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`
`NOSIGVN
`
`IM 9ULS§
`
`is NOLAVG M SIZI
`NISNODSIM I\INn
`
`KIT AHd039 AS01039
`20I6N26/£0/I0 3992950600000
`92
`
`2083 xx 1808 J.2i—H\13 xxxxxxxxxxxx
`
`
`
`Par Pharm., Inc.
`Exhibit 1035
`Page 001
`
`Par Pharm., Inc.
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`

`
`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`SCIENCE
`
`ISSN 0036-8075
`18 JANUARY 1991
`VOLUME 251
`NUMBER 4991
`
`247
`
`249
`
`This Week in Science
`
`Teaching and Research
`
`256
`
`Safety of Bovine Growth Hormone: D. S. KRONFELD; J. C. IUSKEVICH AND
`C. G. GUYER I Interpreting Cancer Tests: 1. D. WILSON; G. W. GRJBBLE I
`Kidney Transplantation: Overlooked Pioneer: G. B. ELION
`
`260 The Rush to Publish I Lessons from Physics
`263 Third Strike for Idaho Reactor
`
`Research News
`
`264 CDC Abandons Plans for AIDS Survey
`Healy Nominated
`
`265 GAO and DOD Get Into a Cat Fight
`266 Science Literacy: The Enemy Is Us I Science’s Top 20 Greatest Hits
`
`268 New Light on Writing in the Americas
`271 Montagnier Pursues the Mycoplasma—AIDS Link
`272 Despite Reports of Its Death, the Big Bang Is Safe
`274 Global Temperature Hits Record Again
`275 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
`
`Efi; 277
`
`Researcl Article
`
`283
`
`288
`
`293
`
`Subsistence Economy of El Paraiso, an Early Peruvian Site: I. QUILTER,
`B. OJEDA E., D. M. PEARSALL, D. H. SANDWEISS, I. G. JONES, E. S. WING
`Chemistry and Biology of the Immunophilins and Their Immunosuppressivc
`Ligands: S. L. SCHREIBER
`
`CCAAT-Enhancer Binding Protein: A Component of a Dilferentiatzion Switch:
`R. M. UMEK, A. D. FRIEDMAN, S. L. MCKNIGHT
`
`An Antimony Sulfide with a Two-Dimensional, Intersecting System of Channels:
`J. B. PARISE
`
`SCIENCE (ISSN 0036-0075) is published weekly on Friday, except the last week in December, by the American
`Association tor the Advancement of Science, 1333 H Street, NW, Washington, DC 20005. Second-class postage
`(publication No. 484460) paid at Washington, DC, and additional mailing offices. Copyright © 1990 by the American
`Association tor the Advancement of Science. The title SCIENCE is a registered trademark ol the AAAS. Domestic
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`Science is 0036-8075/83 $1 + .10. Science is indexed in the Readers Guide to Periodical Literature and in several
`specialized indexes.
`The American Association for the Advancement of Science was founded in 1846 and incorporated in 1874. its
`objectives are to further the work of scientists, to facilitate cooperation among them, to foster scientific freedom and
`responsibility, to improve the etfectiveness 01 science in the promotion of human welfare. to advance education in
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`science in human progress.
`
`SCIENCE, VOL. 251
`
`Par Pharm., Inc.
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`244
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`
`
`COVER Lakes and ponds on the arctic tundra with Itigaknit Mountain in the
`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]
`
`294 Local Structure and Chemical Shifts for Six-Coordinated Silicon in High-Pressure
`Mantle Phases: I. F. STEBBINS AND M. KANZAKI
`298 Arctic Lakes and Streams as Gas Conduits to the Atmosphere: Implications for
`Tundra Carbon Budgets: G. W. KLING, G. W. KIPPHUT, M. C. MILLER
`301 Putative Skeletal Neural Crest Cells in Early Late Ordovician Vertebrates from
`Colorado: M. M. SMITH
`
`303 Altered Perception of Species-Specific Song by Female Birds After Lesions of a
`Forebrain Nucleus: E. A. BRENOWITZ
`
`305 The Effect of Anti-Neoplastic Drugs 011 Murine Acquired Immunodeficiency
`Syndrome: C. SIMARD AND P. ]oLIcoEUR
`308 Evidence for Biased Gene Conversion in Concerted Evolution of Ribosomal DNA:
`D. M. HILLIS, C. MoRrrz, C. A. PORTER, R. J. BARER
`310 The Effect of the Floor Plate on Pattern and Polarity in the Developing Central
`Nervous System: S. HrRANo, S. FUSE, G. S. SOHAL
`313 Regulation of Interleul<in—2 Gene Enhancer Activity by the T Cell Accessory
`Molecule CD28: J. D. FRASER, B. A. IRVING, G. R. CRABTREE, A. WE1ss
`
`316 Microwave Sounding Units and Global Warming: B. L. GARY AND S. I. KELHM;
`R. W. SPENCER AND I. R. CHRISTY I Lipid Flow in Locomoting Cells:
`M. S. BRETSCHER; K. ]ACoRsoN, I. LEE, M. GUSTAFSSON,
`MAGNUssoN I
`Bryozoan Morphological and Genetic Correspondence: What Does It Prove?:
`I. LEVINTON; I. B. C. JACKSON AND A. H. CHEETHAM
`
`324 Authors of Their Own Lives, reviewed by A. SICA I Australian Ecosystems,
`M. LOWMAN I Thalamic Oscillations and Signaling AND Brainstem Control of
`Wakefulness and Sleep, C. KOCH I Books Received
`
`
`
`Technical Comments
`
`
`
`Boo
`
` views
`
`Pro ucts & Materials
`
`328
`
`Protein Immunoblotdng Incubation Rotator I Micromanipulator Table I Leiden
`Mictoincubator I Freezing Stage with Microtome I Data Analysis for the
`Macintosh I Monoclonal Antibodies I Literature
`
`Board of Directors
`Richard C. Atkinson
`Retiring President,
`Chairman
`Donald N. Langenberg
`President
`Leah M. Lederman
`President-elect
`
`Mary Ellen Avery
`Francisco J. Ayala
`Eugene H. Cota-Robles
`Robert A. Frosch
`Joseph G. Gavin, Jr.
`John H. Gibbons
`Beatrix A. Hamburg
`Florence P. Haseltine
`William T. Golden
`Treasurer
`
`Richard S. Nicholson
`Executive Oflicer
`
`Editorial Board
`Charles J. Amtzen
`Elizabeth E. Bailey
`David Baltimore
`Vinlliam F. Brinkman
`E. Margaret Burbidge
`Pierre—Gi||es de Gennes
`Joseph L. Goldstein
`Mary L. Good
`Harry B. Gray
`F. Clark Howell
`Paul A. Marks
`Yasutomi Nishizuka
`Helen M. Ranney
`Howard A. Schneiderman
`Roberi M. Solow
`Edward C. Stone
`James D. Watson
`
`Board oi Reviewing
`Editors
`John Abelson
`Frederick W. Alt
`Don L. Anderson
`Stephen J. Benkovic
`Gunter K-J Blobel
`Floyd E. Bloom
`Henry Fl. Bourne
`James J. Bull
`Kathryn Calame
`Charles R. Cantor
`Ralph J. Cicerone
`John M. Coffin
`Robert Doriman
`Bruce F. Eldridge
`Paul T. Englund
`Fredric S. Fay
`
`Harry A. Fozzard
`Theodore H. Geballe
`Roger I. M. Glass
`Stephen P. Golf
`Corey S. Goodman
`Stephen J. Gould
`Eric F. Johnson
`Stephen M. Kosslyn
`Konrad B. Krauskopt
`Charles S. Levings Ill
`Richard Losick
`John C. McGifl
`Anthony H. Means
`Mortimer Mishkin
`Roger A. Niooll
`William H. Orme-Johnson ill
`Carl 0. Pabo
`Yeshayau Pocker
`
`Dennis A Powers
`Erkki Ruoslahti
`Thomas W. Schoener
`Ronald H. Schwartz
`Terrence J. Sejnowski
`Thomas A. Steitz
`Robert T. N. Tljan
`Emil R. Unanue
`Geerat J. Vermeij
`Bert Vogelstein
`Harold Weintraub
`Zena Werb
`George M. Whitesides
`Owen N. Wiite
`William E. Wood
`Keith Yamamoto
`
`13 JANUARY 1991
`
`TABLE OF CONTENTS
`
`Z45
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` 7KLV PDWHULDO PD\ EH SURWHFWHG E\ &RS\ULJKW ODZ 7LWOH  86 &RGH

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`Par Pharm., Inc.
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`

`
`discodermolide (15) and deoxyspergualin (16), promise to reveal
`new facets of cytoplasmic signaling mechanisms (17) (Fig, 1).
`
`I The Immunophilins
`The predominant CsA—binding protein in T lymphocytes is the
`soluble, cytosolic receptor cyclophilin (18, 19). Cyclophilin is an
`abundant and ubiquitous protein that is found in both prokaryotic
`and eukaryotic organisms. The major isoform of human cyclophilin
`has a mass of 17,737 daltons and an isoelectric point (p1) of 9.3.
`Two groups have independently reported that cyclophilin is identi-
`cal to peptidyl-prolyl isomerase (Z0, 21), an enzyme that catalyzes
`the interconversion of the cis— and tmns—rotamers of the peptidyl—
`prolyl amide bond of peptide and protein substrates, and this
`rotamasc activity is potently inhibited by CsA.
`Shortly after this discovery,
`the predominant FK506-binding
`protein in calf thymus, human spleen, and the T cell line Iurkat,
`termed FKBP, was isolated and characterized in two laboratories
`(Z2, 23). Like cyclophilin, FKBP was shown to have rotamasc
`activity toward a peptide substrate. FK506 inhibits the rotamase
`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 FKBP from Neuroxpom crassa (26) revealed that, despite their
`common enzymatic properties, FKBP and cyclophilin have dissim-
`ilar sequences. Human FKBP has a mass of 11,819 daltons and, like
`cyclophilin, is a basic (p1 = 8.9) (22, 24), cytosolic protein (27). A
`prokaryotic organism, Neisseria nzerringitidis, was found to have an
`open reading frame that encodes an FKB1’—like protein (24). More
`recently, FKBP was shown to be the predominant rapamycin-
`binding protein in yeast, calf thymus, and human T cells (Jurkat)
`(28). Rapamycin (dissociation constant K,, = 0.2 nM) has an even
`higher aflinity for FKBP than does FK506 (Kd = 0.4 nM), and is
`also a potent inhibitor of FKBP’s rotamasc activity (inhibition
`
`constant K, = 0.2 nM) (29).
`Although cyclophilin and FKBP are the only well—characterized
`immunophilins, other members of this family are known to exist and
`are currently being investigated. For example, a CsA-binding phos-
`phoprotein of relative molecular mass (M,) 45,000 has been detect-
`ed in Jurkat cells (30), and phosphoproteins of M, 60,000 and
`80,000 from this same cell line bind to botl1 FK506 and rapamycin
`(28). The m'naA gene of Drosophila (31, 32) and a second cyclo-
`philin-related gene in Sarclmromyres rereuisiae (33) encode proteins
`that show high homology to cyclophilin. Several
`low molecular
`weight, basic proteins that are retained on CsA, FK506, or rapa-
`mycin aflinity matrices have also been noted (22, 28). Partial
`sequence determination of FK506- and rapamycin-binding immu-
`nophilins of M, 30,000 and M, 13,000 has revealed that these
`molecules,
`together with FKBP, are members of a previously
`unknown family of immunophilins (34). Questions concerning the
`biological relevance, the rotamasc activity, and the aflinity to the
`cognate ligands of these low~abundance 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 >5 nM ditritio-
`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 nM. The
`high-aflinity FKBP ligands FK506 and rapamycin, however, inhibit
`T cell proliferation at subnanomolar concentrations (median inhibi-
`tion concentration IC50 ~0.5 nM) (29, 35). Therefore, inhibition of
`the rotarnase activity of FKBP is very likely an insuflicient require-
`ment for mediating the actions of these drugs in T lymphocytes,
`because only a small fraction of the enzyme would be inhibited at
`elfective drug concentrations. This point has been confirmed by
`mechanistic studies of FK506 and rapamycin (see below); likewise,
`investigations of CsA analogs support a similar conclusion regarding
`the rotamasc activity of cyclophilin (12).
`
`
`M8.,.
`
`REM
`
`0Ma Ms
`
`bile
`
`Rapamycin
`
`Molecular Recognition by the Immunophilins
`
`The rotamase activity of these immunophilins and the ability of
`their irnmunosuppressive ligands to act as
`rotamasc inhibitors
`provide an opportunity for exploration of the molecular basis for the
`high—affinity interactions that exist between them. Initial mechanistic
`studies of cyclophilin led to the suggestion that catalysis of the
`interconversion of ri'.v— and trans-rotamers of a peptide substrate is
`achieved by the formation of a covalent bond to the carbonyl of the
`peptidyl-prolyl 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 mutagencsis of
`human recombinant cyclophilin allowed the systematic replacement
`of all four cysteine residues in cyclophilin With alanine. Because all
`four mutants enzymes were fully active in the rotamasc and binding
`assays, cysteine was ruled out as a participating residue in catalysis
`(37).
`Additional mechanistic studies with both cyclophilin (38) and
`FKBP (39) strongly suggest that these enzymes catalyze rotamer
`interconversion by noncovalent stabilization of the twisted amide
`transition state for
`the noncatalyzed isomerization. The amide
`functionality exhibits a strong preference for a planar geometry,
`wherein the nitrogen lone pair is in conjugation with the carbonyl
`Tl’-Cl0lld. The energy cost of the twisted amide structure (Fig. 2A) is
`15 to 20 kcal (40). The structural basis for cyclophilin and FKBP’s
`ability to stabilize this transition-state structure must await further
`structural analyses of rotamasc-peptide (or inhibitor) complexes.
`SCIENCE, VOL. 251
`
`Par Pharm., Inc.
`Exhibit 1035
`Page 005
`
`2
`
`""‘°"*""“‘°”*’“""2
`
`B O
`
`
`i OH
`
`oi:
`,.
`-
`oH ocoNH
`HaN\I/NHlCH2ls
`nu
`Ma‘
`Me Me"
`,
`Me
`0
`NH
`OH
`on
`Discodermolide
`
`Deoxyspergualin
`
`(A) Recently
`Fig. 1. Probe reagents of intracellular signaling pathways.
`investigated immunophilin ligands.
`(B) Immunosuppressive agents with
`unknown mechanisms of T cell inhibition. (Me, methyl.)
`284
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`Par Pharm., Inc.
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`Page 005
`
`

`
`presenting cell results in the activation of a TCR signal transmission
`pathway. The signal
`is transduced through the cytoplasm by an
`unknown mechanism and results in the activation of specific nuclear
`transcription factors, such as nuclear factor of activated T cells
`(NF-AT). These nuclear factors help to regulate the transcription of
`T cell activation genes, such as the gene of the lymphokine inter-
`leukin-2 (IL-2). Translation of the resultant message is followed by
`secretion of IL-2. CsA and FK506 are potent inhibitors of the
`TCR-mediated signal transduction pathway, as evidenced by their
`ability to inhibit the transcription of early T cell activation genes
`(44). CsA (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—I, and partially inhibit transcription
`mediated by NF—KB (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 cell activation involves not only IL-2 secretion but also expres—
`sion of the lymphokine receptor IL-2R on the surface of the cell.
`After the binding of IL-2 to IL-2R, a lymphokine 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 difierent 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 Tcell 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 insuflicient requirement for mediating the actions of
`FK506 and rapamycin (10, 29).
`In addition to their ability to inhibit different T cell activation
`events, rapamycin and FK506, but not CsA, have been shown to be
`mutually inhibitory in a variety of functional assays (29, 47). These
`results suggest a role for either a single immunophilin or separate
`immunophilins that share a common receptor site in mediating the
`actions of FK506 and rapamycin. Furthermore, rapamycin can
`distinguish the biological actions of FK506 and CsA, because it has
`no effect on the actions of CsA.
`
`The mutual inhibition of FK506 and rapamycin was shown to be
`subject to a buffering action by FKBP (29). A concentration 10 to
`100 times the effective drug concentration (IC50 ~ 0.5 nM) of
`
`(1) TCR signal
`
`
`
`
`(2) LKR signal
`
`Fig. 3. Early events of the T cell activation cascade and the sites of inhibitory
`action by CsA, FKSO6, and rapamycin.
`
`cell differentiation
`cell prolileration
`
`ARTICLES
`
`285
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`Exhibit 1035
`Page 006
`
`
`
`Fig. 2. (A) Model of the transition state structure of a twisted peptidyl-prolyl
`amide bond that is stabilized by the rotamase enzymes cyclophilin and
`FKBP. (B) Substructure of FK506 and (C) CsA (both from x-ray) that is
`proposed to mimic the twisted amide bond of a peptide substrate.
`(D)
`Substructure of FKSO6 (R = OMe) and rapamycin (R = H) proposed to
`mimic a twisted leucyl—prolyl amide bond of a peptide substrate.
`(E)
`Leucyl—prolyl fragment indicating structural similarities to immunosuppres—
`sant substructures.
`
`However, the unusual structure of the immunophilin ligands and
`preliminary structural
`investigations of the immunophilin—ligand
`complex suggest a basis for their rotamase inhibitory properties. The
`total synthesis of a BC-labeled FK506 (41) provided a reagent to
`carry out 13C nuclear magnetic resonance (NMR) studies of the
`FK506-FKBP complex (42). It was suggested that
`the ketone
`carbonyl adjacent to the homoprolyl amide bond of FK506 (Fig.
`2B) and rapamycin is a mimic of the amide carbonyl of a peptide
`substrate. Thus, FK506 and rapamycin are transition-state analogs
`in that their ground—state geometry is similar to the transition—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 of CsA to cyclophilin (11, 12), has structural similarity (Fig.
`2C) to the aforementioned transition-state structure (Fig. 2A). This
`side chain may be a diiferent type of surrogate for the twisted amide
`structure. In this regard, the oubranched hydroxyethylene substruc-
`ture of CsA is reminiscent of the hydroxyethylene amide isostere
`found in aspartyl protease inhibitors such as pepstatin.
`The analogy of the or-keto-homoprolyl grouping in FK506 and
`rapamycin to a twisted-amide bond of a peptide substrate was
`extended (39). A substrate containing a leucyl—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 E) suggest these agents may be transition-state analogs of a
`leucyl-(twisted arnide)—prolyl peptide substrate for FKBP.
`
`The Biological Function of Immunophilins
`
`The complex series of events that comprises the T cell activation
`cascade transpires over several days (2). CsA, 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 of an antigen-
`18 JANUARY 1991
`
`Par Pharm., Inc.
`Exhibit 1035
`Page 006
`
`

`
`Immune
`modulation
`
`FKBP/drug
`
`=
`+d”’9 '
`. ‘_ 3:
`
`I
`
`'
`
`Foldase,
` Complex
`I ::> ‘protein translocalion
`etc,
`\ . FKBP/antagonist
`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
`convened to an inhibitory complex on binding of the drug and buffers the
`actions of the antagonist.
`
`either agent is required for inhibition of the actions of the other (29,
`47). Thus,
`the abundant (~5 nM) uncomplexed immunophilin
`sequesters the antagonist. Only after the excess binding sites are
`occupied does the concentration of the antagonizing agent rise
`sufficiently to displace the drug effectively from its biological
`receptor. These findings also implicate the immunophilin-drug com-
`plex as the 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 bulfer 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 affinity for drug
`in order to compete effectively for binding (Fig. 4).
`Invoking the immunophilin—drug complex as the biological effee~
`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 immunosuppressive ligand does it inhibit T cell
`activation. The cellular immunophilin receptor (possibly FKBP),
`bound to either FK506 or rapamycin, may interact with different
`molecules in distinct pathways ofT cell activation. According to this
`hypothesis, the specificity of the factors associated with different
`signaling pathways is determined by the precise geometry of the
`immunophilin-drug complex. Evidence has been presented (48) that
`the cyclophilin-CsA complex, and not CsA, is the agent responsible
`for the toxic actions of CSA in two lower eukaryotes. CSA-sensitive
`strains of N. crassa and S. rereuisiae were grown in the presence of
`CsA. 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 immunosuppressive ligands to elfectors associated with
`two distinct pathways raises the possibility that immunophilins may
`function as general presenting molecules, by analogy to the way that
`MHC molecules present a large number of peptides to the polymor-
`phic TCRs. If endogenous immunophilin ligands exist that function
`similarly to the immunosuppressive natural products,
`then the
`immune system may have used the molecular recognition associated
`with rotamase catalysis for
`the purpose of modulating T cell
`activauon.
`
`In the case of FK506 and rapamycin, the leucyl-(twisted atnide)-
`prolyl peptidomimetic 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 rapamycin as dual
`domain agents was tested with an FKBP ligand designed to contain
`286
`
` O
`
`Ma
`
`0
`
`‘remove
`effector
`element
`
`....
`
`2
`Ma Ma Ma
`
`506BD
`
`GM.»
`'V.'OH
`
`Fig. 5. Domainal analyses of FKBP ligands. (A) FK506 and (B) rapamycin
`binding domain and elfector elements (shaded). (C) Structure of FK506
`(x—ray) with enolate spacer drawn to illustrate scaffolding effect. (D) Removal
`of the outer loop of structure (C) results in 506BD, a high-affinity (K, = 5
`nM) ligand to FKBP.
`
`the putative FKBP-binding domain of FK506 and rapatnycin in the
`conformation found in the solid state of FK506 (Fig. 5C) (10). The
`resultant molecule, 506BD, was found to bind with high aflinity (Kd
`= 20 nM) and to inhibit the rotamase activity (K, = 5 nM) of FKBP
`potently (Fig. 5D). Because 506BD 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, 530631) 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 of the rotamase activity of
`FKBP is an insuflicient 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 (elfector element).
`
`Future Prospects
`
`The presence of cyclophilin and FKBP in many organisms
`suggests that these enzymes may have some general cellular flinc-
`tion. The recent discoveries of proteins that assist in protein folding,
`unfolding, and translocation in vivo provide precedent for a similar
`function for rotamase enzymes (49). A role for an Esrlzerirliia coli
`cyclophilin in the secretory pathway was suggested after the discov-
`ery that
`it was localized in the periplasm (50). Similarly,
`the
`demonstration that an N. mzxsa FKBP catalyzed protein folding and
`the identification of mitochondrial forms of N. rrassa cyclophilin
`and FKBP led to the suggestion that these immunophilins assist in
`the refolding of proteins that have traversed a biological membrane
`(26). The identification and characterization of new immunophilins
`will increase our understanding ofthese molecules. The structures of
`immunophilins and their drug complexes may prove illuminating, in
`regard to both enzyme mechanism and cell signaling inhibition.
`Many questions remain unanswered concerning the mechanisms
`SCIENCE, VOL. 251
`
`Par Pharm., Inc.
`Exhibit 1035
`Page 007
`
`Par Pharm., Inc.
`Exhibit 1035
`Page 007
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`l
`(
`I
`‘
`I
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`‘
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`
`I.
`T.
`I.
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`AT
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`2VP
`
`6IS
`
`. 5
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`I thank co-workers in my laboratory for contributions to the concepts outlined in
`this article, my collaborators S. I. Burakoff, B. E. Bierer, G. R. Crabtree, M.
`Karplus, G. L. Verdine, and C. T. Walsh for stimulating discussions, and the
`National Institute of General Medical Sciences (GM-38627) for financial support
`of immunophilin research.
`
`of inhibition of T cell activation by these immunosuppressants and
`the role of their cytoplasmic binding proteins. To this end, an
`assessment of the relevance of individual immunophilins, such as
`cyclophilin, FKBP, and the low-abundance immunophilins, and
`their drug complexes, as well as the identification of their target
`factor or factors is underway. In the cases of CsA and FK506, it has
`been suggested that a component of the transcriptional apparatus
`that regulates IL-2 transcription is the target of the immunophilin-
`dmg (CsA or FK506) complexes (24). These drugs potently inhibit
`the transcriptional activity of NF—AT but only partially inhibit its
`DNA binding activity (29). Thus, the target factor or factors may
`interact with NF—AT [and perhaps Oct-1, AP-3, and NF-KB (pleio-
`tropism) (51 )] and enhance their transcriptional activation proper-
`ties. The requirement for protein synthesis before IL-2 gene tran-
`scription (52, 53) suggests that translation of a protein, perhaps an
`as yet unidentified activating factor, occurs before transcription of
`the IL-2 gene. This cytoplasmic activity (translation) is a potential
`site of action for the immunophilin-drug complex, which may act as
`a cytoplasmic anchoring protein (54). This putative activating factor
`may be a cellular analog of the herpes virus protein VPI6, which is
`believed to bind to Oct—1 (a transcription factor also sensitive to
`drug) a11d thereby complete the formation of a preinitiation complex
`at the transcription start site (55).
`Although research on these immunosuppressants has focused on
`T lymphocytes, the nephrotoxic properties (8), hepatotrophic effects
`(56), ability to inhibit apoptosis (29) and exocytosis (57), and
`actions in lower eukaryotes (48) of these agents indicate an alternate
`function in other cell types, It is possible that in each of these studies
`the immunophilin ligand is involved in the inhibition of protein
`traflicking. The varying effects of drug may be due to differences in
`the immunophilins found in different sources, or perhaps more
`likely, due to the inter