WO1993025564A1 - Peptides de fixation de phosphoinositides derives des sequences de gelsoline et villine - Google Patents

Peptides de fixation de phosphoinositides derives des sequences de gelsoline et villine Download PDF

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WO1993025564A1
WO1993025564A1 PCT/US1993/005388 US9305388W WO9325564A1 WO 1993025564 A1 WO1993025564 A1 WO 1993025564A1 US 9305388 W US9305388 W US 9305388W WO 9325564 A1 WO9325564 A1 WO 9325564A1
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Prior art keywords
gelsolin
ppi
pip
peptide
actin
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PCT/US1993/005388
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English (en)
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Paul A. Janmey
Helen Yin
Thomas P. Stossel
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Brigham And Women's Hospital
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Publication of WO1993025564A1 publication Critical patent/WO1993025564A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4735—Villin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides
    • G01N2405/06—Glycophospholipids, e.g. phosphatidyl inositol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to the field of cell biology. Specifically the present invention relates to agents which are capable of binding to the gelsolin binding site on phosphoinositides (PPI) or the PPI binding site on gelsolin and thus inhibiting PPI/gelsolin and PPI/enzyme interactions.
  • PPI phosphoinositides
  • Gelsolin and villin are actin filament severing and capping proteins which are activated by Ca 2+ and have profound effects on actin filament organization and assembly.
  • the polyphosphoinositides (PPI) phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5- diphosphate (PIP 2 ) inactivate the actin filament-severing proteins villin and gelsolin and dissociate them from monomeric and polymeric actin.
  • Severing of actin filaments and nucleation of actin polymerization are probably essential for the remodeling of cortical actin networks that accompanies nearly all types of cell activation (Stossel, T.P., J. Biol. Chem. 264: 18261-4 (1989)).
  • the family of proteins structurally related to gelsolin is likely to regulate, in part, transformations of the cytoplasmic actin network because these proteins exert powerful effects on actin filament length and initiate filament formation from actin monomers (Matsudaira and Janmey, Cell
  • PPI membrane polyphosphoinositides
  • PIP and PIP 2 modulate the activity of a number of actin regulatory proteins (Lassing and Lindberg, Nature 574(6010):472-474 (1985); Janmey and Stossel, Nature 325:362-364 (1987); Yin, H.L., et al. , FEBS 264:78-80 (1990); Yu, F.-X., et al. , Science 250: 1413-1415 (1990); Maekawa and Sakai, J. Biol. Chem. 265: 10940-10942
  • gelsolin and profilin bind preferentially to PPI, have much reduced affinity for phosphatidylinositol, and are not inhibited by phosphatidylserine, -ethanolamine or -choline (Lassing and Lindberg, Nature 574(6010): 472-474 (1985); Janmey and Stossel, Nature 525:362-364 (1987); Goldschmidt- Clermont, P.J. , et al. , Science 257: 1231-1233 (1991)).
  • PPI inhibit gelsolin and profilin binding to actin, and these proteins may in turn modulate PIP 2 metabolism by sequestering/releasing PIP 2 (Goldschmidt-Clermont, P. J. , et al. , Science 257: 1575-1578 (1990); Goldschmidt-Clermont, P.J., et al. , Science
  • PPI polyphosphoinositides
  • PIP 2 phosphatidylinositol 4,5-bisphosphate
  • SDS sodium dodecyl sulfate
  • PAGE polyacrylamide gel electrophoresis. 257: 1231-1233 (1991)).
  • gelsolin Hartwig, J.H., et al , J. Cell Biol. 108:467-479 (1989)
  • profilin Harmonic acid
  • gelsolin first attaches laterally to actin filaments through an actin binding site located within its domains II-III ((human plasma gelsolin (GS), residues 150-406)) and then breaks the acti actin bond via another actin binding site located in domain I (GS 1-149; this will be referred to as GS149) (Fig. 12A).
  • actin binding site located within its domains II-III ((human plasma gelsolin (GS), residues 150-406)
  • GS149 human plasma gelsolin
  • Fig. 12A Previously, we have found that PPI prevented domains II-III from binding to actin filaments and therefore hypothesized that PPI inhibits severing through the side-binding domain (Yin, H.L., et al , J. Cell Biol 706:805-812 (1988)).
  • the present study investigated the PPI and F-actin binding properties of synthetic peptides based on the sequences of villin and gelsolin within the domains thought to account for PPI regulation of the parent molecules. Direct binding of peptides and their fluorescently labeled derivatives was assessed by light scattering and fluorescence polarization. Competition between the peptides and native gelsolin for PPIs was measured using functional assays for the actin filament severing activity of gelsolin.
  • domain I of gelsolin is also inhibited by PPI. Its PPI-binding site is distinct from the actin binding site.
  • PPI binding is localized to between residues 135-149 by deletional mutagenesis and competition with a synthetic peptide.
  • the existence of multiple PPI- binding sites as well as actin binding sites allows stringent and differential regulation of gelsolin activity in response to changes in PPI concentrations.
  • the present invention is based on the discovery that short peptide sequences from gelsolin are capable of modulating the interaction of PPI with gelsolin as well as modulatating the interconversion of PPI to various isomers and phosphorylation states. Based on these observation, agents are described which are capable of binding to the gelsolin binding site of PPI or the PPI binding site of gelsolin.
  • the present invention discloses peptides with the following amino acid sequences:
  • CKSGLKYKKGGVASGF (Seq. ID No. 1 corresponding to GS134- 149, hereinafter "SI ")
  • peptides as well as fragments of these sequences, antibodies capable of binding to these peptides, and anti-idiotypic anti-Si or anti-S2 antibodies are capable of inhibiting the interaction of PPI with gelsolin.
  • PPI/gelsolin interaction By inhibiting PPI/gelsolin interaction, the inhibitory effect of PPI on gelsolin mediated actin cleavage can be modulated.
  • the modulation of PPI/gelsolin interaction can be used to further elucidate cellular responses which cause actin turnover.
  • the above agents are capable of inhibiting the enzymes which phosphorylate, dephosphorylate, or isomerize PPI by binding to PPI making them unavailable as an enzyme substrate or as a feedback inhibitor of enzymatic activity.
  • the present invention further discloses methods of inhibiting the interaction of PPI and gelsolin in vivo.
  • PPI/gelsolin interactions in vivo can be modulated by supplying to an individual a pharmaceutically acceptable composition containing one of the agents of the present invention.
  • cells can be engineered through recombinant techniques to inducibly or constitutively express the peptide agents of the present invention.
  • a cell By expressing and producing the peptide agent, a cell can be directed to modulate PPI/gelsolin or PPI/enzyme interaction.
  • the present invention further discloses methods of identifying agents capable of inhibiting PPI/gelsolin interaction.
  • an agent is incubated in a sample with gelsolin, PPI, and pyrene-F-actin and the solution diluted to an actin concentration of 300 ⁇ M.
  • Agents capable of inhibiting PPI/gelsolin interaction increase the rate at which the fluorescence of such sample decreases as the actin depolymerizes.
  • Figure 2 Maintenance of gelsolin' s F-actin severing activity by peptides in the presence of inhibitory amounts of PIP 2 .
  • the ability of peptides to compete with intact gelsolin for PIP 2 was determined by measuring the ability of gelsolin to sever actin filaments in the presence of sufficient PIP 2 to cause approximately 90 % inhibition of gelsolin in the absence of competitive binding by other ligands. Experimental details are described in the Examples.
  • Open triangles villin 84-153; closed triangles: gelsolin 150-169; open circles: villin 140-147; closed circles: villin 133-147; large squares: MARCKS 155-173; open squares: arginine trimers; closed squares: lysine pentamers.
  • Figure 3 Aggregation of PIP 2 micelles by villin 84-153. The total light scattering intensity (600 nm light, 90°) of 30 ⁇ M micellar PIP 2 in water after addition of increasing amounts of villin peptide.
  • Figure 5 Maintenance of gelsolin's F-actin severing activity by peptides in the presence of inhibitory amounts of phosphatidylcholine (PC)/PIP 2 vesicles.
  • PC phosphatidylcholine
  • the ability of peptides to compete with intact gelsolin for PIP 2 in mixed lipid vesicles containing a 10: 1 PC:PIP 2 molar ratio was determined by measuring the ability of gelsolin to sever actin filaments in the presence of sufficient PIP 2 to cause approximately 90% inhibition of gelsolin in the absence of competitive binding by other ligands. Experimental details are described in the Examples.
  • Open triangles profilin; closed triangles: gelsolin 150-169; closed circles: thymosin- -4; large squares: MARCKS 155-173; open squares: arginine trimers; closed squares: lysine pentamers.
  • the large open circles represent the severing activity of 43 nM gelsolin incubated with various amounts of PC:PIP 2 vesicles in the absence of peptide with effective PIP 2 concentrations ranging from 0.5 ⁇ M to 5 ⁇ M.
  • Figure 6 Average hydrodynamic diameter of PC/PIP 2 vesicles in various concentrations of gelsolin or gelsolin 150-169. Increasing concentrations of gelsolin (triangles) or the gelsolin 150-169 peptide (circles) were incubated with 50 ⁇ g/ml of either 10: 1 PC/PIP, vesicles (solid symbols) or control PC vesicles (open triangles), and the apparent diameter determined by QLS as described in the Examples. The vertical arrow denotes the peptide concentration at which the effect of the PC/PIP 2 vesicles on gelsolin is reduced by 90%.
  • Figure 7 Restoration of filament-severing ability of gelsolin inhibited by polyphosphoinositides.
  • Gelsolin/PIP 2 closed circles
  • gelsolin/PIP complexes open circles
  • incapable of severing actin filaments were incubated for ⁇ 5 seconds with gelsolin 150-169 at the molar ratios shown, and reversal of the inhibition measured by the filament-severing assay described in the Examples.
  • FIG. 1 SDS- polyacrylamide gel of purified GS149 and GS134 (lanes 3 and 2, respectively).
  • micellar PIP 2 Effect of PIP on interactions with actin. Increasing concentrations of micellar PIP 2 were added to solutions containing 5.1 ⁇ M pyrene-actin and 2.4 ⁇ M GS149 or 2.8 ⁇ M GS134 and incubated for 16 hr. In the absence of
  • PIP 2 , GS149 and GS134 reduced actin polymerization by half. This was defined as 100% actin binding on the Y-axis. Polymerization in the presence of PIP 2 was expressed as percent of this value, (closed circles) GS149; (squares) GS134.
  • Figure 9 Binding of gelsolin polypeptides to PIP 2 detected by gel filtration chromatography.
  • A Left panel, elution profiles of 25 ⁇ M GS149 in the presence of 0, 35 and 212 ⁇ M PIP 2 micelles. GS149 was incubated with PIP 2 and chromatographed at room temperature on a Superose 12 FPLC column (Pharmacia). Right panel, elution profiles of 26 ⁇ M GS134 in the presence of 0, 53 and 212 ⁇ M PIP 2 . Absorbance at 280 ⁇ was expressed in arbitrary units.
  • B Coomassie blue stained gels of gelsolin polypeptides in column fractions in the absence and presence of PIP 2 .
  • GS149 and GS149 were incubated with 90 ⁇ M PIP 2 and 300 ⁇ l of each column fraction were dried down in a SpeedVac and analyzed by SDS-PAGE. Although the starting materials were relatively pure (Fig. IA and Fig. 6A), the column fractions contained more degradation products probably because they were stored at 4°C for several days before processing for SDS-PAGE. GS134 was incubated with 136 ⁇ M PIP 2 and every other fraction was analyzed by SDS-PAGE. C. The amount of bound gelsolin polypeptide, determined from the decrease in absorbance of the free protein peak, was expressed as a function of PIP 2 concentration.
  • FIG. 10 Circular dichroism. Spectra for GS 149 (A) and GS 134 (B) were determined in the absence (solid line) or presence of 77 ⁇ M PIP 2 (dashed line). The GS149 and GS134 concentrations, determined by amino acid analysis, were 10 and 17 ⁇ M, respectively.
  • Severing activity by gelsolin was measured by following the decrease in pyrene fluorescence after dilution of pyrene F-actin to 0.3 ⁇ M. In the absence of competing peptides, 30 ⁇ M PIP 2 inhibited severing by 90%. None of the competing molecules sever actin filaments, (closed circles) GS149; (open triangles) ⁇ ⁇ (closed triangles) P 2 ; (closed squares) lysine pentamers. The latter two curves were taken from Fig. 5 of Janmey, P. A., et al , J. Biol. Chem. 267(17): 11818-11823 (1992).
  • Figure 12 Existence of basic amino acid motifs among PPI binding proteins.
  • A The structure of the gelsolin N-terminal half (human plasma gelsolin residues 1-406), which contains three semiconserved repeating domains. It severs actin filaments, and severing requires the cooperative interaction between an actin filament side binding site (located in GSII-III) and another actin monomer binding site located in GSI.
  • Pi which is at the C-terminus of GSI, bound PIP 2 .
  • P 2 the contiguous sequence also bound PIP 2 (Janmey, P.A., et al , J. Biol. Chem.
  • FIG. 13 Expression of gelsolin domain I mutants in which a single lysine was substituted by alanine.
  • A SDS polyacrylamide gel. 3 ⁇ g of GS149 (A141), GS149 (A139) and GS149 (lanes 2-4, respectively) were analyzed. Lane 1, M.W. standards of 66, 45, 31, 21 and 14 kDa.
  • B Comparison of gelsolin domain I interactions with actin and PIP 2 . 2.7 ⁇ M pyrene actin was polymerized in the presence of 1.3 ⁇ M gelsolin polypeptide as described in
  • Fig. 1. a, no PIP 2 ; b, 14 ⁇ M PIP 2 ; c, 28 ⁇ M PIP 2 .
  • the present invention is based on the novel observation that peptides whose amino acid sequence are depicted in Seq. ID Nos 1 and 2, as well as fragments thereof, are capable of 1) modulating (inhibiting) the interactions of
  • agents are described which are capable of modulating PPI/gelsolin interaction.
  • PPI/gelsclin interaction can be inhibited.
  • the agents of the present invention will modulate PPI's inhibition of gelsolin mediated actin cleavage.
  • the agents are peptides whose sequences are depicted in Seq. ID Nos. 1 and 2, or fragments of these sequences.
  • the peptide agents of the present invention are capable of binding to the gelsolin binding site on a PPI.
  • peptide agents can readily generate such peptide agents using protein synthesis techniques known in the art.
  • a host can be transformed with DNA sequences capable of constitutively or inducibly expressing the desired peptide in order to produce large quantities of the desired peptide.
  • DNA sequences capable of constitutively or inducibly expressing the desired peptide in order to produce large quantities of the desired peptide.
  • One skilled in the art can readily adapt, without undue 0 experimentation, any host/vector system and protein purification techniques currently available to express and isolate the peptide agents of the present invention.
  • Fragments of Seq. ID Nos. 1 and 2 which possess the ability to bind to the gelsolin binding site on PPI can be readily generated using the above- 5 described procedures.
  • One skilled in the art can utilize the actin severing assay disclosed herein as a means of testing and identifying, without undue experimentation, fragments of Seq. ID Nos. 1 and 2 which possess the desired binding characteristics.
  • the modulating agents are 0 antibodies or peptides (anti-peptide peptides) which are capable of binding to a peptide whose amino acid sequence is depicted in Seq. ID Nos. 1 and 2.
  • the antibody and anti-peptide agents of the present invention are capable of binding to the PPI binding site on gelsolin.
  • the anti-peptide peptides of the present invention can be generated in one of two fashions.
  • the anti-peptide can be generated by replacing the basic amino acid residues found in Seq. ID Nos. 1 and 2 with acid residues, while maintaining hydrophobic and uncharged polar groups.
  • the lysine, arginine, and/or histidine residues found in Seq. ID Nos. 1 or 2 are replaced with aspartic acid or glutamic acid and glutamic acid residues in Seq. ID No. 2 is replaced by lysine, arginine or histidine.
  • the anti-peptides of the present invention can be generated by synthesizing or expressing the peptides encoded by the antisense strand of DNA which encodes peptides of Seq. ID Nos. 1 or 2. Peptides produced in this fashion are, in general, similar to those discribed above since codons complementary to those coding for basic residues generally code for acidic residues.
  • the agents are anti-idiotypic antibodies capable of binding to the antigen binding sites of the anti-Si or anti- S2 antibodies.
  • the anti-idiotypic antibody agents of the present invention are capable of binding the gelsolin binding site on a PPI.
  • Anti-idiotypic antibodies can be generated by any of the methods described above using one of the antibodies of the present invention as an immunogen.
  • One skilled in the art can readily adapt known methods in order to generate the anti-idiotypic antibodies of the present invention.
  • PPI/gelsolin interactions can be inhibited by supplying to a sample (comprising PPI and gelsolin) one of the agents of the present invention which is capable of binding to either the gelsolin binding site on a PPI or the PPI binding site on gelsolin.
  • a sample comprising PPI and gelsolin
  • the PPI mediated inhibition of actin cleavage by gelsolin can be modulate by inhibiting
  • agents capable of binding the gelsolin binding site on PPI modulate, the interconversion of PPI to various isomers and phosphorylation states.
  • Some of the enzymes found to be modulated by the agents of the present invention include, but are not limited to, PI 4-kinase,
  • PI 3-kinase PI(4)P 5-kinase
  • phospholipase C- ⁇ PI 3-kinase, PI(4)P 5-kinase, and phospholipase C- ⁇ .
  • Other enzymes which have been found to be modulated by the agents of the present invention include, but are not limited to other PPI specific phosphatases and other PPI specific kinases.
  • Other reactions which have been found to be modulated by the agents of the present invention include, but are not limited to PI -- > PI(4)P or PI(3)P PI(4)P - > PI(4,5)P 2 PI(4,5)P 2 - > PI(3,4,5)P 3 PI(3)P - > PI(3,4)P 2
  • modulate is defined as the ability of an agent to stimulate or inhibit an enzymatic reaction or a naturally occuring non- enzymatic interaction.
  • PPI inhibition of gelsolin mediated actin cleavage can be modulated.
  • the agents of the present invention modulated th is inhibition by decreasing PPI/gelsolin interaction. This decrease in PPI/gelsolin interaction leads to the stimulation of gelsolin mediated actin cleavage.
  • the type of modulation exerted by the agents of the present invention varies between enzymes which use a PPI as a substrate.
  • an agent can bind PPI, removing it as a substrate and thus inhibiting enzymes which use the particular PPI as a substrate.
  • the agent can bind to PPI and thus stimulate an enzyme which is feedback inhibited by the presence of the PPI.
  • the samples in which the various enzymatic activities can be modulated can either be comprised of living cells or tissues, or can be cell free, comprising components which are either synthesized or derived from living cells.
  • the cells In samples which contain living cells, the cells themself can be modified to produce the protein agents of the present invention. Specifically, one skilled in the art can readily modify a cell using recombinant techniques such that one of the agents of the present invention is either constitutively or inducibly expressed.
  • a cell is transformed with an expression vector capable of directing the expression of an agent of the present invention.
  • Methods for generating such an expression vector and for transforming cells are well known in the art (Sambrook et at., "Molecular Cloning, " Cold Spring Harbor Press (1989).
  • the expression of the agent can be controlled.
  • modulation of the various enzymes can be used to further elucidate biochemical pathways as well as serve as a basis of developing treatments for pathological conditions and disease states which are associated with deficiencies or overexpression of the various modulated enzymes.
  • condition include, but are not limited to condition which lead to cytoskeletal damage and an inflammatory reaction.
  • the present invention further provides methods of treating pathological conditions which are associated with the increased or decrease of PPI/gelsolin or PPI/enzyme interactions.
  • the agents of the present invention can be formulated using known procedures to obtain pharmaceutically acceptable compositions.
  • the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc.
  • the agent is an antibody
  • a dosage of antibody which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) is preferred.
  • the agent is a peptide
  • the agents of the present invention may be administered to patients intravenously, intramuscularly, subcutaneously, enterally, topically or parenterally. When administering the agent by injection, the administration may be by continuous injections, or by single or multiple boluses.
  • the agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to "physiologically effective. " An amount is said to be physiologically effective if the dosage, route of administration, etc. of the agent are sufficient to modulate gelsolin/PPI interaction or to bind the particular PPI.
  • one of the agents of the present invention is provided to a patient for the intention of modulating gelsolin/PPI interaction is said to be physiologically effective if it is provided in sufficient dosage to suppress PPI inhibition of gelsolin mediated actin cleavage.
  • agents of the present invention can be administered either alone or in combination with one or more additional agents disclosed herein.
  • the administration of the agent of the present invention may be for either a "prophylactic” or "therapeutic” purpose.
  • the agents are provided in advance of the onset of the pathological condition, response, or onset of symptoms.
  • the prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent event.
  • the agent When provided therapeutically, the agent is provided at (or shortly after) the onset of the appearance of a symptom or the diagnosis of a pathological condition.
  • the therapeutic administration of the agent serves to attenuate any actual symptom.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
  • Such an agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • the agents of the present invention can be formulated according to known methods of preparing pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed. , Osol, A., Ed., Mack, Easton PA (1980)).
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of an agent of the present invention together with a suitable amount of carrier.
  • the antibodies of the present invention may be humanized, through chimerization or CDR grafting, to become more "pharmacologically acceptable" to a patient.
  • Control release preparations may be achieved through the use of polymers to complex or absorb the agents of the present invention.
  • the rate and duration of the controlled delivery may be regulated to a certain extent by selecting an appropriate macromolecule matrix, by varying the concentration of macromolecules incorporated, as well as the methods of incorporation.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate the agents of the present invention into particles of a polymeric material, such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, by gelatine or poly(methylmethacylate) microcapsulation, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • the invention further includes a pharmaceutical composition comprising one or more agents selected from the group consisting of; (a) a peptide whose sequence is selected from the group consisting of Seq. ID Nos. 1 and 2, or a fragment thereof, (b) an antibody capable of binding to a peptide defined in Seq. ID Nos. 1 or 2, and (c) anti-idiotypic antibodies capable of binding to the antigen binding site of the above antibodies.
  • a pharmaceutical composition comprising one or more agents selected from the group consisting of; (a) a peptide whose sequence is selected from the group consisting of Seq. ID Nos. 1 and 2, or a fragment thereof, (b) an antibody capable of binding to a peptide defined in Seq. ID Nos. 1 or 2, and (c) anti-idiotypic antibodies capable of binding to the antigen binding site of the above antibodies.
  • the present invention further discloses methods of identifying agents capable of inhibiting PPI/gelsolin interaction.
  • agents are identified as being capable of inhibiting PPI/gelsolin interaction by their ability to remove PPI inhibition of gelsolin mediated actin cleavage.
  • an agent is incubated with a sample comprising gelsolin, pyrene-F-actin (preferably diluted to below its critical monomer concentration), and a PPI capable of binding to gelsolin thus inhibiting gelsolin mediated actin cleavage.
  • the sample is then incubated under condition which would allow gelsolin to cleave actin if the reaction was not inhibited by PPI.
  • the rate of fluorescence is monitored.
  • the rate of fluorescence change of the sample is compared to the rate of fluorescence of a sample not containing the agent (negative control), and either a sample not containing the PPI or a sample containing one of the peptide agents of the present invention (positive control) .
  • Agents capable of inhibiting PPI/gelsolin interaction lead to a more rapid decrease in fluorescence intensity of the sample and the rate of decrease is proportional to the degree of inhibition.
  • concentration of the components of the above assay will vary depending on the type and nature of detection employed. In one application of this embodiment, from about 200 to 300 nM pyrene-F actin, from about 33 to 50 nM gelsolin, and from about 10 to 20 ⁇ M PIP ⁇ micelles or mixed lipid vesicles containing various amounts of PIP 2 is employed and fluorescence is measure using a Greg ISS instrument in an L configuration (see example section for details).
  • Actin Spudich and Watt, J. Biol. Chem. 246:4866-4871 (1971)
  • gelsolin Choponnier et al , J. Cell. Biol. 705: 1473-1481 (1986)
  • actin was labeled with either pyrene-iodoacetamine (Kouyama and Mihashi, Eur. J. Biochem. 774:33-38 (1981)) or pyrene-maleimide (Kawasaki et al, Biochim. Biophys. Ada 446:166-178 (1976)) as previously described.
  • G-actin was prepared by dialysis into solutions containing 2 mM Tris, pH 7.4, 0.2 mM CaCl 2 , 0.2 mM dithiothreitol, 0.5 mM ATP (buffer A) and polymerized by addition of 2 mM MgCl 2 and 150 mM KC1 (to form buffer B).
  • Gelsolin was stored frozen in 10 mM Tris, 100 M KC1, 1 mM EGTA, 2 pH 7.4 (TBS- EGTA).
  • Thymosin b-4 (Safer et al , J. Biol. Chem. 266:4029-4032 (1991)) was the generous gift of Daniel Safer and Vivianne Nachmias, University of Pennsylvania. Peptides based on the sequences of yeast ABP-1 (Drubin et al. ,
  • EGTA [ethylenebis(oxyethylene- nitrilo)]tetraacetic acid
  • PPI polyphosphoinositide
  • PIP phosphatidylinositol 4-monophosphate
  • PIP 2 phosphatidylinositol 4, 5 -bis-phosphate
  • PC phosphatidylcholine
  • MARCKS myristoylated, alanine-rich kinase C substrate
  • DLS dynamic light scattering. as a spacer residue.
  • the peptides were labeled with pyrene-maleimide for the polarization experiments or with iodoacetamide to prevent disulfide exchange. All peptides were dissolved in TBS-EGTA or water, and their sequences are summarized in Table 1.
  • PC Dioleoyl-L- ⁇ - phosphatidylcholine
  • PIP phosphatidylinositol 4-monophosphate
  • PIP 2 phosphatidylinositol 4,5-bisphosphate
  • the hydrodynamic diameter of phospholipid vesicles was calculated from the intensity autocorrelation functions measured by dynamic light scattering (DLS) using a Brookhaven Instruments BI30AT apparatus, PC or PC/PIP 2 (10: 1) were diluted to a concentration of 56 ⁇ g/ml in buffer B and centrifuged at 16,000 x g for 15 min to remove dust and aggregates. DLS measurements were made on 1-ml samples in cylindrical scattering tubes at angles from 60 to 120 degrees. The diffusion constant D was calculated from the slope of a plot of the average decay constant, measured by a second order cumulant fit, versus the inverse scattering vector, and the diameter d calculated from the expression
  • Fluorescence Polarization The fluorescence polarization of pyrene- maleimide-labeled peptides was measured using a Greg (Urbana, IL) ISS instrument in an L configuration. A sample containing peptide in the absence of either F-actin or PIP was first measured using buffer as a blank correction.
  • pLcIIFXGS a human plasma gelsolin expression vector (kindly provided by Drs. M. Way and A. Weeds (Way, M. , et al , J. Cell Biol. 709:593-605 (1989))) was linearized at the Hindlll site, blunted and ligated with BamHI linkers. The insert was released by BamHI digestion and subcloned into M13mpl8. Termination codons were inserted at specific sites by oligonucleotide-directed mutagenesis (Amersham version 2 system). The cDNA constructs were sequenced to confirm the mutation.
  • the cDNA was cloned into the BamHI site of pet3a, the T7 RNA polymerase directed expression vector of Sturdier et al. (Studier, F.W., et al , Methods Enzymol. 785:60-89 (1990)). We switched from pLcII to pet vector because the latter gave superior expression.
  • the resultant proteins contained a fusion peptide (MASMTGGQQMGRGSIEGRA (Seq. ID No. 3) at their N-terminus.
  • a gelsolin cDNA encoding domains II-III (GS 150-406) was generated by restriction enzyme digestion and site-directed mutagenesis.
  • BL21 (DE3) pLysS cells transfected with expression constructs were grown in LB medium containing 100-200 ⁇ g/ml ampicillin at 37°C to an OD ⁇ nm of 0.6, and induced by 0.4 mM isopropyl-1-thio-jS-galactopyranoside as described previously (Yu, F.-X., et al , J. Biol. Chem. 266: 19269-19275 (1991)).
  • Gelsolin polypeptides were obtained by solubilizing inclusion bodies with 8 M urea (no detergents), and purified by sequential anion and cation exchange chromatography (Yu, F.-X., et al , J. Biol. Chem. 266: 19269-19275 (1991)). Protein concentration was determined by the method of Bradford (Bradford, M.M., Ann. Rev. Biochem. 72:248-254 (1976)), and protein purity was assessed by electrophoresis on 5-20% acrylamide gradient gels in the presence of SDS.
  • GS149 prevented actin monomers from polymerizing, resulting in a decrease in pyrene-actin fluorescence intensity compared with actin control (Way, M., et al , EMBO J. 9:4103-4109 (1990); Yu, F.-X. , et al , J. Biol. Chem.
  • PIP 2 was incubated with PIP 2 before addition of pyrene actin.
  • PIP (Sigma) micelles were prepared by dissolving PIP 2 in water to a final concentration of 1 mg/ml, and sonicated for 30s. at maximum power (model W185; Heat Systems Ultrasonics, Inc., Farmingdale,
  • Gelsolin was prepared from human plasma (Chaponnier, C , et al., 3. Cell Biol. 705: 1473-1481 (1986)).
  • CKSGLKYKKGGVASGF (Seq. ID No. 1, N-terminal cysteine added) was synthesized by standard solid phase methods and purified by g-HPLC using an acetonitrile gradient in 0.1 % trifluoroacetic acid. Its concentration was determined by amino acid analysis. Circular Dichroism. CD spectra of GS149 and GS134 in the presence or absence of PIP 2 micelles were measured using an Aviv model 60DS spectrometer at 25 °C in 1 mm pathlength cells. Spectra were scanned at 1 nm intervals for 3 s, and three scans were averaged. The proteins were dialyzed against 5 mM Tris-HCl, pH 7.5, centrifuged and filtered before assay.
  • Protein concentrations were determined by amino acid analysis.
  • the apparent stoichiometry is approximately 1 peptide, 5 PIP, and the dissociation constant is less than micromolar.
  • the specificity of the binding is shown by the lack of polarization seen in another labeled peptide, derived from the actin-binding protein yeast ABP-1 (Drubin et al , Nature 545:288-90 (1990)). PIP also had no effect on the fluorescence polarization of either free pyrene-maleimide-SH or pyrene-labeled G-actin (data not shown).
  • Figure 2 shows the ability of various peptides to compete with gelsolin for binding to PPIs as determined by their ability to prevent inhibition of gelsolin' s severing activity by PIP 2 .
  • PIP 2 micelles were incubated with peptides for 5 seconds prior to addition of gelsolin and the commencement of the severing assay by adding pyrene-labeled F-actin.
  • the highly basic sequence 140-147 which is not essential for PIP 2 sensitivity in the homoLogous domain (164-173) of gelsolin, has much less ability to compete with gelsolin for PIP2, although at concentrations nearly equimolar to PIP 2 , this peptide, like penta-lysine and neomycin, which have similar numbers of free amino groups, prevents the inhibition of gelsolin by PIP 2 .
  • the calmodulin-binding domain of MARCKS which contains 7 basic residues in a 25 residue span is at least an order of magnitude less effective than the larger gelsolin/villin peptides, even though it has more net positive charge than gelsolin 150-169.
  • Figure 4 shows that the gelsolin peptide is more effective in preventing gelsolin inhibition by PIP 2 in the presence than in the absence of Mg 2 + . Similarly, more peptide is required to prevent inhibition of gelsolin by PIP, which does not aggregate in Mg 2+ , although both PIP and PIP 2 micelles inhibit gelsolin at approximately equal molar ratios (Janmey, P. A., et al , J. Biol.
  • small mixed lipid vesicles containing PIP 2 in PC were prepared by sonication as described in Materials and Methods.
  • Such PC/PIP 2 vesicles inhibit gelsolin' s severing function as avidly as PIP 2 micelles (Janmey and Stossel, J. Biol. Chem. 264:4825-31 (1989)), and as Figure 5 shows, the gelsolin peptide was able to prevent inhibition of gelsolin by these PC/PIP 2 vesicles.
  • 150-169 peptide and intact gelsolin can be made by comparing the effect of the peptide on severing by a constant amount of gelsolin in the presence of PIP 2 (closed triangles) to the severing activity of a constant amount of gelsolin incubated in various concentrations of PIP 2 (open circles).
  • the molar ratio of gelsolin to PIP 2 at which half of the severing activity is lost is equal to the ratio of peptide to PIP 2 at which half of the severing activity is restored to maximally inhibited gelsolin. This result suggests that the affinity of the gelsolin 150-169 peptide for PIP 2 is very similar to that of native gelsolin.
  • Figure 7 shows that the gelsolin-derived peptide completely reverses the inhibition of gelsolin by PIP and PIP 2 micelles. Higher concentrations of the peptide are required to reverse gelsolin-PPI complexes than are required to prevent their formation, consistent with the highly cooperative nature of these protein- 0 phospholipid complexes.
  • the ability of the peptides to restore severing activity to gelsolin that has been complexed to PPIs suggests that such a regulation in vivo would be readily reversed by displacement of the lipid by the appropriate competitive ligand. In no case was any of the peptides themselves found to sever actin filaments or to inhibit their severing by 5 gelsolin.
  • the F-actin binding of these peptides, and perhaps of the corresponding sites in the intact proteins, 5 is of only moderate affinity and sufficiently labile so that they do not co- sediment with F-actin, nor inhibit the formation of very high affinity bonds consequent to severing and capping of actin filaments by the multiple actin- binding sites of intact gelsolin or villin.
  • actin-binding protein thymosin-/3-4 which contains numerous positive charge clusters is 100 times less effective than gelsolin 150- 169 in competing with gelsolin for PIP 2 in phospholipid bilayers.
  • MARCKS peptide is a potent bundler of actin filaments, and MARCKS activity has not been demonstrated to be affected by PPI's.
  • the actin monomer binding protein profilin also binds PPI's (Goldschmidt, C.P.J. , et al , Science 247: 1575-8 (1990); Lassing and Lindberg, Nature 574(6010):472-474 (1985); Lassing and Lindberg, J. CellBiochem. 57:255-67 (1988)) and is a highly basic protein with an isoelectric point of 9.3.
  • PPI's Goldschmidt, C.P.J. , et al , Science 247: 1575-8 (1990); Lassing and Lindberg, Nature 574(6010):472-474 (1985); Lassing and Lindberg, J. CellBiochem. 57:255-67 (1988)
  • a polybasic domain in the primary structure of a protein appears to promote conformational changes in the rest of the protein when it binds phospholipid.
  • polylysine transforms from a random coil in aqueous solution to a jS-sheet or an ⁇ -helix when it binds phosphatidylserine or phosphatidic acid respectively (Fukushima, K., et al , Biophys. Chem. 54:83-90 (1989)).
  • Synthetic peptides with as little as 20 amino acids bind as avidly as intact gelsolin or villin to PIP and PIP 2 . These peptides are therefore useful for determining the structure of phosphoinositide binding sites in other proteins and can serve as potent pharmacologic inhibitors of PPI turnover, due to their ability to sequester PPI's from proteins that potentially bind them in vivo.
  • Fig. 8A shows the SDS-polyacrylamide gel recombinant gelsolin domain 1 (GS 1-149, referred to as GS149, lane 3) and a further truncated protein GS134 (GS1-134, lane 2).
  • GS149 inhibited actin polymerization by binding actin monomers stoichiometrically, so maximal inhibition was observed at a 1 : 1 mutant/actin molar ratio (Fig. 8B).
  • the presence of a 17 amino acid fusion peptide at the N-terminus of gelsolin domain I did not interfere with its actin binding activity.
  • GS134 was equally effective, confirming previous reports that gelsolin domain I is functional after truncation to residue 126 (Way, M., et al , EMBO J. 9:4103-4109 (1990); Kwiatkowski, D.P., et al , J. Cell Biol. 108: 1717-
  • Fig. 8C shows that PIP 2 inhibited GS149 binding to actin and half-maximal inhibition of 2.4 ⁇ M GS149 was observed at 12 ⁇ M PIP 2 . No inhibition was observed with 100 ⁇ M phosphatidylcholine or phosphatidylserine (data not shown), suggesting that the effect of PIP, was specific, as has been shown previously for gelsolin (Janmey and Stossel, Nature 525:362-364 (1987); Janmey and Stossel, J. Biol. Chem.
  • GS134 was minimally inhibited by 12 ⁇ M PIP 2 . Therefore, the deleted residues 135-149 (KSGLKYKKGGVASGF (Seq. ID No. 1 minus terminal C residue) are required for optimal PIP regulation. Loss of regulation was unlikely to be due to global denaturation, since GSI 34 inhibited actin polymerization as well as GS149. This was supported by the finding by others that an even shorter fragment (GS126) binds actin monomer (Way, M., et al , EMBO J. 9:4103-4109 (1990); Way, M., et al , J. Cell. Biol. 776: 1135-1143 (1992)).
  • GS149 and GS134 were compared by gel filtration analyses. This method had been used to demonstrate high affinity PIP binding by profilin (Goldschmidt-Clermont, P.J., et al , Science 257: 1231-1233 (1991); Machesky, L.M., et al , Cell Reg. 7:937-950 (1990)) and CapZ (Heiss and Cooper, Biochemistry 50:8753-8758 (1991)).
  • Fig. 9A left panel shows that in the absence of PIP, GS149 eluted with a V e (elution volume) of 13.9 ml, and the peak was asymmetric. Addition of PIP, changed the protein elution profile.
  • the amount of GS149 bound to PIP was calculated from the decrease in the original GS149 absorbance peak and plotted against PIP 2 concentration in Fig. 9C. It increased linearly with PIP, concentration until all of the protein was complexed (open circle at 140 ⁇ M PIP 2 ). No difference in the binding of 39 and 25 ⁇ M GS149 was observed, as long as saturation was not reached. The slope of the plot gave a ratio of 1 GS149 to 4 PIP 2 , which is similar to the binding stoichiometry reported for human platelet profilin (139 amino acids and binds 5 PIP 2 (Machesky, L.M., et al , Cell Reg. 7:937-950 (1990))).
  • residues 135- 149 decreased PIP binding affinity and reduced PIP, inhibition of actin binding, establishing that these residues are critically important for the high affinity interaction of gelsolin domain I with PIP,. They are however not required for actin monomer binding, suggesting that the PPI and actin binding sites are located on different residues.
  • Fig. 10 shows the circular dichroism profiles of GS149 in the absence and presence of PIP,.
  • Computer curve fitting indicated that in the absence of PIP 2 , GS149 contained about 55 % jS-sheets, 35 % random coil and 10% ⁇ - helix.
  • the profile was consistent with 25 % random coil, 65 % 3-sheet and 10% ⁇ -helix.
  • PIP did not induce significant conformational change in GSI 34, confirming that the truncated polypeptide did not bind PIP,.
  • GSI 34 had a different conformation than GS149.
  • GS134 had a similar jS-sheet and random coil content as GS149 complexed with PIP,. This may be coincidental, and does not necessarily indicate that GS134 and GS149:PIP, complexes had identical secondary structures. Nonetheless, since the former binds actin while the latter does not. it is not possible to conclude at present that the conformational change in GS149 after PIP, binding accounts for inhibition of actin binding.
  • FIG. 13A shows an SDS-polyacrylamide gel of GS149 and two mutants, GS149(A141) (the mutated residue is designated in parentheses) and GS149(A139).
  • the mutants inhibited actin polymerization and were regulated by PIP, to a similar extent as the wild type GS149 (Fig. 13B). Therefore, the replacement of a single lysine with alanine had little effect on the ability of this domain to bind actin or PIP,. That these mutants bound PIP, was confirmed by gel filtration (data not shown).
  • Gelsolin domain I contains an actin binding site which is necessary for severing actin-actin bonds after gelsolin attaches laterally to actin filaments via domains II-III.
  • the interactions of domain I with actin have been characterized extensively, and residues important for act in binding have been identified by deletional and site-directed mutagenesis (Bryan, J., J. Cell Biol.
  • the region important for PPI binding in domain I was located to within the C-terminal 15 residues of domain I. Deletion of this sequence resulted in substantial loss of PPI binding and regulation of actin binding, whereas a synthetic peptide containing this sequence competed effectively with domain I and gelsolin for PIP,.
  • the PPI binding site is distinct from the actin binding site, because the C-terminal truncated domain I polypeptide bound actin as well as full length domain I.
  • Inhibition of actin binding may result from an induced change in the conformation of the actin binding sites or indirectly through changes in other parts of the molecule to limit access to actin and/or cooperative interactions between different actin binding domains.
  • Human profilin (Ampe, C , et al , FEBS 228: 17-21 (1988)) has a modified motif, with a 6 amino acid spacing between the first two basic residues, and substitution of K with H. Since H is partly charged at neutral pH, human profilin may have lower PIP, binding affinity than gelsolin. Acanthamoeba profilin II which binds PIP 2 but with at least 10 fold lower affinity than human profilin (Machesky, L.M. , et al , Cell Reg. 7:937-950 (1990)) does not have the putative motif (Ampe, C, et al , FEBS 228: 17-21 (1988)). Cofilin has a similar motif in residues 13-22.
  • cofilin residues 13-22 contain another PPI binding site.
  • the PIP, binding motif is not restricted to actin regulatory proteins.
  • Several inositol-specific phospholipase C isozymes (Studier, F.W. , et al , Methods Enzymol 785:60-89 (1990)) have a similar motif (Fig.

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Abstract

L'invention se base sur l'identification de deux fragments peptidiques de gelsoline pouvant inhiber l'interaction PPI/gelsoline et PPI/enzyme par fixation des PPI. Sur la base de ces observations, l'invention décrit des agents pouvant moduler l'interaction PPI/gelsoline et PPI/enzyme par fixation au site de fixation de PPI de la gelsoline et au site de fixation de gelsoline des PPI. L'invention décrit, de plus, des procédés d'identification d'agents capables de moduler l'interaction PPI/gelsoline.
PCT/US1993/005388 1992-06-15 1993-06-08 Peptides de fixation de phosphoinositides derives des sequences de gelsoline et villine WO1993025564A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020887A1 (fr) * 1996-11-14 1998-05-22 Brigham And Women's Hospital, Inc. Peptides se liant aux polyphosphoinositides, pour l'administration intracellulaire de medicaments
US5783662A (en) * 1995-02-22 1998-07-21 Brigham & Women's Hospital, Inc. Polyphosphoinsitide binding peptides for intracellular drug delivery

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017170A1 (fr) * 1990-05-04 1991-11-14 Biogen, Inc. Constructions de fusion de gelsoline multimeres

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017170A1 (fr) * 1990-05-04 1991-11-14 Biogen, Inc. Constructions de fusion de gelsoline multimeres

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ENDOCRINE REVIEWS, Volume 6, Number 1, issued December 1985, N.R. FARID et al., "Antiidiotypic Antibodies as Probes for Receptor Structure and Function", pages 1-23. *
J. BIOL. CHEM., Volume 264, Number 9, issued 25 March 1989, P.A. JANMEY et al., "Gelsolin-Polyphosphoinositide Interaction", pages 4825-4831. *
J. CELL BIOL., Volume 106, issued March 1988, H.L. YIN et al., "Identification of a Polyphosphoinositide-Modulated Domain in Gelsolin which Binds to the Sides of Actin Filaments", pages 805-812. *
J. CELL BIOL., Volume 108, issued May 1989, D.J. KWIATKOWSKI et al., "Identification of Critical Functional and Regulatory Domains in Gelsolin", pages 1717-1726. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5783662A (en) * 1995-02-22 1998-07-21 Brigham & Women's Hospital, Inc. Polyphosphoinsitide binding peptides for intracellular drug delivery
US5846743A (en) * 1995-02-22 1998-12-08 Brigham And Women's Hospital, Inc. Polyphoshoinositide binding peptides for intracellular drug delivery
WO1998020887A1 (fr) * 1996-11-14 1998-05-22 Brigham And Women's Hospital, Inc. Peptides se liant aux polyphosphoinositides, pour l'administration intracellulaire de medicaments

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