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WO1999062494A2 - Ameliorations apportees a l'absorption de substances par des cellules - Google Patents

Ameliorations apportees a l'absorption de substances par des cellules Download PDF

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Publication number
WO1999062494A2
WO1999062494A2 PCT/GB1999/001723 GB9901723W WO9962494A2 WO 1999062494 A2 WO1999062494 A2 WO 1999062494A2 GB 9901723 W GB9901723 W GB 9901723W WO 9962494 A2 WO9962494 A2 WO 9962494A2
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Prior art keywords
molecule
moiety
dynamin
endocytosis
peptide moiety
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WO1999062494A3 (fr
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Harvey Thomas Mcmahon
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Medical Research Council
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Medical Research Council
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Priority to EP99955211A priority patent/EP1084144A2/fr
Priority to CA002333751A priority patent/CA2333751A1/fr
Publication of WO1999062494A2 publication Critical patent/WO1999062494A2/fr
Publication of WO1999062494A3 publication Critical patent/WO1999062494A3/fr
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    • 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
    • C07K14/4722—G-proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a novel molecule and to various uses thereof.
  • Eukaryotic cells secrete substances from membrane-bound vesicles present in the cytoplasm by migration of the vesicles to the cell membrane. Fusion of the vesicle membrane with the cell membrane allows the vesicle contents to escape into the extracellular environment. This process is termed exocytosis.
  • Exocytosis and endocytosis are performed by many different cell types and are involved in many physiological processes (e.g. secretion of digestive enzymes, secretion of neurotransmitters and hormones, cell surface receptor downregulation/internalisation, retrieval of membrane components from vesicles released by exocytosis, etc).
  • endocytosis There are thought to exist several types of endocytosis, one of the more important of which is clathrin-mediated endocytosis which is implicated, inter alia, in the cycling of neurotransmitters at synapses. Molecules destined to be internalised are concentrated in clathrin-coated pits which are then "pinched-off" to form vesicles. Steps leading to this include the synthesis of a curved clathrin lattice, providing a mechanical scaffold for vesicle formation, and the recruitment of a GTPase, dynamin, to the neck of the forming vesicle.
  • Small synaptic vesicles are specialised containers for the controlled release of neurotransmitters in response to nerve depolarisation. With the nerve terminals often far away from the cell body the neuron has become adapted to recycle these transmitter containers multiple times.
  • the pathway for endocytosis is thought to occur predominantly by a clathrin-mediated mechanism [1] . This is supported both morphologically by clathrin coated vesicle profiles after nerve terminal stimulation and by the marked enrichment of the molecular machinery for this pathway (including clathrin, adaptors and dynamin) in nerve terminals [2, 3] .
  • endocytosis for the sustained activity of neurons is highlighted in shibire, a temperature sensitive mutant of dynamin in Drosophila [4, 5]. At the nonpermissive temperature there is an accumulation of endocytosis profiles and a rapid onset of paralysis. In this mutant the electron-dense collars around the necks of endocytosing vesicles resemble the dynamin rings found in vitro [6] or when lysed synaptosomes are treated with GTP ⁇ S [7] . Thus dynamin is likely to be a key protein in the "pinching off" stage of synaptic vesicle endocytosis.
  • dynamin contains a cluster of proline-rich sequences at its C-terminus that have the potential to interact with various Src homology 3 (SH3) domains.
  • SH3 domains are modules commonly found in signal transduction and cytoskeletal proteins. They mediate protein-protein interactions by binding to proline rich sequences that adopt several turns of a type II polyproline helix.
  • the polyproline region of dynamin (PRD) is important both in endocytosis and in its colocalisation with clathrin-coated pits, as has been demonstrated by transfection of deletion constructs into fibroblasts.
  • Amphiphysin a major dynamin binding partner, is proposed to be involved in dynamin recruitment to the necks of coated vesicles [8-11]. Disruption of the interaction between these two proteins in either the lamprey giant reticulospinal synapse [10] or in fibroblasts [11] leads to an inhibition of clathrin-mediated endocytosis, probably through a blockade of dynamin recruitment. Amphiphysin is concentrated in nerve terminals and is present as a phosphorylated heterodimer of two isoforms (1 and 2) both of which bind to dynamin [12].
  • Synaptojanin and endophilin/SH3p4 have also been implicated in synaptic vesicle endocytosis by virtue of their concentration in nerve terminals and their interaction with components of the endocytosis machinery [13- 15].
  • Synaptojanin additionally has a phosphatidyl inositol 5' phosphatase activity that may perform lipid modifications during endocytosis [13, 16] .
  • dynamin In response to nerve terminal depolarisation dynamin is dephosphorylated by the calcium- calmodulin activated protein phosphatase calcineurin (also known as protein phosphatase 2B) [17]. This has led to speculation that phosphorylation/dephosphorylation could regulate synaptic vesicle recycling [18]. In support of a role for calcineurin, this phosphatase is known to be enriched in nerve terminals where it co-localises with amphiphysin and indeed dephosphorylates amphiphysin and synaptojanin in parallel with dynamin [19]. On a wider scale, calcineurin activity in the brain is also implicated in synaptic plasticity, nerve regeneration and brain disease [20-22] .
  • Dynamin binds to the SH3 domain of amphiphysin via the PSRPNR sequence in its polyproline domain.
  • the invention provides a membrane permeable molecule which inhibits dynamin-mediated endocytosis in a eukaryotic cell, the molecule comprising a membrane permeable hydrophobic moiety, and a peptide moiety comprising the amino acid sequence QVPSRPNRAP.
  • the sequence QVPSRPNRAP represents a portion of the prior art peptide, and specifically inhibits amphiphysin binding to dynamin.
  • the amino acid sequence may comprise trivial modifications such as amino acid residue substitutions, preferably of a conservative nature (e.g. substituting threonine for serine, or glutamine for asparagine). Preferably there are no more than 2 substitutions, more preferably no more than one substitution, and most preferably no substitutions.
  • the peptide moiety may also comprise one or more additional amino acid residues at the N- and/or C-terminal. If such additional residues are present, their number is preferably small (e.g. no more than 5 additional residues at either terminal). For example, between 1 and 5 additional amino acid residues may be incorporated at either or both terminals.
  • the peptide moiety may comprise one or more D-amino acids - these are thought not to interfere with the endocytosis-inhibiting activity of the molecule, but render it more resistant to proteases and peptidases.
  • the molecule of the invention inhibits dynamin interactions with amphiphysin but does not affect interactions between dynamin and Grb2.
  • the molecule may comprise one or more labelling moieties, such as a fluorophore and/or a chromophore, or biotin and the like.
  • labelling moieties such as a fluorophore and/or a chromophore, or biotin and the like.
  • the hydrophobic moiety may be joined to a side chain of one of the amino acid residues of the peptide moiety.
  • Serine, arginine, asparagine and glutamine all comprise side chains with groups which may be conveniently derivatised.
  • the molecule may comprise a plurality of hydrophobic moieties, joined to a single peptide moiety.
  • the molecule may comprise a dimer, oligomer or polymer comprising a plurality of the peptide and/or hydrophobic moieties.
  • hydrophobic moiety is conveniently covalently coupled to the peptide moiety.
  • a range of hydrophobic moieties are suitable for inclusion in the molecule of the present invention and include substituted or unsubstituted alkyl, alkenyl, alkoxy or aryl groups. Generally preferred are straight-chain (i.e. unbranched) substituted or unsubstituted alkyl, alkenyl or alkoxy groups, especially those comprising 10-24 carbon atoms, or more preferably 12-18 carbon atoms. In some instances, acetylation of the peptide moiety may be a convenient modification.
  • the molecule of the invention may be formed, at least in part, by a condensation reaction between a suitable hydrophobic fatty acid (e.g. lauric, myristic, palmitic, or stearic acids) and a terminal amino acid of the peptide moiety.
  • a suitable hydrophobic fatty acid e.g. lauric, myristic, palmitic, or stearic acids
  • the hydrophobic moiety is joined to the amino acid residue at the N terminal (as judged by reference to the sequence of dynamin protein) .
  • the molecule comprises the amino acid sequence QVPSRPNRAP, joined at the N terminal via a covalent bond to a myristoyl group.
  • suitable hydrophobic groups are fatty acids which are present in membrane lipids of eukaryotic cells, and lipid moieties such as sphingosine.
  • the inventors have found that the presence of a negative charge on the residue at the C terminal (as judged by reference to the sequence of dynamin) inhibits its downregulatory effect on endocytosis. Accordingly, it is preferred to avoid the presence of a carboxyl group at the "C" terminal. This can be achieved in a number of ways, most conveniently by synthesis of the peptide moiety with an amino group in place of a carboxy group at the C terminal amino acid residue. It is therefore preferred that the peptide moiety has no negative charge at the "C" terminal at physiological pH (e.g. pH 6-8), and in some embodiments that there is no net negative charge on the peptide moiety (at physiological pH).
  • physiological pH e.g. pH 6-8
  • the molecule may comprise a targeting moiety, such that the molecule is targeted to, and preferentially taken up by, particular cell types, such that endocytosis is preferentially inhibited in the target cell relative to non-target cells which may be present.
  • targeting moieties include immunoglobulins or fragments thereof (e.g. Fv, Fab, scFv). Typically these targeting moieties would have a specific binding activity for a receptor molecule (such as a cell surface antigen, tumour-associated antigen or the like) expressed on the surface of a target cell.
  • Alternative targeting moieties are hormones or other ligand molecules, or portions thereof, which retain specific binding activity for the cognate receptor expressed on the surface of a target cell.
  • the molecule of the invention is useful as an inhibitor of clathrin-mediated (specifically, dynamin-mediated) endocytosis.
  • the molecule has considerable usefulness as a research reagent for studying endocytosis, especially in in vitro culture systems (e.g. in cultured neurones or isolated synaptosomes) .
  • the invention provides a method of inhibting endocytosis in a eukaryotic cell in vitro, the method comprising the steps of : providing a culture of eukaryotic cells in vitro; and adding to the extracellular environment a molecule in accordance with the first aspect of the invention defined above.
  • the eukaryotic cells will be mammalian cells, preferably simian, mouse, guinea pig, rat or human cells.
  • the method may further comprise the step of detecting and/or measuring the amount of endocytotic activity in the cells.
  • Means of detecting and/or measuring endocytotic activity are known to those skilled in the art (e.g. as disclosed herein) - the most appropriate means may depend on the type of eukaryotic cells being investigated.
  • the invention provides a pharmaceutical composition for inhibiting endocytosis in a mammalian (preferably human) subject, the composition comprising a molecule in accordance with the first aspect defined above and a physiologically acceptable diluent, carrier or excipient (e.g. saline or phosphate-buffered saline solution).
  • a physiologically acceptable diluent, carrier or excipient e.g. saline or phosphate-buffered saline solution.
  • the composition will typically be substantially sterile, and may comprise additional components, such as liposomes.
  • An effective dose of the composition will be sufficient to provide between 0. l ⁇ g and lOOOmg of the active molecule, typically between lO ⁇ g and 500mg.
  • An optimum dose may be determined by routine trial and error by a person skilled in the art.
  • the molecule could lead to depression of neurotransmitter release: for example, the inventors have found that a molecule in accordance with the invention depresses synaptic transmission in rat brain slices, and may exert a similar effect in man.
  • the molecule could also inhibit receptor sequestration, although the molecule would be unlikely to affect ionotrophic receptors since these are not down regulated by intemalisation.
  • the metabotrophic, G-protein coupled receptors such as some of the glutamate receptors, are internalised and this class of receptor has been implicated in some disorders.
  • the molecule of the invention might be used to inhibit internalisation/downregulation of G-protein coupled receptors and certain other cell surface receptors.
  • the inventors have already found that a molecule in accordance with the invention can inhibit transferrin uptake in COS cells and somatostatin receptor intemalisation (data not shown).
  • the in vivo method of the invention may preferably be performed using a molecule in accordance with the first aspect which comprises a targeting moiety, as defined above. In this way, endocytosis may be inhibited preferentially in target cells in the subject's body.
  • the invention provides a method of inhibiting dynamin-mediated endocytosis in a mammalian subject, the method comprising the steps of: forming a pharmaceutical composition (preferably substantially sterile) comprising a molecule in accordance with the first aspect of the invention; and administering the composition to the subject.
  • a pharmaceutical composition preferably substantially sterile
  • composition may be administered by injection (e.g. intravenous or intramuscular) or transdermally (e.g. by use of patches, dressings and the like).
  • the composition may be administered orally (e.g. in the form of a tablet, capsule or the like).
  • it may be preferred to provide the composition in the form of an enteric-coated capsule for sustained gradual release of the active agent.
  • the invention also provides a method of making a membrane permeable molecule which inhibits dynamin-mediated endocytosis, the method comprising the steps of: forming a peptide moiety comprising the amino acid sequence QVPSRPNRAP; and covalently joining a hydrophobic moiety to the peptide moiety.
  • Figure 1 shows a schematic representation of the structure of amphiphysin, and illustrates the amino acid sequence of a number of peptides corresponding to fragments of dynamin;
  • Figure 2 shows photographs of a number of Coomassie-stained SDS PAGE gels;
  • Figure 3A shows confocal microscopy images of synaptosomes loaded with a fluorescent dye (panel 1) and with the synaptosomes having subsequently released the dye (panel 2);
  • Figure 3B is a schematic representation of the assay method used by the inventors (step 1, loading synaptosomes with lOO ⁇ M FM2-10; step 2, stimulation; step 3, washing and re- polarisation; step 4, second stimulation);
  • Figure 4 is a graph of ⁇ F (arbitary units) against time (seconds), showing the results of an assay as illustrated in Figure 3B.
  • Example 1 Mapping amphiphysin binding site on dynamin
  • At least seven proteins containing SH3 domains bind to dynamin in vitro via different polyproline sequences on the C-terminal ⁇ 100-residue Polyproline Region Domain (Okamoto et al, 1997 J. Biol. Chem. 272, 11629-11635).
  • Isoform 1 of amphiphysin (Amphl) interacts with the sequence PSRPNR at a site partly overlapping the sequence reported to bind to the Grb2 N-terminal SH3 domain (Grabs et al, 1997; Okamoto et al, 1997; cited above).
  • the DNA fragment corresponding to the SH3 domain of Amph2 (corresponding to amino acids 494-588 of Amph2 deposited at Genbank, Y 13380) was amplified by PCR and cloned into the expression vector pET15b (Novagen) and expressed from the E. coli strain BL21 (DE3).
  • residue 494 of the full length Amph2 was defined as residue 1 of the Amph2 SH3 domain.
  • the resulting protein contained an N-terminal His6 tag and thrombin cleavage site, had an apparent molecular weight on SDS PAGE of approximately 14kDa.
  • This fragment was also cloned into a pCMV-MYC eukaryotic expression vector and into pGex4T for expression as an N-terminal GST fusion protein.
  • the sequence encoding residues 595-683 of the SH3 domain of Amphl was cloned and amplified by PCR in essentially the same way using the same vectors.
  • Rat Grb2 N-terminal SH3 domain was obtained by PCR from a rat brain cDNA library using primers as described previously (Gout et al, 1993 Cell 75, 25-36) and cloned into pGex4T and pCMV-MYC.
  • His6Amph2 SH3 single transformants were grown in 2TY medium supplemented with 120 ⁇ g/ml ampicillin at 37°C. Expression was induced by the addition of IPTG to a final concentration of 0.3mM. Cells were harvested after 5 hours growth by centrifugation, resuspended in 30ml buffer per litre of culture (50mM Tris pH 7.5, lOOmM salt, lOmM ⁇ mercaptoethanol O. lmM PMSF O. lmM benzamidine) and lysed by 2 passes through a French pressure cell. Protein was purified at 4°C by NiAgarose affinity, ion exchange (fast-flow Q-sepharose) and gel filtration (Superdex S200) chromatography giving a final yield of 20mg purified protein per litre of culture.
  • Figure 1 is a schematic representation of the common domain structure of Amphl and Amph2, along with the sequence of the polyproline region of dynamin with which both isoforms interact. Sequences PI, P2, P4, P5 and P6 indicate the five peptides used for mapping the binding regions, and the solid bars indicate Grb2 N-terminal and amphiphysin SH3 domain binding sites. The ability of each of peptides P1-P6 to inhibit the interaction of dynamin with the SH3 domains of Amphl, Amph2 or Grb2 was tested in vitro, as described below.
  • FIG. 2 The results of the SDS PAGE analysis are shown in Figure 2.
  • the top panel shows results obtained when recombinant GST-SH3 domain fusion proteins were incubated with rat brain extract and GST agarose beads in the absence of peptide.
  • the panels below show results obtained in the presence of 200 ⁇ M concentrations of peptides PI, P2, P4, P5 and P6 respectively.
  • a molecule in accordance with the invention was prepared as described below.
  • the molecule consisted of a myristoyl group coupled to the N-terminal of a peptide moiety;
  • the Gin residue represents the N-terminal and Proline the C terminal of the peptide moiety, by analogy with the natural sequence of dynamin.
  • the inventors synthesised the peptide with an amino group on the C terminal proline residue, as this was expected to increase the stability of the molecule.
  • it was also found that the presence of an amino group at the C terminal was desirable for optimum membrane permeability:- when the molecule was re-synthesised with a carboxyl group at the C terminal, the molecule was not taken up by cells.
  • the inventors believe that the presence of a negative charge (the carboxyl group is negatively charged at physiological pHs i.e. about pH 6-8) at the C terminal is detrimental to uptake of the molecule by cells, so a carboxyl or other negatively- charged group at the C terminal is best avoided.
  • the synthesis was performed on a NovaSyn 'Crystal' peptide synthesizer using lOO ⁇ mol of Fmoc-NovaSyn-KR resin, which automatically gives a C-terminal amide.
  • the C-terminal proline was double-coupled for 2x2 hours using a 5-fold excess of Fmoc- OPfp ester in the presence of 1 equivalent of HOBt in DMF. Acylation reactions were monitored using UV absorbance monitoring at 304nmn.
  • Arg and Ser were coupled for 1 hour using a 5-fold excess of Fmoc-OBt ester generated in situ from the protected amino-acid, PyBOP, HOBt and Hiinig's base in DMF. Acylation reactions were monitored using UV absorbance monitoring at 348nm.
  • the N-terminus was myristoylated off-column for 2 hours using a 5-fold excess of Myristyl- OBt ester generated in situ from myristic acid, PyBOP, HOBt and H ⁇ nig's base in DMF.
  • the peptidyl-resin was cleaved using 94:3:3 (v/v/v) TFA/iPr3SiH/H20 for 2.5 hours and the crude product dissolved in a 1: 1 mixture of 0.1 %TFA in H 2 O and CH 3 CN containing 10% 0.1 %TFA in H 2 O.
  • the crude product can be purified by reverse-phase preparative hplc performed on a Vydac 208TP1022 C8 200x20mm column, as below.
  • Buffer A 0.1 % TFA/H20.
  • Buffer B CH3CN containing 10% Buffer A.
  • the column was eluted isocratically with 40% Buffer B for 2 minutes then with a linear gradient of 40-90% over 25 minutes. Detection was at 215nm, flow rate lOml/min. Pure fractions were combined and lyophilized.
  • the inventors wished to test the ability of the MyrP4 molecule to inhibit endocytosis in vitro. To do this, they investigated the effect of Myr P4 on synaptic vesicle recycling in rat brain synaptosomes, using a fluorescent dye, FM2-10, to assay endocytosis.
  • Rat brain synaptosomes were prepared essentially as described previously [24] but taking care of the following points.
  • a rat brain was dissected on ice to remove the mid-brain and hind- brain, meninges and as much myelin as possible. This is particularly important as myelin in the synaptosome preparation traps large amounts of FM2-10 resulting in a high background fluorescence and thus a greatly decreased signal to noise ratio.
  • the brain was then homogenised in approximately 20ml of 0.32M Sucrose, 20mM HEPES pH7.3 using 6 strokes with a Potter homogeniser at around 800rpm.
  • the brain homogenate was then centrifuged in a Sorvall SS-34 rotor for 2min at 5000rpm and the pellet discarded. The supernatant was centrifuged again in the same rotor for a further 12min at l l,500rpm. The pellet was resuspended in approximately 8ml of 0.32M sucrose, 20mM HEPES and this was loaded onto percoll gradients (4%, 10%, 23 %) and centrifuged at 18,000rpm for l lmin in a Beckman SW40Ti rotor. The synaptosomes were removed from the interface between the 10% and 23 % percoll, leaving any remaining myelin behind in the 4% percoll layer.
  • the synaptosomes were diluted into HEPES buffered medium (HBM - 140mM NaCl, 5mM KC1, 5mM NaHCO 3 , 1.2mM Na 2 HPO 4 , ImM MgCl 2 , 20mM HEPES, lOmM Glucose, pH7.3), centrifuged for a final time for 14min at 1 l,500rpm in the Sorvall SS-34 rotor and the pellet resuspended in approximately 4ml of HBM.
  • HBM HEPES buffered medium
  • FM2-10 was used to follow endocytosis in isolated nerve terminals
  • FM dyes are highly fluorescent when inserted into lipid membranes but their fluorescence is negligible in aqueous solution (for review see [25]).
  • the dye partitions into the membrane.
  • a stimulus that causes synaptic vesicle endocytosis results in dye-loaded vesicles being internalised.
  • Subsequent exocytosis releases internalised dye and a decrease in fluorescence is observed.
  • Figure 3A shows confocal microscopy images of a synaptosome preparation that has been loaded with FM2-10 on stimulation of vesicle cycling. Active synaptosomes fluoresce due to the labelled vesicles they contain and can be clearly visualised after washing away excess dye (Panel 1). Exocytosis caused by a second depolarising stimulus results in the recycled synaptic vesicles releasing the trapped dye (Panel 2) and consequently the fluorescence is largely lost as the dye rapidly dilutes into the aqueous phase.
  • FIG. 3A The field shown in Figure 3A is a selected one that demonstrates the phenomenon of destaining most effectively. In practice it was hard to obtain such images consistently and reproducibly. Synaptosomes adhere poorly to cover slips, additionally, due to their small size, it was usually difficult to find the same plane of focus on the same synaptosomes before and after any particular treatment. Also varying degrees of myelin contamination presented a problem. For these reasons, quantitative measurements of fluorescence changes were made using a large population of synaptosomes in a spectrofluorimeter as shown schematically in Figure 3B. In essence, synaptosomes were incubated with FM2-10 at 37°C (Step 1) and then vesicle endocytosis was stimulated as described above (Step 2).
  • Step 3 The dye loaded synaptosomes were then washed to repolarise and remove external dye (Step 3). Vesicle cycling was then measured by a further stimulation (Step 4) in which dye labelled vesicles were released.
  • Steps 3 and 4 in Figure 3B correspond respectively to panels 1 and 2 of Figure 3 A.
  • the decrease in fluorescence ⁇ F on dye unloading is a measure of the amount of dye that was endocytosed during the first stimulation (loading).
  • Synaptosomes (500 ⁇ l, prepared as described above) were diluted to 1ml in HBM in a stirred cuvette and incubated with FM2-10(100 ⁇ M) and Ca 2+ (normally 1.3mM) for 3min at 37°C. Vesicle cycling was then stimulated with 30mM KC1 for 45s. The sample was then washed with two short 10s spins, to remove externally bound dye, and resuspended in 1ml fresh HBM to allow repolarisation. The synaptosomes are gently stirred at 37 °C for approximately lOmin to further remove dye from the plasma membrane. Labelled vesicles are then released during a second round of vesicle cycling, stimulated with 30mM KC1, 1.3mM[Ca 2+ ] 0 (external Ca 2+ concentration).
  • the inventors measured the decrease in fluorescence when the dye is exocytosed in a Fluoromax-2 spectrofluorimeter, exciting at 467nM and collecting at 550nM.
  • the decrease in fluorescence ( ⁇ F) on dye unloading is a measure of the amount of dye that was endocytosed during the first stimulation (loading).
  • FM-143 has previously been used to measure exocytosis in synaptosomes [26] but FM2-10 is more easily washed from external membranes giving a lower background signal.
  • the compound was added to the synaptosome preparations, prior to initial stimulation, at a concentration of 50 ⁇ M.
  • a myristoylated scrambled peptide with the same amino acids, but in a different order was also tested.
  • McPherson et al. Nature 1996, 379: 353-357.

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Abstract

L'invention concerne une molécule à perméabilité membranaire destinée à inhiber, dans une cellule eucaryote, une endocytose ayant pour origine la dynamine, cette molécule renfermant un fragment hydrophobe à perméabilité membranaire et un fragment peptidique renfermant la séquence aminoacide QVPSRPNRAP.
PCT/GB1999/001723 1998-06-01 1999-06-01 Ameliorations apportees a l'absorption de substances par des cellules Ceased WO1999062494A2 (fr)

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AU42748/99A AU4274899A (en) 1998-06-01 1999-06-01 Improvements in or relating to uptake of substances by cells
EP99955211A EP1084144A2 (fr) 1998-06-01 1999-06-01 Ameliorations apportees a l'absorption de substances par des cellules
CA002333751A CA2333751A1 (fr) 1998-06-01 1999-06-01 Ameliorations apportees a l'absorption de substances par des cellules

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Cited By (6)

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WO2001081408A3 (fr) * 2000-04-21 2002-07-18 New England Medical Ct Agonistes et antagonistes du recepteur couple a la proteine g (gpcr) et procedes d'activation et d'inhibition de gpcr au moyen de ces angonistes et antagonistes
EP1691800A4 (fr) * 2003-11-21 2009-03-18 Univ Newcastle Res Ass Procedes et agents pour inhiber l'endocytose dependant de la dynamine
US7696168B2 (en) 2000-04-21 2010-04-13 Tufts Medical Center, Inc. G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same
US8440627B2 (en) 2004-11-04 2013-05-14 Tufts Medical Center, Inc. G protein coupled receptor agonists and antagonists and methods of use
US20160264976A1 (en) * 2013-10-18 2016-09-15 Universite De Strasbourg Dynamin 2 inhibitor for the treatment of centronuclear myopathies
WO2024178250A1 (fr) * 2023-02-22 2024-08-29 Radella Pharmaceuticals Llc Inhibiteurs de l'interaction ptp1b/nck1 et leurs procédés de fabrication et d'utilisation

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Publication number Priority date Publication date Assignee Title
WO1995026719A1 (fr) * 1994-04-01 1995-10-12 Arcturus Pharmaceutical Corporation Procede d'introduction de substances biologiquement actives au moyen d'un bioprecurseur thioester ou thioether
US6184205B1 (en) * 1994-07-22 2001-02-06 University Of North Carolina At Chapel Hill GRB2 SH3 binding peptides and methods of isolating and using same

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US8563519B2 (en) 2000-04-21 2013-10-22 Tufts Medical Center, Inc. Methods of activating or inhibiting G protein coupled receptors (GPCRs)
US8389480B2 (en) 2000-04-21 2013-03-05 Tufts Medical Center, Inc. G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same
US6864229B2 (en) 2000-04-21 2005-03-08 New England Medical Center Hospitals, Inc. G protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same
US7696168B2 (en) 2000-04-21 2010-04-13 Tufts Medical Center, Inc. G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same
US8324172B2 (en) 2000-04-21 2012-12-04 Tufts Medical Center, Inc. G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same
US8354378B2 (en) 2000-04-21 2013-01-15 Tufts Medical Center, Inc. G protein coupled receptor antagonists and methods of activating and inhibiting G protein coupled receptors using the same
JP2003530875A (ja) * 2000-04-21 2003-10-21 ニュー イングランド メディカル センター ホスピタル インコーポレイテッド Ｇタンパク質共役型受容体（ｇｐｃｒ）のアゴニストおよびアンタゴニスト、および、それらを用いてｇｐｃｒを活性化および阻害する方法
WO2001081408A3 (fr) * 2000-04-21 2002-07-18 New England Medical Ct Agonistes et antagonistes du recepteur couple a la proteine g (gpcr) et procedes d'activation et d'inhibition de gpcr au moyen de ces angonistes et antagonistes
EP1691800A4 (fr) * 2003-11-21 2009-03-18 Univ Newcastle Res Ass Procedes et agents pour inhiber l'endocytose dependant de la dynamine
US8440627B2 (en) 2004-11-04 2013-05-14 Tufts Medical Center, Inc. G protein coupled receptor agonists and antagonists and methods of use
US11499155B2 (en) 2013-10-18 2022-11-15 Universite De Strasbourg Dynamin 2 inhibitor for the treatment of centronuclear myopathies
US10647986B2 (en) * 2013-10-18 2020-05-12 Universite De Strasbourg Dynamin 2 inhibitor for the treatment of centronuclear myopathies
US20160264976A1 (en) * 2013-10-18 2016-09-15 Universite De Strasbourg Dynamin 2 inhibitor for the treatment of centronuclear myopathies
WO2024178250A1 (fr) * 2023-02-22 2024-08-29 Radella Pharmaceuticals Llc Inhibiteurs de l'interaction ptp1b/nck1 et leurs procédés de fabrication et d'utilisation

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AU4274899A (en) 1999-12-20
EP1084144A2 (fr) 2001-03-21

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