Rotator I Micrornanipulator Table I Leiden
`Microincubator I Freezing Stage with Microtome I Data Analysis for the
`Macintosh I Monoclonal Antibodies I Literature
`
`Teclmietll (ioiiiments
`
`Book Reviews
`
`p!'(lLlllt‘:l:i
`
`l\"l:1leriail.~.'a
`
`Board of Director:
`Flieliard C. Atkinson
`Rafiririg President.
`Cheiir.-nan
`Donald N. Larlgenberg
`President
`Leon M. Laclannan
`President-alacr
`
`Mary Ellen Avery
`Francisco J. Ayala
`Eugene H. Cola-Floblas
`Flnbari A. Frosd1
`Joseph G. Gavin. Jr.
`John H. Gibbons
`Beatrix A. Harntiung
`Florence P, Hasalline
`
`lmlllam T. Gale-an
`Treasurer
`
`Flichard S. Nicholson
`Executive Ofiiclsr
`
`Editorial Board
`Charles J. Alrllzen
`Ellzabalh E. Elallay
`David Ballimora
`Mlllam F. Brlnkman
`E. Margaret Burbidga
`Pierre-Gilles da Gennes
`Joseph L. Goidsleln
`Mary L. Good
`Harry B. Gray
`F. Clark Howell
`Paul A. Marks
`Yasutuml Nishizuka
`Helen M. Flanney
`Howard A. Schnaldernian
`Ruben M. Soles-r
`Edward 0. Stone
`James D. Watson
`
`Board of Reviewing
`Edllara
`Jnhn Abelson
`Frederick W. All
`Don L. Anderson
`Slephen J. Banlrovit:
`Gunter K4 Blobel
`Floyd E. Bloom
`Henry H. Bourna
`James J. Bull
`Kalhryn Calame
`Charles Fl. Canlor
`Flalph J. Cicerone
`John M. Collin
`Robert Dorlrnan
`Bruce F. Eldridge
`Paul T. Englund
`Fredric S. Fay
`
`Harry A. Fozzard
`Theodore H. Gaballe
`Roger I. M. Glass
`Stephen P. Goff
`Carey S. Goa-tlrnan
`Slephan .1. Gould
`Eric F. Johnson
`Stephen M. Kosslyn
`Konrad B. Krttuslmp!
`Charles 5. Lettings Ill
`Richard Loslck
`John C. McGll'f
`Anthony Fl. Means
`Martlmar Mlshltin
`Finger A. Nlooll
`‘Mlllam H. Orrnaa-Johnson Ill
`Carl 0. Pabo
`Yeshayau Packer
`
`Dennis A. Powers
`Erkkl Fluoslahll
`Thomas W. Schoaner
`Ronald H. Schwartz
`Terreriea J. Se]nowsltl
`11-uurnas A. Stall:
`Fluban 1'. N. man
`Emil Fl. Unanue
`Gearal J. Verrnai]
`Elan Vogelsleln
`Harold Welnlraub
`Zena Wail:
`George M.Wl1l1asldes
`Owen N. Wine
`William E. Wood
`Kailh Yarnarnoto
`
`13 JANUARY 1991
`
`'I'ARl.E CIF CON'i'l:'.N'l'S
`
`245
`
`st-Ward Pharm.
`Exhibit 1035
`Page 003
`
`West-Ward Pharm.
`Exhibit 1035
`Page 003
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`. E. I’. Lanning, .l’rm Bljitrc lllr films (Prentice-H.'l.l|, i:‘.I'|glr:w0u-Ll (Hills, NI. 1967).
`_ T, C. Patterson, in Prehistoric .“lq.rt'(lI.‘I'tm.-, S. Streuvcr, E-Id. (Nantral History Press,
`Garden City, NY, 1971}, pp. 181-258.
`_ R. Fung I’.,A_mmIrs Arqrtrol. 2, 11 (1972).
`_
`]'_ II. $tc\v:lrd and L. C. Fermi, Native l’cop.lus afSnml'l'l .“l:llt:rt'c.t {Mc(.ir.uv Hill. New
`York, 1959).
`. M. H. Parsons, Am. /Irrtiq. 35, 292 (I970).
`, D. I. Wilson. Alli. /illlllraprll. 83, 93 (1931).
`.
`I. S. Raymond, /lm. Atttiq. 46, Silo (1931).
`,
`I. Quilter and T. Stacker, Am. Aillltropol. 85, 5-15 (1983).
`.
`I. Quilter, J. Field Arrllnral. 12, 2579 (I985).
`. F. A. Etlgcl,
`Soc. Alli. 55, -*1-3 (1966); Alllll. Cir|Il.l'. Uut':a_ Agnnia 5, 2,4]
`(19:57).
`.r\. 0.'ibt:)1'n,ll1 For Tllmry Blflldltgg I'll/lrt'lrm:nl'u__qy, L. llirlfnrd, lid. (Auclcmic Press,
`New York, 197.7], pp. 15?-243.
`. S. Quilter suggested this possibility during work at the ]’ak:m:r Site in 1976.
`. C. E. Smith,
`in La (Ialgudn Pcm, A Prrrrramfr Clllftlrc ill Tmltsflioll, 'l"ctcncc
`(it-icdcr er al,, Eds. (University nfTt:x:ls l're_:s, J\I.Lsrin, Til, 1983), pp. l2.5—l5l.
`. S. Pozorsld and T. Pozorski, Early Srltlmmit mm‘ Sttbsittailrr in til: Ctilmfl Valley,
`Pm: (University of Iowa Press. Iowa City, 1987).
`. V. Popper, in L0: Gdvilnllrr, Mar,
`.Drn'rr.r1'o y O:I.tl'.s cu in l"h'rfo.|'iut rlcl Holllltrr, D.
`Bunavia, Ed. (Editorial Ausonia, Lima, Peru, 1982), pp. 148-156.
`1‘). S. l'o7.orski and T. Pomrski, Arm. Canlegic :'vl'u.t. Nat. Hist. 4-9, 33? (1979); I. B.
`llllfi, Illllltropnl.
`l"rlp. Am. Mlls. Nrn‘. I-list. 62 (1985), part I.
`, A. Grnhrnan, in Let: Gnlrilamzs, Mar.
`l)r.u‘o1o y Oiisi: cu in Hisloriu rlcl Hamlin;-, I).
`
`.
`
`.
`
`liunavia. lid. (Editorial Ausooin. Lima, Peru, 1982), pp. I57'—l?'9; IL L. Burger
`and N. van dcr Mcnvc. Am. flllllfrllpal. 92, ‘)6 ([990).
`. T. Dillchay. P. Nclhcrly, I. Ros-scn. Am. Autiq. 54, 733 (I939).
`.
`I. Quilter, “Tu lish ill thr: artcrlloolt: Beyond subsistcllcc Ltrlnrlnlics ill the snldy of
`carly Amlclln civilivnlioll,“ paper prcscntc-.l at 51st Altllual Meeting of the Society
`for r\mt:ric:lI1 firchaerlltlgy, New Orlcalls, LA, 23 l'\pril 1986.
`. M. Moseley, l"m-ngrr'cn.‘tttru.l Coastal C:'m‘l'£zr.-lfam in Pam {Carolina Biology Read-
`ers, no. ‘)0, liurlington, NC, l9?R).
`. 1. Quilter, "Core and periphery in Prccctanlic constnl Peru,” paper prcscnted at the
`83th Annual Meeting ofthc Amcricsut J\nthmp-ulog-ical Society, Wzlsllington, DC,
`19 Novcrnbcr I989.
`. R. G. Wilkinson, Pawrry mm‘ i"ragrr.t.s (Pracgcr, New York, 1973).
`. Following tax: listed in V. Alamo V. and V. Vnldivicm ll.-'l., Baler. bur. Mar Perri
`(volulnen cxttaordinario. Callno, Peru, I987].
`. Funding For the El I'ar.1iso research was provided by NSF grant BNS~B3-03630,
`Ripun College Faculty l)cv:|opmcnt Funds, and the Continerltnl Code: Product:
`Company (a wholly owned subsidiary of Quaker Oats}. The excavations were
`carried out under Crcdcncial D38-83-DCIRBM, issued by the fmrimro Narioiml lit‘
`Cnlllma of Peru. We thnrlk A. A. I-Iuntrr (Missouri) who idcn tified the squash seeds
`and A. Price, I. Azttbcrry, and L. Haubrich who hclpcd in sorting and tallying data.
`Additional aid in processing the subsistence l'l:1'!!alIIS iv-as given by N. Salazar and
`M. C. R0dr1'gucz dc Sandwciss in Penn. 1.. Salazar-Burger, assistant field director,
`was essential to the project. The Cmtm Jr iinuesrljgnriniu-5 tilt’ Ztma: Ardias was our
`base rnfnpcrations and analysis and we thank F. A. Engci and M. Vallcjris and many
`other l‘cmvin.n collegugs for support.
`
`Chemistry and Biology of the Immunophilins
`and Their Immunosuppressive Ligands
`
`STUART L. SCI-IREIBER
`
`Cyclosporin A, FK506, and rapamycin are inhibitors of
`specific signal transduction pathways that lead to T lym-
`phocyte activation. These immunosupprcssive agents bind
`with high aflinity to cytoplasmic receptors tcrmcd immu-
`nophilins (immunosuppressant binding proteins). Studies
`in this area have focused on the structural basis for the
`
`molecular recognition of ilnmunosupprcssants by immu-
`nophilins and the biological consequences of their inter-
`actions. Dcfining the biological roles of this emerging
`family of receptors and their ligands may illuminate the
`process of protein trafiicking in cells and the mechanisms
`of signal transmission through the cytoplasm.
`
`E.sEmtc:H DURING THE l’AS'l' [‘1E(JM)l-1 Has (:oN'rluIsU':‘1sn
`
`significantly to our knowledge ofT lymphocyte fimction.
`The identification and functional analysis ol‘T cell surface
`rcccptors (1) and nuclear transcription factors (2) have made Ll'lt'.St:
`components of thc signal transduction apparatus among the best
`understood in biology. This understanding is largely due to the use
`of probe reagents, such as monoclonal antibodies and radiolabclcd
`nucleic acids, that have been developed for the study of surface and
`nuclear phenomena, respectively. However, the mechanisms for the
`transduction of signals through the cytoplasm, the “black box” of
`the signal transduction patltway, remain mysterious.
`A family of natural products has emerged as probe reagents For
`cytoplasmic signaling mechanisms in the T lymphocyte. Thcsc small
`
`The author LI: a professor of Chemistry, Harvard University, Cambridge, MA 01133.
`IH JRNUARY 1991
`
`molecules are immunosupprcssants that appear to exert thcir inhib-
`itory actions distal to early rncmbranoassociatcd events and proxi-
`mal to nuclear processes. Studies on a family of immunosupprcssant
`binding proteins, the immunophilins, have attempted to identify the
`structural rcquircmcnts for high—aHinity interactions between immu-
`nophilins and their immunosupprcssivc ligands and the biological
`consequences of the formation ofimniunophilin-ligand complexes.
`Although then: is much to explore in this avcnuc of research, some
`gcncral principles :lS.‘iOCl:ltC(.l with the intermediary events of signal
`processing are emerging.
`
`The Immunosuppressants
`
`Cyclosporin A (GSA), an inhibitor ofT cell activation, is currently
`the favored therapeutic agent For prevention of graft rejection after
`organ and bone marrow transplantation, and it has been credited
`with initiating a revolution in clinical transplantation (3-5). The
`recently discovered compound FK506 inhibits T cell activation by
`mechanisms that arc similar to thosc ofC.-3A, but FK506 is 10 to 100
`times as potent (6). FK506 has performed remarkably well in initial
`human clinical transplantation trials (7, 8}, dcspitc reports of toxic
`effects in animals (6). Rapamycin inhibits T cell activation at
`concentrations comparable to those of the structurally rclatcd
`FK506, yet with mechanisms that are strikingly dilfcrcnt from those
`mediated by FK506, and thus CsA (9). Only CSA, FK506, and
`rapamycin have bccn uscd For the identification of members of the
`immunophilin class. A nonnarural ligand, 5(}6BD (10), and analogs
`of CSA (1143) have also provided insights into the inhibitory
`mcchanisrns of ln1mLIn0SUpp1'K'.S5aI1L'i. Many recently discovered
`imrnunosupprcssivc agents (14) with undcfinccl mcchanisrns, such as
`AR'['1CL1.‘.S
`283
`
`- st-Ward Pharm.
`Exhibit 1035
`Page 004
`
`West-Ward Pharm.
`Exhibit 1035
`Page 004
`
`

`

`diseodermolide (I5) and cleoxyspergualin (16), promise to reveal
`new facets of cytoplasmic signaling mechanisms (17) (Fig. 1).
`
`The Immunophilins
`
`The predominant CSA-binding protein in T lymphocytes is the
`soluble, cytosolie receptor cyclophilin (18, 1'9). Cyclophilin is an
`abundant and ubiquitous protein that is found in both prokaryotic
`and eukaryotie organisms. The major isoform of human cyclophilin
`has a mass of 17,737 daitons and an isoelectric point (pl) of 9.3.
`Two groups have independently reported that cyciophilin is identi-
`cal to peptidyl—prolyl isomerase (20, 21}, an enzyme that catalyzes
`the interconversion of the rise and traits-rotaincrs of the peptidyl—
`prolyl amide bond of peptide and protein substrates, and this
`rotamase activity is potently inhibited by (EA.
`Shortly after this discovery,
`the predominant FK506-binding
`protein in calf thymus, human spleen, and the T cell line Iurkat,
`termed FKB1’, was isolated and characterivcd in two laboratories
`(22, 23). Like eyclophilin, FKBP was shown to have rotamase
`activity toward a peptide substrate. FK506 inhibits the rotaniase
`activity of FKBP, but not of cyclophilin; likewise, CsA does not
`inhibit the rotamase activity of FKBP. The cloning (24, 25) and
`overexpression (24) of human recombinant FKBP and the cloning
`of an FKBI’ from Nenrospara cram: (26) revealed that, despite their
`common enzymatic properties, FKBP and eyclophilin have dissim-
`ilar sequences. Human FKBP has a mass of 11,819 daltons and, like
`cyclophilin, is a basic {pl = 8.9) (22, 24), cytosolic protein (27). A
`prokaryotic organism, Neisseria meningirirlis, was Found to have an
`open reading frame that encodes an FKBI’—like protein (24). More
`recently, FKBP was shown to be die predominant rapamycin-
`binding protein in yeast, calf thymus, and human T cells (Iurkar)
`(28). Rapamycin {dissociation constant K‘, = 0.2 nM) has an even
`higher affinity for FKBP than does FK506 (K, = 0.4 nM), and is
`also a potent
`inhibitor of Fl(BP’s rotamase activity (inhibition
`
`constant K; = 0.2 nM) (29).
`Although cyciophilin and FKRP are the only well-cliaracterizcd
`immunophilins, other members of this family are known to exist and
`are currently being investigated. For example, a CsA-binding phos-
`phoprotcin of relative molecuiar mass (M,) 45,000 has been detect-
`ed in Iurkat cells (30), and phosphoproteins of M, 60,000 and
`80,000 from this same cell line bind to both FK506 and rapamycin
`(28). The m'rm./l gene of Drosaplifla (31, 32) and a second cyclo-
`philin-related gene in Srrtdmromyres rerevisine (33) encode proteins
`that show high homology to cyclophilin. Several low molecular
`weight, basic proteins that are retained on CsA, FK506, or tapa-
`mycin aflinity matrices have also been noted (22, 28). Partial
`sequence determination of FK506- and rapamycin—binding immu-
`nopliilins of M, 30,000 and M, 13,000 has revealed that these
`molecules,
`together with FKBI’, are members of a previously
`unknown family of immunophilins (34). Questions concerning the
`biological relevance, the rotamase activity, and the aflinity to the
`cognate ligands of these lowvabundance immunophilins should soon
`be answered.
`
`Although the exact cellular concentrations of FKBP and cyclo-
`philin are not known, both are abundant. Saturation binding in the
`cytosol of Iurkat cells was reported to occur at 35 nM diI:ririo-
`FK506 (27). As FKBP is the predominant cytosolic receptor for
`drug, this measurement is largely accounted for by FKBP, and thus
`the cytoplasmic concentration of FKBP may approach 5 rLM. The
`high~alfinity FKBP ligands FK506 and rapamycin, however, inhibit
`T cell proliferation at subnanomolar concentrations (median inhibi-
`tion concentration IC59 ——0.5 r1M) (29, 35). Therefore, inhibition of
`the rotamase activity of FKBP is very likely an insufiicient require-
`ment for mediating the actions of these drugs in T lymphocytes,
`because only a small fraction of the enzyme would be inhibited at
`effective drug concentrations. This point has been confirmed by
`mechanistic studies of FK506 and rapamycin (see below); likewise,
`investigations ol'CsA analogs support a similar conclusion regarding
`the rotamase activity of cyclophilin (12).
`
`Molecular Recognition by the Immunophilins
`
`The rotamase activity of these immunophilins and the ability of
`their immunosuppressive ligands to act as
`rotarnase inhibitors
`provide an opportunity for exploration ofthe molecular basis for the
`high-alfinity interactions that exist between them. Initial mechanistic
`studies of cyclophilin led to the suggestion that catalysis of the
`interconversion of ris- and !.mi'lS-vl'()Ial”l'Ic1'5 of a peptide substrate is
`achieved by the formation of a covalent bond to the carbonyl of the
`peptidyl~pt‘olyl amide with a cysteine—derived thiol (36). Loss of
`amide resonance would be expected to lower the activation barrier
`to rotation about the amide C—N bond. Site-directed mutagenesis of
`human recombinant eyclophilin allowed the systematic replacement
`of all four cysteine residues in cyelophilin with alanine. Because all
`four mutants enzymes were Fully active in the rotamase and bintling
`assays, cysteine was ruled out as a parlzicipating residue in catalysis
`(37).
`Additional mechanistic studies with both cyclophilin (38) and
`FKBP (39) strongly suggest that these enzymes catalyve rotamcr
`interconversion by noncovalent stabilization of the twisted amide
`transition state For
`the noncatalyzed isomerization. The amide
`liuictionality exhibits a strong preference for a planar geometr)’.
`wherein the nitrogen lone pair is in conjugation with the carbonyl
`1r-cloud. The energy cost ofthe twisted amide structure (Fig. 2A) is
`15 to 20 kcal (40). The structural basis For eyclophilin and FKl1l".'i
`ability to stabilize this transition-state structure must await further
`structural analyses of rotamase-peptide (or inhibitor) complexcri.
`sci I-ZNILJI-L, v01.. 25]
`
`West-Ward Pharm.
`Exhibit 1035
`Pae O05
`
`Flapamycln
`
`M:3**m,a
`
`:
`
`'
`
`Moon DCDNII-_,
`
`H
`“Htcmk/tNHLl,NHl'C“:h”“iC“:la““2
`0
`
`H!"
`
`K
`
`Dlscodermollde
`
`Deoxyspergualin
`
`Fig. 1. Probe reagents of intracellular signaling pathways. (A) Recently
`investigated ilnmunophilin ligands.
`(B) lmmnnosupptessive agents with
`unknown mechanisnis ul'T cell inhibition. (Me, methyl.)
`234
`
`West-Ward Pharm.
`Exhibit 1035
`Page 005
`
`

`

`presenting cell results in the activation ofa TCR signal transmission
`pathway. The signal
`is trauisduccd through the cytoplasm by an
`uni-znown mechanism and results in the activation of specific nuclear
`transcription factors, such as nuclear l‘acl'or of activated T cells
`(NF-AT). These nuclear factors help to regulate the transcription of
`T cell activation genes, sucli as the gene of the lymphokine inter-
`leukin-2 (IL-2). Translation of the resuitant message is followed by
`secretion of IL-2. CsA and FK506 are pt)tent inhibitors of the
`TCII-mediated signal transduction pathway, as evidenced by their
`ability to inhibit the transcription of early T cell activation genes
`(44). Csfl (45) and FK506 (29, 46), but not rapamycin, inhibit the
`binding of NF-AT to the IL-2 enhancer and inhibit transcriptional
`activation by NF—AT. CsA and FK506 also inhibit transcription
`mediated by AP-3 and Oct-1, and partially inhibit transcription
`mediated by NF—i<B (45, 46). Another illustration involves the use of
`T cell hybridomas that undergo a suicidal event called apoptosis
`after stimulation of the TCR-CD3 complex. CsA and FK506, but
`not rapamycin, are potent inhibitors of apoptosis induced by an
`antibody to the TCR—CD3 complex (29).
`T ceil activation involves not only IL-2 secretion but aiso expres-
`sion of the lymphokine receptor IL-29. on the surface of the cell.
`After the binding of IL-2 to IL-211, a lympholtine receptor (LKR)
`signal transmission pathway is activated. Transduction of this signal
`again proceeds by an unknown mechanism through the cytoplasm
`and into the nucleus, where a dilferent set of genes is transcribed.
`Whereas rapamycin, despite its structural similarity to FK506, has
`no effect on the production of IL-2, it potently inhibits the response
`of the T cell to IL-2 (29, 35, 47). Rapamycin thus appears to inhibit
`a later LKR—associated signaling pathway (Fig. 3). Because both
`rapamycin and FK506 are potent inhibitors of the rotamase activity
`of FKBP and inhibit distinct signaling pathways,
`these results
`support the suggestion that the inhibition of rotamase activity of
`FKBP is an insufficient requirement for mediating the actions of
`FK506 and rapamycin (I0, 29).
`In addition to their ability to inhibit diflcrent T cell activation
`events, rapamycin and FK506, but not Cm, have been shown to be
`mutually inhibitory in a variety of Functional assays (29, 47). These
`results suggest :1 role for either a single immunophilin or separate
`immunophilins that share a common receptor site in mediating the
`actions of FK506 and rapamycin. Furthermore,
`rapaniycin can
`distinguish the biological actions of FK506 and CsA, because it has
`no effect on the actions of CSA.
`
`The mutual inhibition of I-‘K506 and rapamycin was shown to be
`subject to a buficring action by FKBP (29). A concentration 10 to
`100 times the effective drug concentration (IC5,, -- 0.5 nM) of
`
`(1) TCH signal
`
`Fig. 3. Early events of the T cell activatitill cascade and the sites of inhibitory
`action by CSA, FK506, and rapamycin.
`
`call dlflerentlatlon
`cell proliteralian
`
`AR'!'IC[.[-ES
`
`285
`
`st-Ward Pharm.
`Exhibit 1035
`Page 006
`
`Fig. 2. (A) Model ofthe transition state structure ofa twisted peptidyl-prolyl
`amide bond that is stabilized by the rotamase enzymes cyclophilin and
`FKBP. (B) Substructure of FK506 and (C) CL-iA (both From x-ray) that is
`proposed to mimic the twisted amide bond of a peptide substrate. (D)
`Substructure of FKSU6 (R = ()Me) and rapamycin (R = H) proposed to
`mimic a twisted leucyl-prolyl amide bond of a peptide substrate.
`(E)
`l_A.'lICyi'PI'0I)‘l fragment indicating structural similarities to itmnunosuppres-
`S'.‘IIl{ substructures.
`
`However, the unusual structure of the immunophilin ligands and
`preliminary structural
`investigations of the immunophilin-ligand
`complex suggest a basis for their rotamasc inhibitory properties. The
`total synthesis of a l3C-labeled FKSU6 (41) provided a reagent to
`carry out “C nuclear magnetic resonance (NMR) studies of the
`FKSDG-FKBP complex (42). It was suggested that
`the ltetonc
`carbonyl adjacent to the homoprolyl amide bond of FK506 (Fig.
`28) and rapamycin is a mimic of the amide carbonyl ofa peptide
`substrate. Thus, FK506 and rapamycin are transition-state analogs
`in that their ground-state geometry is similar to the tr-ansition—state
`structure of a peptide substrate (Fig. 2, A and B). Also, the side
`chain of the unusual amino acid N—methyl-butenylthreonine
`(MeBmt) of CSA, which is known to be essential for high—aflinity
`binding ofCsA to cyclophilin (11, 12), has structural similarity (Fig.
`2C) to the aforementioned uansition-state structure (Fig. 2A). This
`side chain may be a different type ofsurrogate for the twisted amide
`structure. In this regard, the oi-branched hydroxyethylcne substruc-
`ture of CsA is reminiscent of the hydroxyethylene amide isostere
`found in aspartyl protease inhibitors such as pepstatin.
`The analogy of the ct-keto-homoprolyl grouping in FK506 and
`rapamycin to a twisted-amide bond of a peptide substrate was
`extended (39). A substrate containing a lcucy!-prolyl dipeptide was
`found to be optimal for FKBP (39, 43). The structural similarities of
`FK506 and rapamycin to a twisted leucyl-amide bond (Fig. 2, D
`and B) suggest these agents may be transition-state analogs of a
`lcucyl-(twisted amide)—prolyl peptide substrate for FKBI’.
`
`The Biological Function of Immunophilins
`
`The complex series of events that comprises the T cell activation
`cascade transpires over several days (2). (ISA, FK506, and rapamycin
`act within the first hours of the process (Fig. 3). Stimulation of the
`T cell receptor (TCR) by Foreign antigen presented by a major
`histocompatibility (MHC) molecule on the surface oi’ an antigen-
`lli IANUARY 199i
`
`West-Ward Pharm.
`Exhibit 1035
`Page 006
`
`

`

`|
`
`m.'.’ll3.‘;l:Zn <33‘
`
`FI<B?!dn.ig
`complex
`+dnJg
`
`‘mi
`
`antagonist O
`
`O FKBP!antagonis1
`complex
`
`Fig. 4. Schematic illustrating the relative abundance of receptor and ligands.
`The abundant FKBP, which may have a cellular function as a foldase,
`is
`converted to an inhibitory complex on binding of the drug and buliers the
`actions of the antagonist.
`
`either agent is required for inhibition ofrhe actions of the other (29,
`47). Thus, die abundant
`(---5 uM) uneomplexcd immunophilin
`sequesters the antagonist. Only after the excess binding sites are
`occupied does the concentration of the antagonizing agent
`rise
`sufiicielltly to displace the drug effectively from its biological
`receptor. These findings also implicate the immunophilin-drug com-
`plex as Lhe inhibitor of T cell activation. Although a role For FKBP as
`the mediator of the biological actions of rapamycin and FK506 has
`not been shown, the buffer effect of FKBP evident in the studies of
`reciprocal inhibition should also be operative with competing cellular
`receptors for these drugs. The low-abundance immunophilins must
`overcome the high abundance of FKBP and its high alfinity for drug
`in order to compete eliectively for binding (Fig. 4-).
`Invoking the immunophilin-drug complex as the biological effec-
`tor addresses the issue of how the ubiquitous cyclophilin and FKBP
`could be involved in T cell activation. One possibility is that these
`proteins have a more general function, perhaps assisting in protein
`folding in vivo by acting as foldases. Only when the immunophilin
`combines with its immunosuppressivc ligand does it inhibit T cell
`activation. The cellular immunophiiin receptor (possibly FKBP),
`bound to either FKSD6 or rapamycin, may interact with diflerent
`molecules in distinct pathways of’? cell activation. According to this
`hypothesis, the specificity of the factors associated with different
`signaling pathways is determined by the precise geometry of the
`immunophilinvdrug complex. Evidence has been presented (48) that
`the cyclophilin-CsA complex, and not (L-:A, is the agent responsible
`for the toxic actions of CSA in two lower eukaryotes. CSA-sensitive
`strains of N. crassrt and S. rereufsiae were grown in the presence of
`(1-:A. Analysis of the CsA-resistant mutant strains that resulted
`revealed that either they no longer produced cyclophilin or, if they
`did, the cyclophilin of the mutant strains did not bind CsA (-48}.
`The common biological receptor site implied by the mutual
`inhibition of FK506 and rapamycin suggests that the immunophilin
`may present multiple ligands to cytoplasmic components of signal
`transmission pathways. The ability of a single immunophilin to
`present two immunosupprcssive ligands to effectors associated with
`two distinct pathways raises the possibility that immunophilins may
`filnction as general presenting molecules, by analogy to the way that
`MHC molecules present a large number ofpcptides to the polymor-
`phic TCRs. Ifcndogenous immunophilin ligands exist that function
`similarly to the imnmnosuppressive natural products,
`then the
`immune system may have used the molecular recognition associated
`with rotamase catalysis for
`the purpose of modulating T cell
`activation.
`
`In the case of FK506 and raparnycin, the lcucyl-(twisted amide)-
`proiyl pcptidomimetic fragment shared by these drugs constitutes
`the structural element largely responsible for binding to FKBP. This
`common immunophilin binding domain is then fused to distinct
`effector elements that, after presentation by the immunophilin,
`determine the signaling pathway with which the drug will interfere
`(Fig. 5, A and B). This view of FK506 and rapaliiycin as dual
`domain agents was tested with an FKBI’ ligand designed to contain
`136
`
`alfector
`element
`
`50630
`
`Fig. 5. Domainal analyses of FKBI’ ligands. (A) I-‘K506 and (B) rapamycin
`binding domain and effector elements (shaded). (C) Structure of FKSU6
`(x-ray} with enolate spacer drawn to illustrate sealiiilding efiizct. {D} Removal
`of the outer loop of structure (C) results in 506131), a high-allinity (K, = 5
`nM) ligand to FKBP.
`
`the putative FKBP-binding domain of FK506 and raparnycin in the
`conformation found in the solid state of FK506 (Fig. SC) (10). The
`resultant molecule, 506313, was found to bind with high affinity (K,(
`= 20 nM) and to inhibit the rotamase activity (K, = 5 nM) of FKBI’
`potently (Fig. 5D}. Because 506151)
`lacks the putative effector
`elements of either FK506 or rapamycin,
`it was not expected to
`inhibit either the TCR or LKR signaling pathways associated with
`T cell activation. Indeed, 5061'-l-D does not inhibit T cell activation
`by either mechanism, even at high concentrations. However, this
`immunophilin ligand inhibits the actions of both FK506 and
`rapamycin at concentrations that would be anticipated given the
`relative aflinity of these agents to FKBP and the buffer effect (10). In
`addition to illustrating that the inhibition ofthe rotamasc activity of
`FKBP is an insufficient requirement For mediating the actions of
`FK506 and rapamycin, these studies support the view that these
`immunosuppressants are composed of two domains, one important
`for binding to immunophilin (binding element) and one essential
`for biological action (efiector element).
`
`Future Prospects
`
`The presence of cyclophilin and FKBP in many organisms
`suggests that these enzymes may have some general cellular func-
`tion. The recent discoveries of proteins that assist in protein folding.
`unfolding, and translocation in vivo provide precedent For a similar
`function for rotarnase enzymes (49). A role For an Esrherfcliin roli
`cyclophilin in Lhe secretory pathway was suggested after the discov-
`ery that
`it was localized in the pcriplasm (50). Similarly,
`the
`demonstration that an N. rrnssn FKBP cataiyaed protein folding and
`the id