JP2001519765A - Regulation of epithelial-mesenchymal interaction - Google Patents

Abstract

  (57) [Summary] A method of modulating the development of skin appendages, comprising modulating FGF-2 activity in one or both of the dermis or epithelium.

Description

DETAILED DESCRIPTION OF THE INVENTION                        Regulation of epithelial-mesenchymal interaction                                Background of the Invention   Epithelial-mesenchymal interactions are described in Skin appendages (Sengel, P. Morphogenesis of Skin. Cambridge University Press, Cambridge, UK (1976)) (feathers, scales and Hair), extremities (Fallon, J.F. Lopez, A. Ross, M.A., Savage, M.P., Olwin, B.O. And Simandl, B.K., Science 264: 104-107 (1994), 8, 9), teeth (Vainio, S. Karavanova) , I., Jowett, A. and Thesleff, I., Cell 75: 45-58 (1993)), kidney (Patterson, L. T. and Dressler, G.R., Curr. Opin. in Gen. and Dev. 4: 696-702 (1994)), lungs ( Peters, K., Werner, S._,Liao X., Wert, S., Whitsett, J. and Williams, L. EMBO J . 13: 3296-3201 (1994)) and the mammary gland (Cuhna, G.R., Cancer 74: 1030-1044 (1994)) Plays an essential role in morphogenesis. Feather or scale formation of chicken embryo skin In the area, the epithelial primordia is a prediction of the initial morphogenesis of the site of appendage formation ( Sengel, P .; Morphogenesis of Skin. Cambridge University Press, Cambr, UK idge in (1976)). Atypical and metachronous recombination of the ectoderm and mesoderm components of the skin Figure 4 shows that primordium formation is determined by mesodermal signals. Skin signals Also determine whether scale or wings will occur, and if wings occur, Determine the exact hexagonal pattern arrangement. These primordia are furthermore directly The signal gives rise to the formation of dermal condensation below (Sengel, P. Morphogenesis of Skin. Cambridge University Press, Cambridge, UK ( 1976)). In the wing formation area, the cells of skin condensation formation and the epithelial primordia of individual wing buds Continuous interaction between things, initially radially symmetric and then growing backwards Causes bud growth. The direction of wing development is determined by the ectoderm (Novel , G.J., Emryol. Exp. Morph. 30: 605-633 (1973)). Much research has been done on cell adhesion molecules Suggests that extracellular matrix molecules and transcription factors may be involved in skin morphogenesis (Chuong, C-M., BioEssays 15: 513-552 (1993), Noven, A., Jiang, T-X, Ting -Bcrreth, S.A. and Chuong, C-M., J. Invest. Derm. 104: 711-719 (1995)). However Naga To date, no particular molecular mechanism has been identified to date.                                Summary of the Invention   In general, the present invention relates to a method of modulating epithelial-dermal interaction, or epithelial-dermal phase. It is characterized by processes or products resulting from the interaction. Such interactions include, for example, Primordium formation, dermal condensation formation, FGFR-1 induction, or skin appendages such as hair, Includes scale or feather development. The method is: one or both of the dermis and epidermis Modulating (eg, promoting or inhibiting) FGF-2 activity in .   In a preferred embodiment, FGF-2 activity is enhanced. FGF-2 activity is an example For example, exogenous FGF-2 or a fragment thereof, or a peptide or non-peptide It can be facilitated by administration of nalog. FGF-2 activity also indicates that FGF-2 By administering a nucleic acid encoding a fragment thereof, or a peptide analog thereof, Alternatively, it can be promoted by promoting endogenous FGF-2 activity.   In a preferred embodiment, FGF-2 activity is inhibited. FGF-2 activity is an example For example, an FGF-2 binding molecule such as an anti-FGF-2 antibody or its FGF-2 binding block By administration of a fragment, or an FGF-2 binding fragment of the FGF-2 receptor; Compounds that competitively or non-competitively inhibit the binding of FGF that binds to a sceptor but lacks biological activity (eg, signal transduction activity) -2 fragment, which binds to a receptor but has a biological activity (eg, signal transduction activity) ) Lacking a peptide or non-peptide analog or small molecule mimetic of FGF-2 Encoding an antisense molecule that inhibits FGF-2 expression Can be inhibited.   In a preferred embodiment, the method is performed in vivo and the patient is a vertebrate animal. Birds, or mammals, such as rodents, such as mice or rats, or primates For example, a non-human primate or a human. In a preferred embodiment: the patient is a bird, Other than rodents such as mice or rats, or non-human primates.   In a preferred embodiment, the method is performed in vivo and: Wild-type for one or more loci that regulate the development of the genus Is wild-type for the scale-deficient locus; the patient controls the development of skin appendages. Heterozygous, hemizygous, or mutation at one or more loci Homozygous, for example, the patient has one or more mutated pairs of scale-deficient loci. Have progeny; patients may be placed at one or more loci that regulate the development of skin appendages Transgenic that is heterozygous, hemizygous or homozygous for the mutation For example, the patient has one or more mutated pairs of scale-deficient loci. It has a progeny.   In a preferred embodiment, the method is performed in vitro, for example, in tissue or cell metabolism. It is performed on cultured cells.   In a preferred embodiment, a compound that modulates FGF-2 activity is added at least once. Preferably at least 2, 4 or 10 doses are given. Duration of administration is 2, 4, 10 or May be longer than 30 days and less than 1 year.   In preferred embodiments, modulates FGF-2 activity in the dermis (eg, increases Or decrease).   In a preferred embodiment, one or both of the dermis or epidermis is the fetal dermis or epithelium. Yes; adult dermis or epithelium.   In another aspect, the invention provides for the generation of skin appendages such as hair, scales or feathers. It features a method of adjusting. The method is: one or both of the dermis or epidermis Modulating (eg, promoting or inhibiting) FGF-2 activity.   In a preferred embodiment, FGF-2 activity is enhanced. FGF-2 activity, for example, For example, exogenous FGF-2 or a fragment thereof, or a peptide or non-peptide thereof. It can be facilitated by administering a peptidic analog. FGF-2 activity It also encodes FGF-2 or a fragment thereof, or a peptide analog thereof. Is also enhanced by the administration of nucleic acids or by enhancing endogenous FGF-2 activity. Can be   In a preferred embodiment, it inhibits FGF-2 activity. FGF-2 activity is, for example, , FGF-2 binding molecules such as anti-FGF-2 antibodies or FGF-2 binding thereof Administration of an FGF-2 binding fragment of the FGF-2 receptor; Compounds that competitively or non-competitively inhibit binding to its receptor, such as Binds to a puter but lacks biological activity (eg, signal transduction activity) FGF-2 fragments that bind to receptors but have biological activity (eg, signal alterations) Or non-peptidic analogs of FGF-2 lacking Administration of a mimetic; a nucleic acid encoding an antisense molecule that inhibits FGF-2 expression Can be inhibited.   In a preferred embodiment, the method is performed in vivo and the patient is a vertebrate For example, birds, or mammals, such as rodents, such as mice or rats, or primates Or non-human primates or humans. In a preferred embodiment, the patient is a bird, Any other than a rodent, such as a mouse or rat, or a non-human primate.   In a preferred embodiment, the method is performed in vivo and: Wild-type for one or more loci that regulate the development of the genus Is wild-type for the scale-deficient locus; the patient controls the development of skin appendages. Heterozygous, hemizygous, or mutation at one or more loci Homozygous, for example, the patient has one or more mutated pairs of scale-deficient loci. Have progeny; patients may be placed at one or more loci that regulate the development of skin appendages Transgenic that is heterozygous, hemizygous or homozygous for the mutation For example, the patient has one or more mutated pairs of scale-deficient loci. It has a progeny.   In a preferred embodiment, the method is performed in vitro, for example, on a tissue or cell. For example, it is performed on cultured cells.   In a preferred embodiment, the compound that modulates FGF-2 activity is at least once Preferably, it is administered at least 2, 4 or 10 times. The administration period is 2, 4, 10 or May be longer than 30 days and less than 1 year.   In a preferred embodiment, FGF-2 activity is modulated in the dermis (eg, Increase or decrease).   In a preferred embodiment, one or both of the dermis or epidermis is the fetal dermis or epidermis. Yes; adult dermis or epidermis.   In another aspect, the invention relates to a skin accessory in tissue or a patient, such as hair, It features a method of promoting the development of scales or wings, the method comprising providing a therapeutically effective amount outside the range. Including administering the causative FGF-2 or a fragment thereof to a tissue or patient.   In a preferred embodiment, the patient is a vertebrate, such as a bird, or a mammal, such as a rodent Species such as mice or rats, or primates such as non-human primates or humans is there. In a preferred embodiment, the patient is a bird, rodent, such as a mouse or rat. Or a non-human primate.   In a preferred embodiment, the exogenous FGF-2 or a fragment thereof is directly, preferably To the patient, e.g., locally, by injection or local administration to a given site, or systemically For example, it is given by parenteral or oral administration.   In another aspect, the invention relates to the ability to bind an FGF-2 polypeptide; Modulates FGF-2 / FGFR-1 interaction or modulates FGF-2 activity And a method for evaluating a compound for its ability to This compound, for example, Ripeptides or non-peptidic compounds, such as natural ligands of FGF-2 polypeptides E.g., a fragment of an FGFR-1 polypeptide, e.g., an FGFR-1 polypeptide. May be. The method comprises contacting the compound with an FGF-2 polypeptide; Interacting with FGF-2 and FGF-2 polypeptides, eg, binding or forming a complex The ability of the compound to inhibit the FGF-2 / FGFR-1 interaction Including evaluating. This method may be used in vitro, for example, in a cell-free system, or in vitro. Performed in vivo, for example, in a two-hybrid interaction trap assay. Can be. Using this method, FGF-2 agonists or antagonists Certain compounds, such as fragments or analogs of FGFR-1, can be identified. This The method further comprises applying the evaluated compound to an animal, tissue or cell and treating its epithelium- Effects on dermal interaction or processes or processes resulting from epithelial-dermal interaction Adults such as primordium formation, dermal condensation formation, FGFR-1 induction or skin appendages (eg, For example, further evaluating the occurrence of hair, scales or feathers). .   In another aspect, the present invention relates to a method for preparing a first compound, such as an FGF-2 polypeptide. A second compound such as a second polypeptide or a non-peptide compound such as FGF-2 Binds a natural ligand of the polypeptide, eg, FGFR-1 or a fragment thereof It features a method of evaluating ability. This method involves converting a first compound into a second compound. Contacting with the compound: and the ability of the first compound to form a complex with the second compound Including evaluating power. This method can be used in vitro, for example in cell-free systems, or Is performed in vivo, eg, in a two-hybrid interaction trap assay be able to. Using this method, an agonist or antagonist of FGF-2 For example, a fragment or analog of GFG-2 can be identified. This Using the method described above, a compound that is an agonist or antagonist of FGF-2 For example, a fragment or analog of FGFR-1 can be identified. This method is The evaluated compound is applied to an animal, tissue, or cell to allow its epithelium-dermis interaction. Effects on action, or processes or products arising from epithelial-dermal interaction Primordium formation, dermal condensation formation, FGFR-1 induction or skin appendages (eg, hair, A further step of assessing the occurrence of scales or wings can be included.   In yet another aspect, the invention relates to a method for synthesizing a compound, for example, with a second polypeptide. Ability to modulate the interaction, eg, a second polypeptide of FGF-2, eg, polypeptide A peptide, such as a natural ligand of an FGF-2 polypeptide, such as FGFR-1 or It features a method of evaluating its ability to inhibit interaction with the fragment. this The method comprises the steps of (i) a second polypeptide (preferably a purified sample), FGF-2 Peptides (preferably purified preparations) and compounds, for example, in the absence of the compound Interacts with an FGF-2 polypeptide such as a second polypeptide (eg, Combining under conditions capable of combining or forming a complex); and (ii) ) Its interaction, including, for example, the second polypeptide and the FGF-2 polypeptide. Detecting the formation (or dissociation) of the complex. In the presence of the compound Changes in complex formation (compared to those seen in the absence of the compound) (eg, , Decrease or increase) is the interaction between the second polypeptide and the FGF-2 polypeptide. (Eg, inhibition or promotion). In a preferred embodiment, the second polypeptide The peptide and FGF-2 polypeptide are combined in a cell-free system and contacted with the compound. Selecting a cell-free system from the group consisting of a cell lysate and a reconstituted protein mixture; FG The F-2 polypeptide and the second polypeptide are co-expressed in one cell and Of cells with this compound, for example, in an interaction trap assay (eg, two hybrid Contact). Using this method, an agonist of FGF-2 or Identify compounds that are antagonists, such as fragments or analogs of FGFR-1 Can be The method further comprises the step of translating the evaluated compound into an animal, tissue or cell. To the epidermis-dermis interaction and the processes resulting from the epidermis-dermis interaction or Products such as primordium formation, dermal condensation formation, FGFR-1 induction or skin appendages ( Further estimating its effect on the development of eg hair, scales or feathers) Can be included.   The molecule has the properties of FGF-2 if it has at least one of the following properties: Has biological activity: (1) It is a sc / sc chicken as described herein When applied to tissue, results from epithelial-dermal or epithelial-dermal interaction Process or product such as primordium formation, dermal aggregation formation, FGFR-1 induction or Induces the development of skin appendages (eg, hair, scales or feathers); It is mitogenic; (3) it binds to the FGFR-1 receptor; ( 4) It is an antagonist or agonist of at least one of the above properties You.   The practice of the present invention will be, unless otherwise indicated, cell biology, cell culture, molecular biology, Conventional techniques of transgenic biology, microbiology, recombinant DNA and immunology (this These are within the skill of the art). Such techniques are described in the literature. You. For example, Molecular Cloining A Laboratory Manual, 2nd edition (Sambrook, Fr. edited by itsch and Maniatis) (Cold Spring Harbor Laboratory Press: 1989); DNA Clon ing, Vol. I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J.Gai t ed., 1984); Mullis et al., US Pat. No. 4,683,195; Nucleic acid Hybrid (B.D.Hames and S.J.Higgins eds., 1984); Transcription And Translation ( B.D.Hames and S.J. Higgins eds., 1984); Culture Of Animal Cells (R.I. Freshney Alan R .; Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 198). 6); Perbal, A Practical Guide To Molecular Cloning (1984); Dissertation, Methods  In Enzymology (Academic Press, Inc., New York); Gene Transfer Vectors For M ammalian Cells (edited by J.H. Miller and M.P. Calos, 1987, Cold Spring Harbor Lab) oratory); Methods In Enzymology, Vol. 154 and 155 (Wu et al.), Immunochemical Methods In Cell And Molecular Biology (edited by Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, Vol.I-IV (D.M.Weir and And C.C. Blackwell, 1986); Manipulating the Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York , 1986).   Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Let's be.                                Detailed descriptionFGF-2 and morphogenesis   Epithelial appendages such as the development of wings, scales and hair, the ectoderm and mesoderm of eradicated skin (Scngel, P. Morphogencsis of Skin. Cambr idge University Press, Cambridge, UK (1976)). At the morphological level, these phases Interaction occurs first in the appearance of ectodermal primordia, and then in the form of dermal condensation directly beneath the primordia. Is reflected by Wings and absence of scales in scale-deficient (sc / sc) mutations (Abbott, U.K., and Asmundson, V.S., J. Hered. 18: 63-67 (1957)) (Goetinck, P.F. and Abbott, U.K., J. Exp.Zool. 154: 7-19 (1963), Se ngel, P. and Abbott, U.K., J. Hered. 54: 254-262 (1963), Song, H.-K. and Sawyc r, R.H., Dev. Dyn. In Press (1995)) and such embryos have ectodermal areas on their skin. Groups cannot occur (Goetinck, P.F. and Sekellick, M.J., Dev. Biol. 28: 636-648). (1972)). FGF- in epithelial-mesenchymal signaling at the onset of wing development The role of 2 was investigated. Here, the genes encoding FGF-2 and FGFR-1 in normal embryo skin were We report spatially and temporally restricted patterns of offspring transcription. FGF-2 expression Is restricted to epithelial primordium, whereas FGFR-1 expression is restricted to dermal condensation You. Transcription of these genes could not be detected in sc / sc skin. Treatment of sc / sc skin with FGF-2 caused feather formation at the site of application of this growth factor. Occurs. In addition, FGFR-1 transcripts showed that feathers induced in the treated mutant skin Evidence in mesoderm. Thus, FGF-2 is the epithelium that occurs during wing development. It was identified as an early ectoderm signal in mesenchymal interactions. This growth factor The observation that rescue of the mutant phenotype of sc / sc embryos lacking epithelial primordia was confirmed by FGF- 2 is or is a signal that mutant ectoderm cannot be produced Suggests that it is in the flow.Transcription of FGF-2   To examine the role of FGF-2 in wing development, we examined the transcription of the FGF-2 gene. Spatial and temporal patterns (Zuniga, A. Mejia, B., Meijers, C. and Zeller, R. et al. Dev. Biol. 157: 110-118 (1993)). Examined by the Stage 33 of a normal embryo (Hamburger, V. and Hamilton, H .; L., J. Morphol. 88: 49-92 (1951)) In the skin of (E8), FGF-2 transcription is Was detected in all the wing primordia. Hexahara dyeing is hexagonal Appears in a perfect circular arrangement in the pattern. In the spinal cord region, FGF-2m RNA could be detected at the very end of the zone in Hanehara, where The formation of epithelial primordia precedes the formation of dermal condensation (Sengel, P. Morphogenesis of  Skin. Cambridge University Press, Cambridge, UK (1976)). Stained embryo Cross-sections show that FGF-2 mRNA expression is restricted to epithelial primordia of wing primordia . Thus, FGF-2 expression is spatially restricted to epithelial primordia and temporally true. Precedes the formation of skin condensation. In more developed feather buds, FGF-2 mRNA is It was found at the end of growing buds. The full length of a feather bud as it grows into a barb , Express FGF-2. At the base of the barb, FGF-2 mRNA Could not be detected. At all stages examined, FGF-2 It was not present in the area between the buds or wings.   Transcription of FGF-2 occurs in the skin of sc / sc embryos at a comparable stage of development Could not be detected. In rare cases, sc / sc embryos may develop wings slightly. Determine if FGF-2 is expressed in these exceptional wings This means that the hybridization signal can be found in the highly colored mutant feathers. It was difficult because it could not be visualized.   All mount-in-situ hybridizations are essentially Riddle, R . D., Johnson, R.L., Laufer, E. And Tabin, C., Cell 75: 1401-1416 (1993) Performed as described in. The FGF-2 RNA probe was replaced with alt-FGF-2. (Zuniga, A. Mejia, B., Meijers, C. and Zeller, R. Dev. Biol. 157: 110-118 (1993).Ectodermal signaling involves FGF-2   FGF-2 transcripts are restricted to the ectoderm primordium of normal wing primordium, and their structure is Incomplete signaling in the ectoderm of mutants is absent in / skin skin It has been hypothesized to include FGF-2. To test this hypothesis, FGF-2 was applied to skin obtained from the dorsal area of 8 day old sc / sc embryos. FGF -2 supported heparin beads on skin supported by Millipore filter chamber And cultured on chorioallantoic membrane of 10-day-old chick embryos for 3 or 5 days.   After 3 days of culture, feather buds are clearly visible on the mutant skin to which the beads are attached. Call In many cases, feather buds all developed around the beads. Table 1 4 shows the correlation between bead attachment sites and induction of feather buds. The center of these skins 83% of the feather buds found correlated with the presence of beads. On the side of the explant The correlation between bead placement and feather development was 95%. Differences between parts Is the chance of few feathers along the center of the dorsal feather in full sc / sc skin Can reflect the occurrence. Such simulated wings develop on the side of the dorsal area of the sc / sc skin. Nevertheless, it was never allowed in vivo. Soak in PBS used as control The beads did not stimulate any wing development in sc / sc skin. Feather arrangement No hexagonal pattern in the position was observed. Most wing primaries are highly colored And they were variable in size and shape.                                   Table 1 Spatial correlation between wing formation site and arrangement of FGF-2 beads -A total of 7 skins were examined after 3 days of culture on chorioallantoic membrane.   A cross-sectional view of the wings on the skin cultured for 5 days shows a normally placed barnacle ridge and Shows winglet cells, indicating that they are normally developed wings . The length of these wings is relatively uniform, but their size and shape are irregular Can be Such irregularities are never observed in normal wings. this Most of these unusual feathers are a fusion of feather buds found close to each other on day three. Seems to arise from this. Fusion occurs such that the barb has a separate base or tip. Can get. Subsequent fusion of the molds can result in fork-like structures. Another type of abnormal barb is 2 Includes a central fusion and separate base and tip of one barb. One of these fused feathers Two cross-section observations show that the fused wings are outlined by a single sheath but the diaphragm is This shows that the internal structure is divided into two separate groups. Serial sections Indicated the presence of separated vesicles. Therefore, it is expressed in the epithelial primordia of the wing primordia of normal embryos. FGF-2 induces wing growth in sc / sc skin lacking epithelial primordium be able to. Identification of FGF-2 as an ectodermal signal in wing development Similar to its role in limbs {FGF-2 and FGF-4 are involved in limb growth Can substitute for the ectoderm crest (Fallon, J.F. Lopez, M.A., Savage, M.P., Olwin , B.O. and Simandl, B.K., Science 264: 104-107 (1994), Niswander, L., Ticle, C ., Vogell, A., Booth, I. And Martin G.R., Cell 75: 579-587 (1993))｝.   The wing orientations induced in these mutant skins were random. Therefore, positive Uniform anterior-posterior orientation in normal embryos is induced by FGF-2 in sc / sc skin Not seen on led wings. The direction of the anterior and posterior direction of the wing primordium is determined by the ectoderm (Novel, G.J., Emryol. Exp. Morph. 30: 605-633 (1973)) The inability of the sc / sc wings to turn backwards indicates that Enzyme mutant ectoderm defects can also result from.   These experiments were performed essentially as follows. Heparin beads (Bio-Rad, California , Hercules) in an FGF-2 solution (1 mg / ml PBS, bovine FGF base). Chic, Minnesota, Minneapolis, R & D Systems) for 2 hours and 3 times with PBS Washed and applied to the sc / sc skin surface. Culture of these skins on chorioallantoic membrane Performed as previously described (Noveen, A., Jiang, T-X, Ting-Berrcth, S.A. and Chu. ong, C-M., J. Invest. Derm. 104: 711-719 (1995)).Expression of FGFR-1   Pattern of transcription of FGFR-1 (Pasquale, E.G. and Singer, J.S. Proc. Natl. . Acad. Sci. USA 86: 5449-5454 (1989)) was also examined in developing skin. FGFR-1 transcripts can be detected in normal embryo feather primordia and developing feathers. I was able to. FGFR-1 expression was restricted to dermal aggregate formation in these wings. these Observations were made by other researchers (Nopji, S., Koyama E., Myokai, F., Nohno, T., Ohuc). hi, H., Nishikawa, K. and Taniguchi, S., Progress in Clin. and Biol. Rcs. 38 3B: 645-654 (1993)). In contrast, in sc / sc embryo skin, FGFR-1 mRNA could not be detected. FGF in sc / sc skin When wings are induced by -2, the FGFR-I transcripts are true of the developing wing. It can be detected in the skin. FGFR-1 gene is untouched This observation indicates that FGF-2 induction is not transcribed in natural sc / sc skin. Suggests that the response to FGFR-1 involves activation of the FGFR-1 gene in the dermis ing. Induction of the receptor by the ligand is triggered by the activin receptor cActR. IIB (Stern, C.D., Yu, R.T., Kakizuka, A., Kintner, C.R., Mathews, L.S., Vale, W.W., Evans, R.M. and Umesono, K. Dev. Biol. 172: 192-205 (1995)) and c ActR-IIa (Levin, M., Johnson, R.L., Stern, C.D., Kuehn, M. and Tabin, C. , Cell 82: 803-814 (1995)) also reported induction by the application of exogenous activin. It has been tell.   All mount-in-situ hybridizations are essentially performed by Riddle, R. D., Johnson, R.L., Laufer, E. and Tabin, C., Cell 75: 1401-1416 (1993) Went as it was. The probe for FGFR-1 was cloned into a chicken cDNA clone (P asquale, E.G. and Singer, J.S. Proc. Natl. Acad. Sci., USA 86: 5449-5454 (1989)) Led from. This RNA probe was used to bind the 5 'end of FGFR-1 and the first immunoglobulin. It was a 490 bp fragment encoding the Roblin-like domain.Induction of wings by FGF-2   Induction of wings by exogenous FGF-2 in sc / sc skin resulted in the development of mutant skin Depends on the raw stage. Feathers are found on sc / sc skin from 7 and 8 day old embryos Induced by FGF-2 but not in skin from 11-day-old embryos Was. These results indicate that the various stages of the sc / sc dermis were normal on day 11 outside the foot Tissue recombination experiments recombined with germ layers (Noveen, A., Jiang, T-X, Ting-Berreth, S .A. And Chuong, C-M., J. Invest. Derm. 104: 711-719 (1995)) Is similar to The wings are transformed when the foot ectoderm is recombined with the dorsal mesoderm. Occurs (Rawles, M.E., J.Embryol. Exp. Morph. 11: 765-789 (1963)). 8 days sc / The sc dermis may be associated with normal ectoderm in wing formation, but this ability Increase Is gradually lost from the growing mutant mesoderm (Noveen, A., Jiang, T-X, Ting-Berreth .S. A. and Chuong, C-M., J. Invcst. Derm. 104: 711-719 (1995). These results Mutant mesoderm responds to both FGF-2 signaling and cues from normal ectoderm Indicate that there are developmental windows that can be answered, Derived FGF-2 is an important signal in normal wing development, Is consistent with the suggestion that sc / sc mutants lacking ectoderm primordium are not present in skin I do.   Skin culture and preparation of beads were performed as described in the above examples.   In this study, we investigated the development of wing primordia in genetically normal and The role of FGF-2 in epithelial-mesenchymal signaling during initiation was examined. Wings Bile scales develop in embryos that are homozygous for the recessive scale deficiency. None (Abbott, U.K., and Asmundson, V.S., J. Hered. 18: 63-67 (1957)). these Embryonic skin does not develop ectodermal primordia (Goetinck, P.F. and Sekellick, M.J., Dev. Biol. 28: 636-648 (1972)). Thus, this scale-deficient mutation is associated with the ectoderm of the mutant skin. Affects the normal sequence of tissue interactions in such a way that it is disrupted. Either the early dermal signal cannot be received by the ectoderm or the ectoderm It's not clear if he can't respond. This mutant mesoderm is genetically normal What is involved in wing and scale development when recombined with ectoderm is well documented. (Goetinck, P.F. and Abbott, U.K., J. Exp. Zool. 154: 7-19 (1963), Scngcl, P. and Abbott, U.K., J. Hered. 54: 254: -262 (1963), Song, H.-K. and Sawyer, R.H. , Dcv. Dyn. InPress (1995)).FGF-2 signaling is sufficient to form dermal condensation; Is direct   In the development of genetically normal chicken embryo wing primordia, fibroblast growth factor ( FGF) -2 and fibroblast growth factor receptor (FGFR) -1 There are spatially and temporally restricted expression patterns of genes. FGF-2 expression While restricted to epithelial primordia, FGFR-1 expression, on the other hand, is restricted to dermal condensation. Transcription of these genes is homozygous (sc / sc) for recessive gene scale deficiency. Was not detectable in the skin of the mutant embryo. Such mutated embryos are No wings can develop as a result of leaf loss. FGF- of sc / sc skin 2 results in the formation of wings at the site of application of this growth factor and their induction The wings expressed express FGFR-1 in their dermal condensation. From these, The role of FGF-2 in epithelial wing primordia formation as an epithelial signal has been established. Observation that FGF-2 can rescue the mutated phenotype of sc / sc embryos Indicates that FGF-2 is a signal that sc / sc mutant ectoderm cannot produce Suggests that it is downstream. The data of this relief was FGF-2 signal Mean that dermal act directly on the mesoderm to form dermal condensation, or Mesoderm before the F-2 treatment directs the ectoderm to form a dermal condensate in the mesoderm Can be interpreted to mean initiating a series of interactions between Came.   The FGF-2 signal is sufficient to form dermal condensation in the underlying mesoderm To test the hypothesis that there is, the exposed mutant mesoderm was exposed to FGF-2. did. This mesoderm is associated with wing development when combined with genetically normal ectoderm Mutations that are competent and inexperienced in what they can The ectoderm was defective and thus was exposed to any ectodermal signals It is unlikely that it will. Place FGF-2 beads on exposed mutant mesoderm At that time, formation of dermal condensation was observed in the mesoderm. These condensations FGFR-, two molecular markers normally expressed by cells that make dermal condensation 1 and positive for BMP-2. Therefore, FGF-2 signaling is true Enough to form skin condensation, this signaling is straightforward.Formulations and methods for administration of FGF-2   FGF-2, an FGF-2 fragment or analog can be obtained by any suitable means. Or directly (eg, by injection or local administration to a tissue site, locally) or Can be given to the patient systemically (eg, parenterally or orally). This Parenteral administration, e.g., intravenously, subcutaneously, intramuscularly, or by aerosol administration When given by the compound of the invention, the compound of the invention is preferably Make up the part. This solution may be used in addition to delivering the desired compound of the invention to the patient. Make sure that this solution does not adversely affect the patient's electrolyte and volume balance. Is acceptable. Thus, an aqueous solution for this FGF-2 or fragment thereof The medium was normal saline (9.85% NaCl, 0.15 M), pH 7-7. . 4).   Solutions useful for parenteral administration include any of the methods well known in the pharmaceutical arts, for example,Remington's Pharmaceutical Science (Gennaro, A., ed.), Mack Pub., 1990 It can be prepared by the method described. The formulation is, for example, a polyalkylene Glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalene, etc. Can be included. In particular, formulations for direct administration include glycerol and other Contains high viscosity components to help maintain compounds of this invention in desired locations be able to. Biocompatible, preferably bioresorbable polymers (eg, Alluronic acid, collagen, tricalcium phosphate, polybutyrate, Including tide and glycolide polymers, and lactide / glycolide copolymers) Are useful excipients for controlling the in vivo release of the compounds of this invention. Can get. Other potentially useful parenteral delivery systems for the compounds of this invention include: Ethylene vinyl acetate copolymer particles, osmotic pump, implantable leach cis And liposomes. Formulations for inhaled administration may include as excipients, e.g., Containing lactose or, for example, polyoxyethylene-9-lauryl ether, It may be an aqueous solution containing lycocholate and deoxycholate.   Formulations for topical administration to the skin surface will allow the compounds of this invention to be tolerated by the skin By dispersing in a carrier such as a lotion, cream, ointment or soap. Can be prepared. Cover skin to limit application and block movement Carriers capable of forming a film or layer are particularly useful.Generation of fragments and analogs   The inventors have found that epithelial-dermal interaction modulates the activity of FGF-2 (eg, (Promoting or inhibiting). Once this shows If so, one skilled in the art would be able to construct the FGF-2 structure, for example, to generate fragments or analogs. Alternatively, the newly created structure can be tested for activity. fragment Examples of prior art methods that enable the generation and testing of analogs and analogs are described below. Discuss. Using these or similar methods, FGF-2 binding to FGFR-1 Polypeptide fragments and analogs can be made and screened. Similarly, FGF-2 binding to FGF-2 polypeptides using these methods Generating fragments and analogs of polypeptide ligands such as FGFR-1 it can.   Fragment generation   Protein fragments can be obtained in several ways, for example, by recombination, by enzymatic digestion, Alternatively, it can be produced by chemical synthesis. Internal or terminal fragments of the polypeptide At one or both ends (in the case of a terminal fragment) of the nucleic acid encoding the polypeptide. (In the case of partial fragments) by removing one or more nucleotides from Can be. Expression of the mutated DNA produces polypeptide fragments . Therefore, digestion with "end-nibbling" endonucleases is DNA can be generated that encodes the alignment of the pieces. Encodes a protein fragment DNA can also be produced by random shearing, restriction digestion, or a combination of the above methods. can do.   Fragments can also be prepared by techniques known in the art, such as conventional Merrifield solid phase f-Moc or Chemical synthesis can also be performed using t-Boc chemistry. For example, the peptide of the present invention Can be freely separated into non-overlapping fragments of the desired length or overlapping fragments of the desired length. Can be split.Analog generation: generation of altered DNA and peptide sequences by random methods   Amino acid sequence variants of a protein may be proteins or specific domains of the protein or Can be prepared by random mutagenesis of the DNA encoding the region . Useful methods include PCR mutagenesis and saturation mutagenesis. run A library of dumb amino acid sequence variants is a library of degenerate oligonucleotide sequences. It can also be produced by the synthesis of a kit (protein variants in a mutant library). Methods for screening quality are described elsewhere herein).   PCR mutagenesis   In PCR mutagenesis, the reduced fidelity of Taq polymerase is used. Introduce random mutations into the cloned DNA fragment (Leung et al., 1989, Te chnique 1: 11-15). This is a very powerful and relatively fast random mutagenesis method is there. The DNA region to be mutated is excised by Taq DNA polymerase. For example, using a dGTP / dATP ratio of 5 under conditions that reduce the fidelity of DNA synthesis And Mn²⁺Polymerase chain reaction (PCR) Amplify using Insert the amplified DNA fragment into an appropriate cloning vector To give a random mutant library.   Saturation mutagenesis   Saturation mutagenesis is a rapid method for multiple single base substitution taloned DNA fragments. Enables introduction (Mayers et al., 1985, Science 229: 242). This technology is a mutation , Eg, sudden alteration due to chemical treatment of single-stranded DNA in vitro or irradiation Includes the generation of heterologous and complementary DNA strands. Mutation frequency determines the intensity of treatment. By adjusting, essentially all the possible base substitutions Obtainable. Since this procedure does not involve genetic selection for mutations, Both neutral and functionally altering substitutions are obtained. The distribution of point mutations is , It is not deflected towards conserved sequence elements.   Degenerate oligonucleotides   Homolog libraries are also generated from sets of degenerate oligonucleotide sequences. Can be Chemical synthesis of degenerate sequences can be performed with an automated DNA synthesizer. And then ligating these synthetic genes into an appropriate expression vector be able to. The synthesis of degenerate oligonucleotides is known in the art (eg, , Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1981) Recombinant DNA, Proc 3 rd Cleveland Sympos. Macromolecules, Editing AG Walton, Amsterdam: Elsevier 2 73-289; Itakura et al. (1984) Annu. Rev. Biochcm. 53: 323; Itakura et al. (1984) Scienc e 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477). Heel Techniques have been used in the directed evolution of other proteins (eg, Scott et al. (1990). ) Science 249: 386-390; Roberts et al. (1992) PNAS 89: 2429-2433; Devlin et al. (1990) Sc ience 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; and U.S. Pat. 23,409, 5,198,346 and 5,096,815. No).   Analog Production: Altered DNA and Peptide Sequences by Site-Directed Mutagenesis Generate columns   Using techniques of non-random or site-directed mutagenesis, Specific sequences or displacements can be provided. Using these technologies, the public It is possible to create variants containing, for example, deletions, insertions or substitutions of residues of a known amino acid sequence. it can. Sites for mutations can be stored individually or consecutively, for example (1) first With more radical choices, depending on the results achieved (2) by removing the target residue, or By inserting adjacent residues of the same or a different class, or It can be modified by a combination of options 1 to 3.   Alanine scanning mutagenesis   Alanine scanning mutagenesis is a suitable location for mutagenesis or A useful method for identifying certain residues or regions of a desired protein that is a domain. Cunningham and Wells (Science 244: 1081-1085, 1989). Alanine Scanin Grouping of residues or target residues (e.g., charged residues, e.g., Arg, Asp, His, Lys and Glu), neutral or negatively charged amino Substituted by an acid (most preferably alanine or polyalanine). Amino acid Substitutions affect the interaction of amino acids with the surrounding aqueous environment both inside and outside the cell obtain. Domains that are functionally sensitive to these substitutions are then Or by introducing another mutant at the substitution site. Therefore, amino acids Although the site for introducing a sequence change is predetermined, the nature of the mutation itself is not There is no need to decide. For example, to optimize the ability of a mutation at a given site Alanine scanning or random mutagenesis to target codons or regions The desired protein subunit mutant expressed can be Screen for the best combination.   Oligonucleotide-mediated mutagenesis   Oligonucleotide-mediated mutagenesis involves DNA substitution, deletion and insertion mutations. It is a useful method for preparing products, for example, see Adelman et al. (DNA 2: 183, 1983). I want to be illuminated. Briefly, the desired DNA is replaced with the oligonucleotide encoding the mutation. Altered by hybridizing leotide to a DNA template ( This template is the unmodified or untouched DNA sequence of the desired protein. Is a single-stranded form of a plasmid or bacteriophage). Hybridization After completion of the template, the complete second complementary strand of the template is Thus, the strand incorporates the oligonucleotide primer and the desired protein is synthesized. Encodes selected changes in white matter DNA. Generally, at least 25 nucleos Use oligonucleotides of a tide length. The optimal oligonucleotide will 12 which is completely complementary to the template on either side of the encoding nucleotide It has １５15 nucleotides. This is because the oligonucleotide is a single-stranded DNA template. Ensure proper hybridization to plate molecules. These oligonu Nucleotides can be obtained by techniques known in the art, such as Crea et al. (Proc. Natl. Acad. Sci. USA, 75 : 5765 [1978]) can be easily synthesized.   Cassette mutagenesis   Another method for preparing variants, cassette mutagenesis, is described by Wells et al. It is based on a more described technique (Gene, 34: 315 [1985]). The starting material is mutated A plasmid (or other vector) containing the DNA of the protein subunit to be deleted It is. Identify the codons in the protein subunit DNA to be mutated. Identification Without unique restriction endonuclease sites on either side of the mutated site No. If such restriction sites do not exist, replace them with the oligonucleotides described above. Produced using nucleotide-mediated mutagenesis, they can be used to generate the desired protein It can be introduced at an appropriate position in the buunit DNA. Plasmid restriction site After introduction into the plasmid, the plasmid is cut at those sites to linearize it. This Double-stranded oligonucleotides encoding the DNA sequence between these restriction sites but containing the desired mutation Nucleotides are synthesized using standard procedures. These duplexes are synthesized separately And then hybridized using standard techniques. This double-stranded oligonucleotide Nucleotides are referred to as cassettes. This cassette is directly transformed into a linearized plasmid. 3 'and 5' ends comparable to the ends of the plasmid so that it can be ligated directly Design to have This plasmid now contains the mutated desired protein Contains subunit DNA sequences.   Combinatorial mutagenesis   Mutants can also be generated using combinatorial mutagenesis. For example, one Aligning the amino acid sequences for homologues of the group or other related proteins, preferably Improve the highest possible homology. All addresses that appear at a given position in the aligned sequence The amino acids can be selected to create a degenerate set of combinatorial sequences. Mutant Are generated by combinatorial mutagenesis at the nucleic acid level, Encoded by a diverse gene library. For example, synthetic oligonucleotides Mixture of enzymes is enzymatically ligated to the gene sequence to generate a degenerate set of potential sequences. As individual peptides or larger fusion proteins containing a set of degenerate sequences It can be expressed as a set.   Primary high-through screening of libraries of peptide fragments or homologs Put method   In this field, various methods for screening the generated mutant gene products have been developed. The technology is known. For screening large gene libraries Techniques often include cloning a genetic library into an replicable expression vector. Transforming appropriate cells with the resulting vector library, The genes are then detected for the desired activity (eg, in this case, FG F-2 or FGFR-1) encodes the gene for which the product was detected. Expression under conditions that facilitate relatively easy isolation of the vector. Each of the techniques below describes, for example, a number of sequences created by random mutagenesis techniques. Is amenable to high-throughput analysis for screening   Two hybrid system   A two-hybrid assay, for example, using the system described above (other screens described herein) GFFR-1 that binds to the intracellular domain of FGFR-1 Fragments or analogs of the two polypeptides can be identified. These are the Agoni (FGFR-1 domain) Is used as a bait protein, and a library of FGF-2 mutants is Expressed as quality). In a similar manner, the two-hybrid assay (here Coupled to other FGF-2 polypeptides). Matching fragments and analogs of FGFR-1 can be found.   Display library   In one approach to screening assays, candidate peptides are A specific cell or virus particle is displayed on the surface of a cell or virus particle. Ability to bind to the appropriate receptor protein via the displayed product of Detect by "panning assay". For example, a gene library The resulting fusion protein can be cloned into the gene for the surface membrane protein of White matter can be detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio / Technology 9: 1370-1371; and Goward et al. (1992) TIBS 18: 136-14. 0). In a similar manner, potentially functional with a detectably labeled ligand Can be evaluated. Fluorescently labeled ligands such as Recept Can be used to detect homologs that retain ligand binding activity. firefly The use of photolabeled ligands can be determined by visually examining the cells and analyzing them under a fluorescence microscope. Release and, if cell morphology permits, use a fluorescence activated cell sorter. To separate them.   Expression of a gene library as a fusion protein on the surface of virus particles Can be. For example, in a phage on fiber system, foreign peptide delivery Can be expressed on the surface of infectious phage, thereby conferring two benefits. it can. First, these phages were added at a rate of 10 per milliliter.¹³Greatly exceeds Can be loaded on the affinity matrix at different concentrations. Can be screened at once. Second, each infectious phage Displays gene products on the surface, so that if a particular phage -If the phage were recovered in a low yield due to entrapment, the phage could be transferred by another round of infection. Can be amplified. E. coli filamentous phage M13, fd And fl are most often used for phage display libraries. Using each of the phage g3. Or g8. Coat proteins, the fusion protein is It can be produced without breaking the packaging of the loose particles. Foreign episodes A top can be expressed at the NH2 terminus of p3. Phage can be recovered from a large excess of phage lacking this epitope ( Ladner et al., PCT published WO90 / 02909; Garrard et al., PCT published WO92. Marks et al. (1992) J. Biol. Chem. 267: 16007-16010; Griffiths et al. (1993) EMBO J 12: 725-734; Clackson et al. (1991) Nature 352: 624-628; and Barbas et al. ( 1992) PNAS 89: 4457-4461).   A common approach is the maltose receptor of E. coli (outer membrane protein LamB). As a peptide fusion partner (Charbit et al. (1986) EMBO 5,3029-3037 ). Insert the oligonucleotide into the plasmid encoding the LamB gene Produced a peptide fused to one of the extracellular loops of the protein. these Peptides can be used to bind ligands, such as antibodies, and their When the cells are administered to an animal, they can elicit an immune response. Other cell surfaces Proteins such as OmpA (Schorr et al. (1991) Vaccines 91, 387-392), PhoE (A gterberg et al. (1990) Gene 88, 37-45) and PAL (Fuchs et al. (1991) Bio / Tech 9,1369-). 1372) and large bacterial surface structures act as vehicles for peptide display I came. Pili, which is a conduit for the exchange of genetic information between bacteria, by polymerizing peptides Can be fused to pilin, a protein that forms (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells In addition, cilia provide useful support for the presentation of peptides to the extracellular environment. Peptide Another large surface structure used in displays is the flagella, a bacterial activator . The fusion of the peptide to the subunit protein flagellin is (Kuwajima et al. (1988) Bio / Tech. 6, 1080-10). 83). Surface proteins of other bacterial species have also served as peptide fusion partners. An example Include Staphylococcus protein A and Neisseria outer membrane protease IgA (Hansson et al. (1992) J. Bacteriol. 174, 4239-4245 and Klauser et al. (1990) E MBO J. 9, 1991-1999).   In the filamentous phage system and LamB system described above, the peptide and its Physical association between the DNA and the coding DNA is caused by particles (cells) having the peptide on the surface. Or phage). Capturing the peptide Capture the offspring and the DNA in them. Another scheme uses the DNA binding protein LacI. To form a bond between the peptide and DNA (Cull et al. (1992) PNAS USA 89:18). 65-1869). This system has an oligonucleotide cloning site at the 3 'end. A plasmid containing the LacI gene having is used. By arabinose Under controlled induction, a LacI-peptide fusion protein is produced. This fusion Binds to a short DNA sequence known as the LacO operator (LacO). Retain the natural ability of LacI. Two copies of LacO in expression plasmid By placing, the LacI-peptide fusion will allow the plasmid encoding it to Tightly coupled to the pad. These plasmids in each cell are a single oligonucleotide Since they contain only the otide sequence and each cell expresses only a single peptide sequence, these Binds specifically and stably to the DNA sequence that directs its synthesis. This Gently lyse the cells of the library and immobilize the peptide-DNA complex. Exposure to the receptor matrix to recover complexes containing active peptides I do. The bound plasmid DNA is then used for amplification and DNA sequencing. To determine the identity of the peptide ligand. Practical utility of this method In order to show the properties, a large random library of 12-mer peptides was created, Selected with monoclonal antibody raised against opioid peptide dynorphin B I chose. A panel of peptides was recovered, all consisting of the six residue portion of dynorphin B Are related by a consensus sequence corresponding to (Cull et al. (1992) Proc. Natl. Acad .Sci.U.S.A.89-1869)   This scheme (often called a peptide on the plasmid) has two important In that it differs from the phage display method. First, the peptide is a fusion protein. Live as a peptide with a free carboxy terminus attached to the C-terminus of white matter This results in a rally member display. Filamentous phage coat protein pIII And pVIII are fixed to phage by their C-terminus and The peptide is located in the outwardly extending N-terminal domain. For some designs The phage-displayed peptide has a right at the amino terminus of the fusion protein. Is given. (Cwirla et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382) The difference between the two is that biology affects the population of peptides actually present in the library. It is a set of objective deflection. LacI fusion molecules are trapped in the host cell cytoplasm. It is. Phage coat fusions are briefly exposed to the cytoplasm during migration, but are C-terminally isolated. The periplasmic zone is rapidly passed through the membrane while remaining fixed in the membrane by the aqueous domain. Secreted into the fraction, the N-terminus contains the peptide (in the periplasm Protruding), waiting for assembly into phage particles. LacI and fur The peptides in the dilibrary are the result of their exposure to various proteolytic activities. As significantly different. Phage coat proteins are transported across the inner membrane and Requires the use of signal peptidase as a prelude to I need it. Certain peptides have deleterious effects on these processes, and Not sufficiently displayed in the blurry (Gallop et al. (1994) J. Med. Chcm. 37 (9): 1233-12 51). These particular deflections are not a factor in LacI display systems. No.   The number of small peptides available in recombinant random libraries is huge . 10⁷-10⁹A library of independent clones is routinely prepared. 10¹¹Although a large library has been created, this size It's close to the practical limits on ballying. There is no limit to this library size Transforming the DNA containing the randomized segment into the host bacterial cell Occurs in the steps. In order to circumvent this limitation, the polysomy of nascent peptides In-vitro systems based on displays in system complexes have recently been developed. Was. This display library method uses currently available phage / phage Generate libraries 3 to 6 orders of magnitude larger than mid or plasmid libraries There is a possibility. Furthermore, construction of these libraries, expression of peptides and Screening is performed in a completely cell-free format.   In one application of this method (Gallop et al. (1994) J. Med. Chem. 37 (9): 1233-125. 1), 10¹²To construct a molecular DNA library encoding the 10-mer peptide of In a transcription / translation system coupled in E. coli S30 in vitro, Was expressed. Conditions so that the ribosome is stuck on the mRNA To cause a significant proportion of the RNA to accumulate in the polysomes and still Complexes containing nascent polypeptides associated with the RNA that encodes them Was produced. These polysomes are used to display more traditional recombinant peptides. Receptors immobilized in much the same way as screening ray libraries Sufficiently strong for affinity purification at low temperature. R from the bound complex The NA is recovered, converted to cDNA, amplified by PCR, and Generate templates for synthesis and screening of the compounds. This polysome The spray method can be combined with a phage display system. A few lau After screening the cDNA, the cDNA from the polysome-enriched pool was It was cloned into a dimid vector. This Better was fused to the coat protein As a peptide expression vector for displaying peptides and for peptide identification Function as both vectors for DNA sequencing. Polysome-derived Expression of the peptide on phage continues this form of affinity selection procedure. Peptides on individual clones can be used in phage ELISA Binding activity or binding specificity in a complete phage ELISA (Barret et al. (1992) Anal. Biochem 204, 357-364). To identify the sequence of the active peptide, the D The NA is sequenced.   Second screening   After the high-throughput assay described above, to further identify biological activity, For example, a second method that allows one of skill in the art to distinguish an agonist from an antagonist. Can be screened. The second type of screening used is , Depending on the desired activity that needs to be tested. For example, the proteins of interest and their respective Primary screening as described above using the ability to block interactions with Gand Identifying an antagonist from a group of peptide fragments isolated by one of the following: Assays that can be developed.   Therefore, methods for producing fragments and analogs and activating them Methods for testing for are known in the art. Once the core sequence of interest has been identified Once obtained, obtaining analogs and fragments is routine for those skilled in the art. is there.   Peptide mimetic   This invention also provides for the reduction of the protein binding domain of an FGF-2 polypeptide. Produce a mimetic, eg, a peptide or non-peptide agent. These peptidomimetics The product can be, for example, a natural paired ligand, such as an FGF-2 polyprotein that binds to FGFR-1. In the case of a peptide, it can disrupt the binding of FGF-2 to its paired ligand You. (This invention also provides an FGF that blocks the binding of FGFR-1 to FGF-2. Also encompasses mimics of R-1. ) Involved in molecular recognition of FGFR-1 polypeptide Determining critical residues of a subject FGF-2 polypeptide, FGF- which competitively or non-competitively inhibits binding to FGFR-1 polypeptide 2 can be used to generate peptidomimetics. (For example, " Peptide inhibitors of human papillomavirus protein binding to retinoblas toma geneprotein "European Patent Applications EP-412,762A and EP-B31,0 80A ". ) For example, using scanning mutagenesis, F Specific FGF-2 polypeptides involved in binding to GFR-1 polypeptides The amino acid residue can be mapped to determine the binding to the FGFR-1 polypeptide. To mimic those residues and therefore the FGF-2 polypeptide to the FGFR-1 polypeptide. Binding of the repeptide can be prevented, thereby allowing FGF-2 or FGFR- A peptidomimetic compound (eg, diazepine or Isoquinoline derivatives). Hydrolysis of critical residues Impossible peptide analogs are identified as benzodiazepines (eg, Freidinger et al., Peptid es: Chemistry and Biology, edited by G.R.Marshall, ESCOM Publishing: Leiden, Netherlands, 1988 Azepine (for example, Huffman et al., Peptides: Chemistry and Biology, G.R.M. arshall, ESCOM Publishing: Leiden, The Netherlands, 1988), gamma lactate replaced Garvey et al., Edited by Pcptides: Chemistry and Biology, G.R.Marshall, ESCOM Edition: Leiden, The Netherlands, 1988), Keto-methylene pseudopeptide (Ewenson et al. (1986) J Med Chem 29: 295; and Ewenson et al., Peptidcs: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Illinois , 1985), β-turn dipeptide core (Nagai et al. (1985) Tetrahedron Lett 26: 647). (1986) J Chem Soc Perkin Trans 1: 1231), and β-aminoal Cole (Gordon et al. (1985) Biochem Biophys Res Commun 126: 419; and Dann et al. (198 6) It can be produced using Biochem Biophys Res Commun 134: 71).Drug screening assays   Modulates the activity of FGF-2 (eg, promotes or inhibits epithelial-dermal interaction) The present invention provides that FGF- Function of the two polypeptides (in this case) in normal cells, or epithelial-dermal interaction Drugs that are either agonists or antagonists of that role in An assay is provided that can be used for leaning. In one specific example The assay uses FGF-2 polypeptide and a natural ligand (eg, FGF The ability of the compound to modulate the binding between R-1) is evaluated. Modification of assay format Is sufficient and will be understood by those skilled in the art in light of the present invention.   Many drug screening programs to test libraries of compounds and natural extracts To maximize the number of compounds examined in a given time period within a program, A throughput assay is desirable. Cell-free system (purified or partially purified protein) Assays performed on white matter) can often be described as "primary screening" (They are useful for the alteration of molecular targets mediated by test compounds). Can be generated to give rapid deployment and relatively easy detection). Further testing The effect of the compound on cytotoxicity and / or bioavailability is generally The system can be ignored; on the contrary, this assay is primarily Focus on the effect of the drug on offspring targets (it is the binding affinity with other proteins) Or changes in the enzymatic properties of this molecular target).FGF-2 peptide analog   The peptide analog of the FGF-2 polypeptide is preferably 150, 130 , 110, 90, 70 amino acids in length, preferably less than 50 amino acids in length And most preferably less than 30, 20, or 10 amino acids in length. Preferred examples Wherein the peptide analog of the FGF-2 polypeptide has at least about 10, It is 20, 30, 50 or 100 amino acids in length.   The peptide analog of the FGF-2 polypeptide is preferably a native FGF- 2 polypeptides and at least about 50%, 60%, 80%, 85%, 90%, 9% Has 5% or 99% homology or sequence similarity.   Peptide analogs of FGF-2 polypeptide are native FGF-2 polypeptides. And at least 1, 2, 5, 10 or 20 amino acid residues; However, preferably, they have less than 15, 10 or 5 amino acid residues. Differs from native FGF-2 polypeptide.   Useful analogs of FGF-2 polypeptides are agonists or antagonists It may be. Antagonists of FGF-2 polypeptides include receptors such as Binds to the FGFR-1 receptor but has certain additional biological activities, such as sig It may be a molecule that lacks a null transformation. Antagonists interact with the epithelium-dermis interaction, Or processes or products resulting from epithelial-dermal interaction, such as primordium formation, dermal coagulation Shrinkage, FGFR-1 induction, or skin appendages (eg, hair, scales or feathers) Development can be inhibited. Agonists exert an epithelial-dermal interaction or Processes or products resulting from dermal interactions, such as primordium formation, dermal condensation formation, Promotes FGFR-1 induction or development of skin appendages (eg, hair, scales or feathers) It is a derivative that can proceed.   Many analogs of FGF-2 are known in the art. This series of knowledge , Will provide those skilled in the art with guidance for making new FGF-2 analogs.   Using site-directed mutagenesis, human fibroblast growth factor 2 (FGF-2) Of the four cysteines at amino acid residues 26, 70, 88 and 93 of the mature protein Each was individually converted to serine (Seno et al., "Stabilizing Basic Fibroblast" Growth Factor Using Protein Engineering ",Biochemical and Biophvsical R esearch Communications, (1988), Vol. 151 (2): 701-708). FGF-2 protein When the cysteine residue at position 70 or 88 is replaced with serine, the biological activity and And heparin binding capacity was retained. This finding indicates that cysteines at these positions It is not essential for the expression of biological activity. These positions, especially 88 Substitution at the position indicated that FGF-2 eluted from the heparin affinity column was Heterogeneity recognized as several peaks after oxidation with hydrogen peroxide Even because the cysteines at these positions are exposed on the surface of the molecule. To form disulfide bonds that induce heterogeneous conformations. Suggests. Furthermore, under acidic conditions, these modified FGF-2s It has been shown to be more stable in retaining their activity.   The structural analog of human fibroblast growth factor 2 (FGF-2) is synthetic FGF-2 Three putative heparin bindings prepared by site-directed mutagenesis of the gene The effect of domain amino acid substitutions on the biological activity of FGF was examined. Heath et al. (1991) Biochemistry 30: 5608-5615 discloses several such analogs. ing. After expression in E. coli, the mutant protein is Purification using homography to homogenize DNA in human foreskin fibroblasts They were analyzed for their ability to stimulate synthesis. Recombinant human FGF-21-14 6 and [Ala⁶⁹, Ser⁸⁷FGF-2 has two of the four cysteines An analog substituted by lanine and serine, which is equivalent to standard bovine FGF-2. Had a strong effect. Of aspartic acid-19 in the first heparin binding domain Replacement with arginine resulted in higher total fibroblast cell counts compared to FGF-2. Molecules have been generated that stimulate the somatic mitogenic response. Further, in the second domain Glutamic acid of arginine-107 or glutamine-123 in the third domain Displacement resulted in compounds that were 2- and 4-fold more potent than FGF-2. In contrast, Substitution of luginin-107 with isoleucine reduced the activity of this molecule 100-fold . [Glu^107.123] FGF-2 and [Arg¹⁹, Lys^123.126] FGF-2 Combinations of domain substitutions that produce variants result in any additional alterations to biological activity. No differences were noted. Changes in the biological activity of these analogs are dependent on the site of modification and the species. Depended on both kind. Increased positive charge in the first domain and the second and The increased negative charge in the three domains has increased biological potential. these The altered activity of the derivative is due, in part, to a change in the affinity of the analog for heparin. Seems like for. All three changes in the putative heparin binding domain are Conclusion that GF-2 results in altered mitogenic activity and heparin interaction Was done.   Springer et al. (1994) The Journal of Biological Chemistry 269: 26879-26884 Uses site-directed mutagenesis based on the protein structure of FGF-2 to signa FGFR junctions on FGF-2 that act to initiate cooperative transformation The position was identified. Both FGFR binding surfaces are heparin sulfate proteoglycan binding domains. It is a different thing. Major high affinity binding interactions include solvent Hydrophobic amino acids (Tyr-24, Tyr-103, Leu-140) exposed to And Met-142) and two polar residues (Arg-44 and As) n-101). These hydrophobic contacts govern the main binding interactions, Gives about 75% binding affinity. The second FGFR binding site on FGF-2 is about It has a 250-fold lower affinity and has a type I β-turn (previously putative receptor binding Exposed surface amino acids Lys-100, Tyr -111 and Trp-114.   Zhu et al. (1995) The Journal of Biological Chemistry 37: 21869-21874 Based on the peptide mapping of F-2 and molecular dynamics calculations of the three-dimensional structure, Using position-directed mutagenesis, positions 107, 109-114 and 96 of FGF-2 The effect of changing the residues on the receptor binding affinity was investigated. All pokes The mutated protein was cloned and expressed in E. coli, and heparin-sepharose was expressed. Purified to homogeneity using a column and evaluated for receptor binding affinity. 1 Substitution of the residues at positions 07 and 109-114 with alanine or phenylalanine is It was found that there was almost no effect on receptor binding affinity compared to wild-type FGF-2. This indicates that residues 109-114 of FGF-2 have a low affinity binding site. Consistent with previous evidence of inclusion. Under contract, place Glu-96 in alanine The exchange resulted in a molecule with about 0.1% affinity for wild-type FGF-2. Corresponding The affinities of lysine and glutamate muteins were 0.3 and 10 respectively. %Met. These findings indicate that residues 106-115 on FGF-2 are FGF-2 Consistent with the notion of representing a low affinity binding site above. Furthermore, Glu-96 is a fiber Identified as a key residue for binding to fibroblast growth factor receptor-1 .   Therefore, the cysteines at residues 26, 70, 88 and 93 of mature FGF-2 are not Plays a role in disulfide bond formation leading to homogeneous conformation Can be summarized. Residues Asp-19, Arg-107 and Glu-12 3 appears to play a role in heparin binding, but residues Tyr-24, T yr-103, Leu-140, Met-142, Arg-44 and Asn-1 01 plays a role in receptor binding. 107 and 109-114 Residues have little effect on the receptor binding affinity of FGF-2 Although not likely, Glu-96 is a key residue for binding to FGFR-1. is there.Equivalent   One skilled in the art will recognize many equivalents to the particular procedures described herein or will Can be verified using experimental experiments. Such equivalents are within the scope of this invention. And is covered by the claims set forth below.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/14 A61P 43/00 111 43/00 111 A61K 37/02 (72) Inventor Wong, Yuin United States of America 02115 Massachusetts, Boston, Sun Stephen Street 109

Claims (1)

[Claims] 1. Modulating FGF-2 activity in one or both of the epithelium and dermis , A method of regulating epithelial-dermal interaction. 2. 2. The method of claim 1, wherein the method promotes FGF-2 activity. 3. 2. The method of claim 1, wherein the method promotes FGF-2 activity in the dermis. 4. The above-mentioned interaction can be determined by primordium formation, dermal condensation formation, FGFR-1 induction or skin 2. The method of claim 1, wherein the method is selected from the occurrence of a genus. 5. 2. The method according to claim 1, wherein said interaction is hair development. 6. Promoting FGF-2 activity by administration of exogenous FGF-2 or a fragment thereof, The method of claim 1. 7. FGF-2 activity by an anti-FGF-2 antibody or an FGF-2 binding fragment thereof, FGF-2 binding fragment of FGF-2 receptor, or inhibits FGF-2 expression 2. The method of claim 1, wherein the method is inhibited by administration of an antisense molecule. 8. 2. The method of claim 1, wherein the method is performed in vivo and the patient is a mammal. 9. 2. The method of claim 1, wherein the method is performed on a tissue or cell in vitro. 10. Modulating FGF-2 activity in one or both of the dermis and epithelium How to control the generation of skin appendages. 11. 11. The method of claim 10, wherein the appendage is hair. 12. 11. The method of claim 10, wherein the method promotes FGF-2 activity. 13. 11. The method of claim 10, wherein FGF-2 activity is promoted in the dermis. 14． The method according to claim 10, wherein the skin appendage is hair. 15. FGF-2 activity is enhanced by administration of exogenous FGF-2 or a fragment thereof The method of claim 10, wherein 16. An FGF-2 antibody or an FGF-2-binding fragment thereof Inhibits FGF-2 binding fragment of FGF-2 receptor, or FGF-2 expression 12. The method of claim 10, wherein the inhibition is by administration of an antisense molecule. 17． 11. The method of claim 10, wherein the method is performed in vivo and the patient is a mammal. 18. 11. The method of claim 10, wherein the method is performed on a tissue or cell in vitro. 19. A method of producing hair in a tissue or patient, comprising a therapeutically effective amount of exogenous The method comprising administering FGF-2 or a fragment thereof to a tissue or patient. 20. 20. The method of claim 19, wherein the method promotes FGF-2 activity. 21. 20. The method of claim 19, wherein FGF-2 activity is promoted in the dermis. 22. FGF-2 activity is enhanced by administration of exogenous FGF-2 or a fragment thereof 20. The method of claim 19, wherein 23. 20. The method of claim 19, wherein the winning patient in vivo is a mammal. 24. Ability to bind FGF-2 polypeptide and FGF-2 / FGFR-1 phase Evaluate compounds for their ability to modulate interactions or modulate FGF-2 activity Contacting the compound with an FGF-2 polypeptide; The ability of the compound to interact with FGF-2; , Applied to a tissue or cell to produce an epithelium-dermis interaction or an epithelial-dermis interaction The above process, including further evaluating its effect on the process or product Law.