WI
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`MADISON
`1215 W DAYTON ST
`UNIV WISCONSIN
`GEOLOGY GEOPHY LIB
`00000905836SC 01/03/92N9103
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`Par Pharm., Inc.
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`Page 001
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`AMERICAN
`AssociATION FOR THE
`Anv AN CEMENT OF
`SCIENCE
`
`• CIENCE
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`-
`
`ISSN 0036-8075
`18 JANUARY 1991
`VoLUME 251
`NUMBER 4991
`
`247 This Week in Science
`
`MDtJft[.JQm- 249 Teaching and Research
`Letters
`256 Safety of Bovine Growth Hormone: D. S. KRoNFELD; J. C. JusKEVICH AND
`C. G. GuYER • Interpreting Cancer Tests: J. D. WILSON; G. W. GRIBBLE •
`Kidney Transplantation: Overlooked Pioneer: G. B. ELION
`
`News & Comment
`
`260 The Rush to Publish • Lessons from Physics
`263 Third Strike for Idaho Reactor
`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 • Science's Top 20 Greatest Hits
`Mdtij$iijijN~(@i- 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 Briiftngs: Radiation Research Shake-Up • Private Initiative on Fetal Research •
`U.K. Antes Up for Telescopes • George Mason to Set Up Think Tank
`
`Articles
`
`Research Article
`
`Reports
`
`·
`
`277 Subsistence Economy of El Paraiso, an Early Peruvian Site: J. QuiLTER,
`B. OJEDA E., D. M. PEARSALL, D. H. SANDWErss, J. G. JoNEs, E. S. WING
`283 Chemistry and Biology of the Immunophilins and Their Immunosuppressive
`Ligands: S. L. SCHREIBER
`
`288 CCAAT-Enhancer Binding Protein: A Component of a Differentiation Switch:
`R. M. UMEK, A. D. FRIEDMAN, S. L. McKNIGHT
`
`293 An Antimony Sulfide with a Two-Dimensional, Intersecting System of Channels:
`J. B. PARISE
`
`•
`
`•
`
`SCIENCE (ISSN 0036-8075) Is published weekly on Friday, except the last week In December, by the American
`Association lor the Advancement of Science, 1333 H Street, NW, Washlng1on, DC 20005. Second-class postage
`(publication No. 484460) paid at Washington, DC, and additional mailing offices. Copyright © 1990 by the American
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`
`244
`
`SCIENCE, VOL. 251
`
`Par Pharm., Inc.
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`Page 002
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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: J. 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. KlPPHUT, 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 on Murine Acquired Immunodeficiency
`Syndrome: C. SIMARD AND P. JoLICOEUR
`308 Evidence for Biased Gene Conversion in Concerted Evolution of Ribosomal DNA:
`D. M . HILLIS, C. MORITZ, C. A. PORTER, R. J. BAKER
`310 The Effect of the Floor Plate on Pattern and Polarity in the Developing Central
`Nervous System: S. H!RANo, S. FusE, G. S. SoHAL
`313 Regulation of Interleukin-2 Gene Enhancer Activity by the T Cell Accessory
`Molecule CD28: J.D. FRASER, B. A. IRVING, G. R. CRABTREE, A. WEISS
`
`316 Microwave Sounding Units and Global Warming: B. L. GARY AND S. J. KEIHM;
`R. W. SPENCER AND J. R . CHRISTY • Lipid Flow in Locomoting Cells:
`M.s. BRETSCHER; K. JACOBSON, J. LEE, M. GUSTAFSSON, K.-E. MAGNUSSON.
`Bryozoan Morphological and Genetic Correspondence: What Does It Prove?:
`J. LEVINTON; J. B. c. JACKSON AND A. H. CHEETHAM
`
`324 Authors of Their Own Lives, reviewed by A. SICA • Australian Ecosystems,
`M. LoWMAN • Thalamic Oscillations and Signaling AND Brainstem Control of
`Wakefulness and Sleep, C. KocH • Books Received
`
`328 Protein Immunoblotting Incubation Rotator • Micromanipulator Table • Leiden
`Microincubator • Freezing Stage with Microtome • Data Analysis for the
`Macintosh • Monoclonal Antibodies • Literature
`
`Technical Comments
`
`Book Reviews
`
`Products & Materials
`
`Board of Directors
`
`Richard C. Atkinson
`Retiring President,
`Chairman
`
`Donald N. Langenberg
`President
`
`Leon M. Lederman
`President-elect
`
`Mary Ellen Avery
`Francisco J. Ayala
`Eugene H. Cola-Robles
`Robert A. Frosch
`Joseph G. Gavin, Jr.
`John H. Gibbons
`Beatrix A. Hamburg
`Florence P. Haselline
`
`William T. Golden
`Treasurer
`
`Richard S. Nicholson
`Executive Officer
`
`Editorial Board
`
`Charles J. Arntzen
`Elizabeth E. Bailey
`David Baltimore
`William F. Brinkman
`E. Mafgare~ Burbidga
`Pierre-Gilles 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
`Robert M. Solow
`Edward C. Stone
`James D. Watson
`
`Board of Reviewing
`Editors
`
`John Abelson
`Frederick .W. All
`Don L. Anderson
`Stephen J. Benkovic
`Gunter K-J Blobel
`Floyd E. Bloom
`Henry R. Bourne
`James J. Bull
`Kathryn Calame
`Charles R. Cantor
`Ralph J. Cicerone
`John M. Collin
`Robert Dorfman
`Bruce F. Eldridge
`Paul T. Englund
`Fredric S. Fay
`
`Harry A. Fozzard
`Theodore H. Geballe
`Roger I. M. Glass
`Stephen P. Goff
`Corey S. Goodman
`Stephen J. Gould
`Eric F. Johnson
`Stephen M. Kosslyn
`Konrad B. Krauskopf
`Charles S. Levings Ill
`Richard Losick
`John C. McGiff
`Anthony R. Means
`Mortimer Mishkin
`Roger A. Nicoll
`William H. Orme-Johnson Ill
`Carl 0. Pabo
`Yeshayau Packer
`
`Dennis A. Powers
`Erkki Ruoslahti
`Thomas W. Schoener
`Ronald H. Schwartz
`Terrence J. Sejnowski
`Thomas A. Steitz
`Robert T. N. Tijan
`Emil R. Unanue
`Geerat J. Vermeij
`Bert Vogelstein
`Harold Weintraub
`Zena Werb
`George M. Whitesides
`Owen N. Witte
`William B. Wood
`Keith Yamamoto
`
`18 JANUARY 1991
`
`TABLE OF CONTENTS
`
`245
`
`Par Pharm., Inc.
`Exhibit 1012
`Page 003
`
`

`
` 7KLV PDWHULDO PD\ EH SURWHFWHG E\ &RS\ULJKW ODZ 7LWOH  86 &RGH

`
`4. E. P. Laruung, Pem Bifore the lucas (Prentice-Hall, Englewood Cliffs, NJ, 1967).
`5. T. C. Patterson, in Prehistoric Agriwlture, S. Streuver, Ed. (Natural History Press,
`Garden City, NY, 1971), pp. 181-208.
`6. R. Fung P., Aptmtes Arqueol. 2, ll (1972) .
`7. J. H. Steward and L. C. Faron, Native Peoples '![Soutlt America (McGraw Hill, New
`York, 1959).
`8. M. H. Parsons, Am. Autiq. 35, 292 (1970).
`9. D.] . Wilson, Am. Authropol. 83, 93 (1981).
`10. J. S. Raymond, Am. Autiq. 46, 806 (1981).
`11. J. Quilter and T. Stocker, Am. Authropol. 85, 545 (1983).
`12. J. Quilter, ]. Field Archaeol. 12, 279 (1985) .
`13. F. A. Engel, ]. Soc. Am. 55, 43 (1966); Aual. Cieut. Uuiv. Agraria 5, 241
`(1967).
`14. A. Osborn, in For 71teory Buildiug iu Archaeology, L. Binford, Ed. (Academic Press,
`New Y~rk, 1977), pp. 157- 243.
`15. S. Quilter suggested this possibility during work at the Paloma Site in 1976.
`16. C. E. Smith, in La Galgada Pem, A Precernmic Culture iu Trni!Sitiou, Terence
`Grieder et nl., Eds. (University of Texas Press, Austin, TX, 1988), pp. 125- 151.
`17. S. Powrski and T. Powrski, Early Settlemeut aud S ubsisteuce iu the Cnsma Valley,
`Pem (University of Iowa Press, Iowa City, 1987).
`18. V. Popper, in Los Gavilaues, Mar, Desierto y Oasis eu In Historin del Hombre, D.
`Bonavia, Ed. (Editorial Ausonia, Lima, Peru, 1982), pp. 148- 156.
`19. S. Powrski and T. Powrski, Atm. Cnmegie Mus. Nat. Hist. 49, 337 (1979); ]. B.
`Bird, Autltropol. Pap. Am. Mus. Nat. Hist. 62 (1985), part 1.
`20. A. Grobman, in Los Gnvilnues, Mar, Desierto y Oasis eu Ia Historin del Hombre, D .
`
`Bonavia, Ed. (Editorial Ausonia, Lima, Peru, 1982), pp. 157-179; R. L. Burger
`and N. van der Menve, Am. A uthropol. 92, 96 (1990).
`21. T. Dillehay, P. Netherly, ]. Rossen, A m. Autiq. 54, 733 (1989).
`22. J. Quilter, "To fish in the afternoon: Beyond subsistence economics in the study of
`early Andean civilization," paper presented at 51st Annual Meeting of the Society
`for American Archaeology, New Orleans, LA, 23 April 1986.
`23. M. Moseley, Pre-ngriwlturnl Coastal C ivilizntious iu Pem (Carolina Biology Read(cid:173)
`ers, no. 90, Burlington, NC, 1978) .
`24. J. Quilter, "Core and periphery in Preceramic coastal Peru," paper presented at the
`88th Annual Meeting of the American Anthropological Society, Washington, DC,
`19 November 1989.
`25. R. G. Wilkinson, Poverty nud Prog ress (Praeger, New York, 1973).
`26. Following taxa listed in V. Alamo V. and V. Valdivicw M., Bolet. lust. Mar Penl
`(volumen extraordinario. Callao, Peru, 1987).
`27. Funding for the El Parafso research was provided by NSF grant BNS-83-03680,
`Ripon College Faculty Development Funds, and the Continental Coffee Products
`Company (a wholly owned subsidiary of Quaker Oats). The excavations were
`carried out under Credencial 038-83-DCIRBM, issued by the l11Stituto Nncioual de
`Cttlturn of Peru. We thank A. A. Hunter (Missouri) who identified the squash seeds
`and A. Price, J. Atteberry, and L. Haubrich who helped in sorting and tallying data.
`Additional aid in processing the subsistence remains was given by N. Salazar and
`M. C. Rodriguez de Sandweiss in Peru. L. Salazar-Burger, assistant field director,
`was essential to the project. The Ceutro de luvestigncioues de Zouns Ardins was our
`base of operations and analysis and we thank F. A. Engel and M. Vallejos and many
`other Peruvian collegucs for support.
`
`Chemistry and Biology of the Immunophilins
`and Their Immunosuppressive Ligands
`
`STUART L. SCHREIBER
`
`Cyclosporin A, FK506, and rapamycin are inhibitors of
`specific signal transduction pathways that lead to T lym(cid:173)
`phocyte activation. These immunosuppressive agents bind
`with high affinity to cytoplasmic receptors termed immu(cid:173)
`nophilins (immunosuppressant binding proteins). Studies
`in this area have focused on the structural basis for the
`molecular recognition of immunosuppressants by immu(cid:173)
`nophilins and the biological consequences of their inter(cid:173)
`actions. Defining the biological roles of this emerging
`family of receptors and their ligands may illuminate the
`process of protein trafficking in cells and the mechanisms
`of signal transmission through the cytoplasm.
`
`R ESEARCH DURING THE PAST DECADE HAS CONTRIBUTED
`
`significantly to our knowledge ofT lymphocyte function.
`The identification and functional analysis ofT cell surface
`receptors (1) and nuclear transcription factors (2) have made these
`components of the 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 radiolabeled
`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 pathway, remain mysterious.
`A family of natural products has emerged as probe reagents for
`cytoplasmic signaling mechanisms in the T lymphocyte. These small
`
`The author is a professor of Chemistry, Harvard University, Cambridge, MA 02138.
`
`molecules are immunosuppressants that appear to exert their inhib(cid:173)
`itory actions distal to early membrane-associated events and proxi(cid:173)
`mal to nuclear processes. Studies on a family of immunosuppressant
`binding proteins, the immunophilins, have attempted to identify the
`structural requirements for high-affinity interactions between immu(cid:173)
`nophilins and their immunosuppressive ligands and the biological
`consequences of the formation of immunophilin-ligand complexes.
`Although tl1ere is much to explore in this avenue of research, some
`general principles associated witl1 the intermediary events of signal
`processing are emerging.
`
`The Immunosuppressants
`Cyclosporin A (CsA), an inhibitor ofT cell activation, is currently
`tl1e favored therapeutic agent for prevention of graft rejection after
`organ and bone marrow transplantation, and it has been credited
`witl1 initiating a revolution in clinical transplantation (3- 5). The
`recently discovered compound FK506 inhibits T cell activation by
`mechanisms that are similar to tl1ose of CsA, but FK506 is l 0 to l 00
`times as potent (6). FK506 has performed remarkably well in initial
`human clinical transplantation trials (7, 8), despite reports of toxic
`effects in animals (6). Rapamycin inhibits T cell activation at
`concentrations comparable to those of the structurally related
`FK506, yet with mechanisms tl1at are strikingly different from those
`mediated by FK506, and thus CsA (9). Only CsA, FK506, and
`rapamycin have been used for tl1e identification of members of the
`irnmunophilin class. A nonnatural ligand, 506BD (10), and analogs
`of CsA ( 11- 13) have also provided insights into the inhibitory
`mechanisms of immunosuppressants. Many recently discovered
`immunosuppressive agents (14) with undefined mechanisms, such as
`
`18 JANUARY 1991
`
`ARTICLES
`
`283
`
`Par Pharm., Inc.
`Exhibit 1012
`Page 004
`
`

`
`discodermolide (15) and deoxyspergualin (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, 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 (pi) of 9.3.
`Two groups have independently reported that cyclophilin is identi(cid:173)
`cal to peptidyl-prolyl isomerase (20, 21), an enzyme that catalyzes
`the interconversion of the cis- and trans-rotamers of the peptidyl(cid:173)
`prolyl amide bond of peptide and protein substrates, and this
`rotamase 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 Jurkat,
`termed FKBP, was isolated and characterized in two laboratories
`(22, 23). Like cyclophilin, FKBP was shown to have rotamase
`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 Neurospora crassa (26) revealed that, despite their
`common enzymatic properties, FKBP and cyclophilin have dissim(cid:173)
`ilar sequences. Human FKBP has a mass of 11,819 daltons and, like
`cyclophilin, is a basic (pi = 8.9) (22, 24), cytosolic protein (27). A
`prokaryotic organism, N eisseria meningitidis, was found to have an
`open reading frame that encodes an FKBP-like protein (24). More
`recently, FKBP was shown to be the predominant rapamycin(cid:173)
`binding protein in yeast, calf thymus, and human T cells (Jurkat)
`(28) . Rapamycin (dissociation constant K d = 0.2 nM) has an even
`higher affinity for FKBP than does FK506 (Kd = 0.4 nM), and is
`also a potent inhibitor of FKBP's rotamase activity (inhibition
`
`constant Ki = 0.2 nM) (29).
`Although cyclophilin and FKBP are tl1e only well-characterized
`immunophilins, otl1er members of mis fanlliy are known to exist and
`are currently being investigated. For example, a CsA-binding phos(cid:173)
`phoprotein of relative molecular mass (Mr) 45,000 has been detect(cid:173)
`ed in Jurkat cells (30), and phosphoproteins of Mr 60,000 and
`80,000 from mis same cell line bind to born FK506 and rapamycin
`(28). The ninaA gene of Drosophila (31, 32) and a second cyclo(cid:173)
`philin-related gene in Saccharomyces cerevis iae (33) encode proteins
`mat show high homology to cyclophilin. Several low molecular
`weight, basic proteins mat are retained on CsA, FK506, or rapa(cid:173)
`mycin affinity matrices have also been noted (22, 28) . Partial
`sequence determination of FK506- and rapamycin-binding immu(cid:173)
`nophilins of M r 30,000 and Mr 13,000 has revealed mat mese
`molecules, together wim FKBP, are members of a previously
`unknown fanlliy of immunophilins (34) . Questions concerning me
`biological relevance, me rotamase activity, and me affinity to me
`cognate ligands of mese low-abundance immunophilins should soon
`be answered.
`Almough me exact cellular concentrations of FKBP and cyclo(cid:173)
`philin are not known, born are abundant. Saturation binding in me
`cytosol of Jurkat cells was reported to occur at > 5 nM ditritio(cid:173)
`FK506 (27). As FKBP is me predominant cytosolic receptor for
`drug, mis measurement is largely accounted for by FKBP, and tlms
`me cytoplasmic concentration of FKBP may approach 5 nM. The
`high-affinity FKBP ligands FK506 and rapamycin, however, inhibit
`T cell proliferation at subnanomolar concentrations (median inhibi(cid:173)
`tion concentration IC50 -0.5 nM) (29, 35). Therefore, inhibition of
`me rotamase activity of FKBP is very likely an insufficient require(cid:173)
`ment for mediating me actions of mese 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 ofCsA analogs support a similar conclusion regarding
`me rotamase activity of cyclophilin (12) .
`
`CsA
`
`50680
`
`OMo
`
`OH
`
`FK506
`
`Rapamycin
`
`B
`
`0
`
`H
`
`OH
`
`0
`OH
`H,NYNH(CH,),.JLNH~NH(CH2),NH(CH2),NH2
`
`NH
`
`0
`
`Discodermolide
`
`Deoxyspergualin
`
`Fig. 1. Probe reagents of intracellular signaling pathways. (A) Recently
`investigated immunophilin ligands. (B) Immunosuppressive agents with
`unknown mechanisms ofT cell inhibition. (Me, metl1yl. )
`
`284
`
`Molecular Recognition by the Immunophilins
`The rotamase activity of mese immunophilins and me ability of
`tl1eir immunosuppressive ligands to act as rotamase inhibitors
`provide an opportunity for exploration of me molecular basis for me
`high-affinity interactions mat exist between mem. Initial mechanistic
`studies of cyclophilin led to me suggestion mat catalysis of tl1e
`interconversion of cis- and trans-rotamers of a peptide substrate is
`achieved by me formation of a covalent bond to tl1e carbonyl of tl1e
`peptidyl-prolyl amide wim a cysteine-derived miol (36). Loss of
`amide resonance would be expected to lower me activation barrier
`to rotation about me amide G-N bond. Site-directed mutagenesis of
`human recombinant cyclophilin allowed me systematic replacement
`of aU four cysteine residues in cyclophilin wim alanine. Because all
`four mutants enzymes were fully active in tl1e rotamase and binding
`assays, cysteine was ruled out as a participating residue in catalysis
`(37).
`Additional mechanistic studies wim born cyclophilin (3 8) and
`FKBP (39) strongly suggest mat mese enzymes catalyze rotamer
`interconversion by noncovalent stabilization of me twisted amide
`transition state for the noncatalyzed isomerization. The amide
`functionality exhibits a strong preference for a planar geometry,
`wherein me nitrogen lone pair is in conjugation wit11 me carbonyl
`1r-cloud. The energy cost of me twisted amide structure (Fig. 2A) is
`15 to 20 kcal (40). The structural basis for cyclophilin and FKBP's
`ability to stabilize tl1is transition-state strucn1re must await further
`structural analyses of rotamase-peptide (or inhibitor) complexes.
`
`SC I ENCE, VO L. 251
`
`Par Pharm., Inc.
`Exhibit 1012
`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(cid:173)
`Ieukin-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 ofNF-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-KB ( 4 5, 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(cid:173)
`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 difFerent set of genes is transcribed.
`Whereas rapamycin, despite its structural similarity to FK506, has
`no effect on the production ofiL-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 botl1
`rapamycin and FK506 are potent inhibitors of the rotamase activity
`of FKBP and inhibit distinct signaling pathways, these results
`support tl1e suggestion that the inhibition of rotamase activity of
`FKBP is an insufficient 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 eitl1er a single immunophilin or separate
`immunophilins that share a common receptor site in mediating tl1e
`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
`
`IL·lR
`
`ICsAl
`~ X ..
`
`~ • •
`
`••• IL-l
`
`TCR I rapamycin J
`X·
`
`(2) LKR signal
`
`-
`
`Flg. 3. Early events of the T ceU activation cascade and the sites of inhibitory
`action by CsA, FK506, and rapamycin.
`
`cell differentiation
`cell proliferation
`
`c
`
`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 FK506 (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(cid:173)
`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 13C-Iabeled 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-affinity
`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 different type of surrogate for the twisted amide
`structure. In this regard, the a-branched hydroxyethylene substruc(cid:173)
`Ulre of CsA is reminiscent of the hydroxyethylene amide isostere
`found in aspartyl protease inhibitors such as pepstatin.
`The analogy of the a-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-an1ide bond (Fig. 2, D
`and E) suggest these agents may be transition-state analogs of a
`leucyl-(twisted amide)-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-
`
`IS JANUARY 1991
`
`ARTICLES
`
`285
`
`Par Pharm., Inc.
`Exhibit 1012
`Page 006
`
`

`
`Immune ¢::::J
`modulation
`
`FKBP/drug
`complex
`+~9
`H /
`antagonist Q
`
`q
`
`Foldase,
`protein translocation
`etc.
`
`FKBP/antagonist
`complex
`
`Fig. 4. Schematic iUustrating the relative abundance of receptor and ligands.
`The abundant FKBP, which may have a ceUular function as a foldase, is
`converted to an inhibitory complex on binding of the drug and buffers the
`actiohs of the antagonist.
`
`either agent is required for inhibition of the actions of the other (29,
`47) . Thus, the abundant ( -5 nM) uncomplexed immunophiJin
`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(cid:173)
`plex as the inhibitor ofT 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 immw1ophilins 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 effec(cid:173)
`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 witl1 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. cerevisiae were grown in the presence of
`CsA. Analysis of the CsA-resistant mutant strains that resulted
`revealed that eitl1er 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 ofFK506 and rapamycin suggests that tl1e immunophilin
`may present multiple ligands to cytoplasmic components of signal
`transmission pathways. The ability of a single immunophilin to
`present two immunosuppressive ligands to effectors associated with
`two distinct pathways raises the possibility that immunophilins may
`function as general presenting molecules, by analogy to the way tl1at
`MHC molecules present a large number of peptides to the polymor(cid:173)
`phic TCRs. If endogenous immw10philin ligands exist that function
`similarly to the immtmosuppressive natural products, tl1en the
`immune system may have used the molecular recognition associated
`witl1 rotamase catalysis for the purpose of modulating T cell
`activation.
`In the case of FK506 and rapamycin, the leucyl-(twisted amide)(cid:173)
`prolyl peptidomimetic fragment shared by these drugs constitutes
`tl1e structural element largely responsible for binding to FKBP. This
`common immunophilin binding domain is then fused to distinct
`effector elements that, after presentation by tl1e immunophilin,
`determine tl1e signaling pathway with which tl1e drug will intetfere
`(Fig. 5, A and B). This view of FK506 and rapamycin as dual
`domain agents was tested witl1 an FKBP ligand designed to contain
`
`A
`
`B
`
`50680
`
`Fig. 5. Domainal analyses ofFKBP ligands. (A) FK506 and (B) rapamycin
`binding domain and effector elements (shaded). (C) Structure of FK506
`(x-ray) with enolate spacer drawn to illustrate scafiolding effect. (D) Removal
`of the outer loop of stmcture (C) results in 506BD, a high-affinity (K; = 5
`nM) ligand to FKBP.
`
`the putative FKBP-binding domain ofFK506 and rapamycin in the
`conformation found in the solid state ofFK506 (Fig. 5C) (10) . The
`resultant molecule, 506BD, was found to bind with high affinity (Kd
`= 20 nM) and to inhibit the rotamase activity (K; = 5 nM) ofFKBP
`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, 506BD 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 affinity 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 insufficient requirement for mediating the actions of
`FK506 and rapamycin, tl1ese 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 (effector element) .
`
`Future Pros