EP1064015A2 - Peptides derives de recepteurs agissant en tant que modulateurs de l'activite de ces recepteurs - Google Patents

Peptides derives de recepteurs agissant en tant que modulateurs de l'activite de ces recepteurs

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Publication number
EP1064015A2
EP1064015A2 EP99908383A EP99908383A EP1064015A2 EP 1064015 A2 EP1064015 A2 EP 1064015A2 EP 99908383 A EP99908383 A EP 99908383A EP 99908383 A EP99908383 A EP 99908383A EP 1064015 A2 EP1064015 A2 EP 1064015A2
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Prior art keywords
seq
receptor
activation
ligand
cell
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German (de)
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Lennart Olsson
Tatjana Naranda
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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the field of this invention is the modulation of activity of cell surface receptors, including both modulation of internalization of the receptor and modulation of activation of the receptors in the presence or absence of ligand.
  • the complex regulatory balance between hormones, receptors and responding cells is critical to the correct functioning of multicellular organisms. Subtle environmental and genetic factors can disrupt this balance, sometimes resulting in disease.
  • the advent of molecular biology has meant that medically important hormones can be made available in therapeutically useful amounts. Among them are human growth hormone, EPO, TPO, insulin-like growth factor, insulin, epidermal growth factor, and numerous others.
  • insulin resistance is an essential feature of a great variety of clinical disorders, such as diabetes mellitus, obesity and certain types of hypertension.
  • Individuals with non-insulin dependent diabetes present with insulin resistance in peripheral tissues. They have a subnormal glucose utilization in skeletal muscle, where glucose transport across the cell membrane of skeletal muscle is the rate limiting step in glucose metabolism. It is possible that a defect exists in insulin-dependent glucose transport in skeletal muscle in diabetic states, where decreased levels of the glucose transporter 4 protein (GLUT4) have been observed.
  • GLUT4 glucose transporter 4 protein
  • insulin stimulates a rapid and dramatic increase in glucose uptake, primarily by promoting the redistribution of the GLUT4 glucose transporter from its intracellular storage site to the plasma membrane.
  • Insulin resistance may also be attributed to a defect in insulin action at the cellular level.
  • the insulin receptor is activated by binding of insulin to the alpha-subunit of the receptor, which causes autophosphorylation of the intracellular beta-subunit region.
  • the activated insulin receptor couples to cytosolic receptor substrates that can affect signaling cascades, resulting in the pleiotropic hormone response.
  • Most proteins involved in the signal transduction pathway are not known yet, but each of them might play a role in the various forms of insulin resistance.
  • the heterogeneous nature of insulin resistance makes treatments that can act "upstream" of the signal transduction pathways very attractive, because a number of different pathologies could be treated with a single drug.
  • MHC-I major histocompatibility complex class I protein
  • Stagsted et al. (1993) J. Biol. Chem. 268:22809-13 demonstrate that such peptides inhibit the internalization of glucose transporters (GLUT4) and insulin-like growth factor II (IGF-II) receptors in insulin-stimulated cells.
  • GLUT4 glucose transporters
  • IGF-II insulin-like growth factor II
  • the present invention provides methods of modulating the activity of a type 2 cell-surface receptor containing an activation sequence comprising binding an exogeneous compound to the activation sequence.
  • the binding is done in the absence of any exogeneous ligand which normally activates the cell-surface receptor.
  • the binding is done in the presence of a ligand which normally activates the receptor, wherein the level of activation is greater with a combination of ligand and exogeneous compound than with the same amount of ligand alone.
  • the present invention provides methods wherein the exogeneous compound is an oligopeptide comprising at least about 8 amino acids and less than about 40 amino acids that has an amino acid sequence corresponding to an activation sequence of the extracellular domian of a cell surface receptor.
  • the invention provides methods of modulating the activity of a type 2 cell-surface receptor containing an activation sequence on the surface of a mammalian cell comprising binding an exogeneous compound to the activation sequence.
  • the invention provides methods of treating a patient with a disorder associated with a ligand that normally activates a type 2 cell surface receptor, comprising administering an exogeneous compound which will bind to the activation sequence of said receptor. This may be done in the absence of exogeneous ligand, or in the presence of exogeneous ligand, wherein the level of activation is greater with the combination of ligand and exogeneous compound than with the same amount of ligand alone.
  • the invention provides methods of screening comprising adding a candidate bioactive agent to a target comprising an activation sequence of a cell-surface receptor and determining whether said candidate bioactive agent is bound to the activation sequence.
  • the invention provides methods of screening for a bioactive agent capable of modulating the activation of a type 2 cell surface receptor comprising adding a candidate bioactive agent to a mammalian cell comprising a type 2 cell surface receptor with an activation sequence and determining whether the candidate bioactive agent binds to said activation sequence and modulates the activation of the receptor.
  • FIGS 1 A and IB Synergistic effect of EPO receptor derived peptide (EPO-Rp, SEQ ID NO: 11) on the effect of EPO as measured by phosphorylation of the EPO receptor
  • EPO-R IP, immunoprecipitation
  • WB Western blot.
  • Figures 1 A and IB are digital images of western blots. Cells were incubated for 20 minutes with EPO and/or EPO- Rp prior to lysis and IP and WB. The panels show that EPO-Rp in itself stimulates the EPO-R, but also that EPO-Rp has a strong synergistic effect as 0.01 U/ml EPO alone has an effect, 10 nM EPO-Rp a weak effect, but that 0.01 U/ml EPO plus 10 nM EPO-
  • Figure 2 Kinetics for the effect of EPO and the EPO receptor derived peptide (EPO- Rp, SEQ ID NO: 11).
  • Figure 2 is a digital image of a western blot. Doses for the two compounds were chosen to give a strong signal on their own. The EPO-induced signal is faster than EPO-Rp (5 minutes with peak at 15 minutes versus 15 minutes and with a duration of at least 90 minutes for EPO-Rp). Thus, the duration of the effect of EPO-Rp is much longer than that of EPO.
  • Figure 3 Demonstration of the stimulation of EPO-R with EPO-Rp. The cells were stimulated for 20 minutes.
  • Figure 3 is a digital image of a western blot. Growth hormone receptor derived peptide (GHRp) was used as control in a concentration of 10 ⁇ M that strongly enhances growth hormone receptor activity. 2.5 U/ml EPO in activity corresponds to about 30-100 nM EPO-Rp.
  • GHRp Growth hormone receptor derived peptide
  • Figure 4 Dimerization of EPO-R is assumed to induce intracellular association between JAK2 and EPO-R. Using an antibody to JAK2 as precipitating antibody, both EPO and EPO-Rp results in association of EPO-R with JAK2.
  • Figure 4 is a digital image of a western blot.
  • FIG. 5 depicts that EPO-Rp selectively activates EPO-Receptor.
  • TF1 cells were stimulated for 20 min with O.lU/ml of EPO, 10 nM TPO, different concentrations of EPO-Rp, and combinations of TPO or EPO (at the same concentrations) together with EPO-Rp.
  • Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibody (PY).
  • Western blot analysis was performed by using anti-EPOR antibody that specifically recognizes EPO-R and anti-JAK2 antibody that will recognize a kinase that specifically associates only with activated receptors.
  • Figure 5 is a digital image of western blot.
  • EPO and EPO-R peptide acts synergistically in activating EPOR (bottom panel) and JAK2 kinase (upper panel). TPO together with EPO-R peptide does not enhance the signal; activation of JAK2 with TPO and EPORp is additive (top panel), because TPO activates its own receptor that also associates with JAK2. As well, bottom panel shows that TPO with EPORp does not activate the EPOR, as the signal is due to presence of the peptide only.
  • Figure 6 depicts the inhibition of EPO-R internalization with EPO-Rp.
  • Internalization of [ 125 ] EPO was measured in TF1 cells in the presence or absence of EPORp. Cells were preincubated for 30 min at 37°C, followed by addition of [ 125 ]-EPO and 15 ⁇ M peptide concentration (EPORp). Cells were further incubated at 37 °C for different times and internalized ligand was measured by the method of acid wash. Cells were spun through oil mixture to separate bound from unbound ligand. Internalized ligand is calculated as percent of [ 125 ]-EPO resistant to acid wash (intracellular) versus the total mount of hormone bound to the cells. Thus, EPORp inhibits internalization of EPO-Receptor and therefore extends its cell surface time.
  • FIG. 7 demonstrates the activation (phosphorylation) of JAK2 kinase by TPO receptor derived peptide (TPO-Rp; SEQ ID NO: 10).
  • TPO-Rp TPO receptor derived peptide
  • Different concentrations of peptide (as indicated) were added to cells for 20 min and IP with anti-PY antibody was performed, followed by western blot using anti-JAK2 antibody.
  • This kinase will get activated only if it can bind to dimerized (biologically active) TPO-Receptors.
  • TPORp enhances signaling through TPO-Receptor.
  • FIG 8 shows that TPORp selectively activates TPO-Receptor.
  • Cells were stimulated for 20 min with O.lU/ml of EPO, 10 nM TPO, different concentrations of TPOR-peptide, and combinations of TPO or EPO (at the same concentrations) together with TPO-R peptide.
  • Cells were lysed and immunoprecipitated with anti-PY antibody.
  • Western blot analysis was performed by using anti-EPOR antibody that specifically associates only with activated receptors (EPOR or TPOR).
  • Figure is a digital image of western blot.
  • TPO and TPO-R peptide act synergistically in activating JAK2 kinase through the TPO-R (upper panel).
  • EPO together with TPO-R peptide does not enhance the signal in the same way as activation of JAK2 with EPO and TPORp as additive.
  • EPO with TPORp does not activate the EPOR, as the signal seen on the bottom panel is due to presence of the EPO only.
  • Figure 9 demonstrates that TPORp activates JAK2 kinase through TPO-Receptor. Experiment was performed as on Figure 8, except that cells were IP with anti-JAK2 antibody. WB probed with anti-PY antibody (top panel) shows that TPORp activates
  • JAK2 kinase in two ways: mimicking TPO activation and acting synergistically with TPO.
  • WB and anti-JAK2 antibody shows that all immunoprecipitates contained the same amount of JAK2 protein.
  • Figure 10 depicts the activation of Leptin Receptor (OB-R) signaling with Leptin Receptor derived peptide (LRp).
  • OB-R Leptin Receptor
  • LRp Leptin Receptor derived peptide
  • COS-1 cells were transiently transfected with long form of Leptin Receptor (OB-R,) and stimulated for 20 min with 6 nM Leptin, 15 ⁇ M LRp or combination thereof.
  • Leptin Receptor was confirmed by western blot analysis using specific anti-OBR antibody and by binding of [ 125 ]-Leptin to the transfected cells. Cells were lysed and immunoprecipitation was performed with anti-JAK2 antibody. WB with the same antibody (left panel) shows that equal amount of proteins are present in all the immunoprecipitates. WB with anti-PY antibody (right panel) demonstrates that LRp activates JAK2 through Lepin Receptor, because there is identical enhancement of protein phosphorylation signal in both cases; cells stimulated with Leptin or LRp.
  • middle band represents phosphorylated STAT5 molecule (both specific proteins in a signaling pathway of OB-R), and bottom band is an unidentified protein whose phosphorylation does not change between non- stimulated and stimulated cells.
  • Two additional peptides were used as a control of specificity: Insulin receptor derived peptide (IRp; SEQ ID NO:3) a peptide that at 15 ⁇ M concentration strongly activates signaling through the Insulin receptor, and Cp, a peptide derivative of Leptin Receptor as well, but without any similarity to MHC -I peptide. None of these peptides activated JAK2 or STAT5 proteins.
  • Figure 11 demonstrates that the LRp mimics Leptin and shows a synergistic effect together with Leptin in activation of Leptin Receptor signaling.
  • COS-1 cells were transfected with long form of Leptin receptor and stimulated for 20 min with different concentrations of Leptin (OB), LRp and the lowest dose of OB (1 ng/ml) together with different peptide concentrations, as indicated on the figure. IP was performed with anti-JAK2 antibody.
  • Bottom panel WB shows that all the immunoprecipitates contain the same amount of JAK2 protein.
  • WB with anti-PY shows activation (phosphorylation) of JAK2 and STAT5 by OB and LRp.
  • Both, natural ligand and the peptide activate JAK2 to the same extent. When combined, there is a synergistic effect between them; e.g., the activation of JAK2 is stronger when 1 ng/ml of OB and 30 ⁇ M LRp were added together (16 arbitrary units), than when the cells were stimulated with the same compounds but separately (2 and 8 units).
  • CP and IRp showed no effect on protein phosphorylation when alone or combined with OB.
  • Numbers in the box below the panel show quantification of western blots. For that purpose, the membranes were scanned and intensity of the protein bands was quantitated using the NTH 1.5 Image program. Upper values are corresponding to JAK2 protein, lower to STAT5.
  • Figure 12 shows the effect of LRp on activation of STAT5 through Leptin Receptor.
  • STAT5 is transcription factor that specifically will bind to activated JAK2, which previously needs to be phosphorylated by interacting with dimerized receptors.
  • anti-STAT5 antibody was used instead of JAK2.
  • WB with anti-PY antibody shows that LRp mimics the action of Leptin (OB) and when combined with it acts synergistically.
  • Lower panel shows that all the immunoprecipitates contain the same amount of STAT5 protein.
  • Figure 13 demonstrates the effect of LRp on the signaling of the Leptin Receptor short form (OB-R j ).
  • COS-1 cells were transfected with short form of Leptin receptor and stimulated for 20 min with different concentrations of Leptin (OB), LRp and two different doses of OB (1 ng/ml and 10 ng/ml) together with different peptide concentrations, as indicated on the figure.
  • IP was performed with anti-OBR antibody and subsequent blot was probed with JAK2 antibody.
  • WB on the upper panel shows that JAK2 protein associates with Leptin receptor only when the receptor has been activated.
  • LRp shows that same effect as natural ligand.
  • LRp and Leptin together can activate short form of leptin receptor (form of the receptor that is expressed in db/db animals), the effect that is not observed when only one of the compound is added.
  • Figure 14 demonstrates the activation of Growth Hormone Receptor (GHR) by GHR- derived peptide (GH-Rp; SEQ ID NO:9).
  • GHR Growth Hormone Receptor
  • GH-Rp GHR- derived peptide
  • IM9 cells were stimulated for 20 min with 10 nM GH, 30 ⁇ M GHRp and combination thereof. IP was performed with anti-JAK2 antibody.
  • WB and JAK2 antibody shows the same amount of the protein present in all the immunoprecipitates.
  • WB with anti-PY demonstrate equal activation (phosphorylation) of JAK2 by GH and GHR-peptide.
  • Figure 15 shows the kinetics for the effect of GH and GHRp. Doses for the two compounds are as indicated on the top of the panels. Upper panel shows that GH and
  • GHRp phosphorylate JAK2 to the same extent; most likely by inducing dimerized conformation between GH-Receptors (receptor dimerization is assumed to induce intracellular association between JAK2 and GHR).
  • the GH-induced signal of JAK2 phosphorylation is faster than GHRp (1 min with duration of approximately 30 min, versus 5 min and with duration of at least 40 min for GHRp).
  • the duration of the effect of GHRp is much longer than that of GH.
  • Figure 16 demonstrates that GHRp (P) broadens the range of GH action.
  • IM9 cells were stimulated with 1 nM, 10 nM and 2 ⁇ M GH in the presence or absence of GHRp. Those concentrations of GH were use, because it is known that GH action has the bel- shaped dose response curve with an IC 50 at 2 ⁇ M GH concentration.
  • IP was performed with anti-JAK2; WB with the same antibody (right panel) or with anti-PY (left panel). Blot probed with anti-JAK2 shows that the same amount of protein is present in all immunoprecipitates.
  • Anti-PY WB demonstrates that peptide enhances JAK2 activation approximately three times in the presence of 1 nM GH. In the GH inhibitory concentration of 2 ⁇ M GHRp has increased natural hormone response approximately two times. Therefore, GHRp broadens the range of GH action by increasing its activity at low concentrations and extending the activity to higher hormone concentrations.
  • Figure 17 demonstrates synergistic effect of GHRp on the GH action.
  • Cells were stimulated for 20 minutes with different concentrations of the GHR-peptide alone (top panel), or in combination with 1 nM (middle panel) and 0.1 nM GH (bottom panel).
  • IP was performed with anti-JAK2 and WB with anti-PY antibody.
  • GHRp shows strong synergistic effect with both concentrations of GH; (i) no effect on JAK2 phosphorylation is observed with low (up to 5 ⁇ M) peptide concentrations, but when combined with 1 nM GH, activation of JAK2 is significantly increased (1 versus 12 arbitrary units); (ii) 0.1 nM concentration of GH shows no effect on JAK2 phosphorylation, but when combined with low 0.3 ⁇ m GHRp (that as well has no effect on its own), JAK2 activation is increased approximately four times.
  • Figure 18 demonstrates that GHRp enhances the phosphorylation of STAT5.
  • STAT5 is molecule downstream of JAK2 in a signaling pathway of the GH. The putative mechanism is that the receptors dimerize (or multimerize), JAK2 associates with the multimerized receptors and becomes phosphorylated. JAK2 can then associate with STAT5, which also becomes activated by phosphorylation.
  • Cells were stimulated with different concentrations of GHRp or GH as indicated at the top of the panel, IP with anti-STAT5 and then WB with anti-PY (upper panel) and anti-STAT5 (lower panel). Increasing phosphorylation of STAT5 is observed as the dose of added GHRp was increasing. The same band of phosphorylation was observed when cells were stimulated with GH indicating specific signaling through GHR. Lower panel demonstrates that all the immunoprecipitates contained the same amount of STAT5 protein.
  • Figure 19 demonstrates that GHRp together with GH has a synergistic effect on activation (phosphorylation) of STAT5.
  • Stimulation of the cells with 0.2 nM GH in the presence or absence of GHRp shows significant increase in STAT5 phosphorylation, e.g., no phosphorylation is observed with 0.2 nM GH or 1.25 ⁇ M
  • GHRp alone, but the signal is increased when both compounds are present.
  • a weak signal of STAT5 protein phosphorylation with 2.5 ⁇ M peptide is strongly increased by presence of 0.2 nM GH.
  • Activity was measured with anti-PY antibody (upper panel). The lower panel demonstrates that the same amount of STAT5 is present in all the immunoprecipitates.
  • GHRp has very strong synergistic effect on GH, measured by STAT5 phosphorylation.
  • Figure 20 shows the selectivity of GHRp for the GH-Receptor.
  • Internalization of [ 125 ]- GH was measured in IM9 cells in the presence of GHRp, IGF- IRP (peptide selective and active on IGF-1R; SEQ ID NO:5) and Pl-Rp (peptide specific for Prolactin receptor, SEQ ID NO:35; the prolactin receptor has a high level of identity (approximately 50%) to the GHR).
  • Cells were preincubated for 30 min at 37°C, followed by addition of [ 12 I]-GH and different peptide concentrations as indicated. After the incubation of 20 min at 37 °C, internalized ligand was measured by the method of acid wash.
  • the complete mRNA sequence encoding the human insulin responsive glucose transporter (GLUT4) has the Genbank accession number M2O747, published by Fukumoto et al. (1989) J. Biol. Chem. 264:7776-7779.
  • the complete mRNA sequence encoding the human insulin receptor has the Genbank accession number
  • the complete mRNA sequence encoding the human leptin receptor has the Genbank accession number U43168, and was published by Tartaglia et al. (1995) Cell 83 : 1263-1271.
  • the DNA sequence encoding the human granulocyte colony stimulating factor (G-CSF) receptor has the EMBL accession numbers M5982O,
  • Additional Swissprot accession numbers are as follows, with the remainder of the numbers easily obtained: interleukin-6 receptor, P08887; interleukin-8 receptor-B, P25025; interleukin-8 receptor- A, P24024; interleukin- 11 receptor, U32324; interleukin- 12, P42701; interleukin- 17 receptor, U31993 ; EPO receptor, P 19235 ; and TPO receptor, P40238.
  • the sequences for other cell surface receptors are known, and easily ascertainable by those in the art.
  • HLA and H-2 alleles may be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest. N.I.H. publication no. 91-3242, vol. 1, pp. 738-740, 761, 770-771, 779-780, 788-789 and 802-804.
  • cell surface receptors of interest are internalized or are recycled into the cytoplasm in response to ligand binding.
  • the present invention is based on the initial discovery that sequences on the extracellular portion of cell surface receptors, termed “internalization sequences" or “activation sequences” herein, are involved in modulation of receptor responses. Fortuitously, the activation sequences are not directly involved in ligand binding; that is, the ligand binding site is separate from the activation sequence of the receptor.
  • NIDDM Non-insulin dependent diabetes mellitus
  • the activation sequence can be important in the activation of the receptor, i.e. the activation of the signalling pathway of the receptor.
  • oligopeptides corresponding to a receptor's activation sequence can actually replace the requirement for the ligand, and will cause receptor activation even in the absence of ligand. That is, even though the binding site for the hormone ligand and the activation sequence are different, the addition of the activation sequence oligopeptide will cause receptor activation.
  • type 2 cell surface receptors occur as monomeric units, each with a distinct ligand binding site and an internalization or activation site.
  • the present invention reveals that this dimerization and subsequent receptor activation can occur upon the binding of the activation sequence oligopeptide, in the absence of ligand.
  • the effect of the ligand and the effect of the activation oligopeptide is signficantly synergistic, and can allow maximum signal using reduced concentrations of each.
  • These oligopeptides can thus be utilized either as a ligand replacement, or to increase the response of a given amount of ligand.
  • the present invention also allows the creation of antagonists to receptor signalling.
  • an internalization or activation peptide will be derived from the sequence of the receptor that is to be modulated.
  • the sequence of interest corresponds to the region of the receptor on the extracellular surface, but usually is not directly involved in ligand binding, i.e. contact is not made with the ligand (that is, there is no effect on the K D of the ligand).
  • the activation sequences from different receptors are highly specific; in general, an activation sequence from one receptor will not activate a different receptor. Sequences of receptors, and positioning of the receptors in the cell membrane are known in the art. Such information may be accessed through public databases, as previously cited.
  • sequences and receptors described herein are from humans; although as will be appreciated by those in the art, the present invention may be useful in the creation and elucidation of animal models of human disease. Accordingly, sequences and receptors from rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (cows, sheep, pigs, goats, etc.) may also be used. In addition, sequences from one organism may be tested in other organisms; for example, rat sequences may be tested in humans, etc.
  • the present invention provides regulatory oligopeptides comprising activation sequences that have an amino acid sequence at least substantially identical to the sequence of a portion of a cell surface receptor extracellular domain.
  • these oligopeptides also have sequence similarity to bioactive oligopeptides of the major histocompatibility locus class I antigens (described in U.S. Patent no. 5,385,888, herein incorporated by reference).
  • the oligopeptides modulate the effect of ligand binding to the corresponding receptor, thereby enhancing the physiological effect of the ligand, and may, as outlined above, act to replace the ligand requirement entirely, depending on the characterization of the receptor.
  • the activation sequences are initially identified by homology to the sequence of an ⁇ , -domain of an MHC Class I antigen.
  • MHC Class I antigens include human MHC Class I antigens and mammalian equivalents thereof, such as Class I antigens of the H-2 locus of mice, in particular H-2 D and K.
  • Human MHC Class I antigens include HLA-A, B and C.
  • amino acid sequences in the polymorphic regions of the ⁇ -1 domain more particularly amino acids 55 to 90, usually 60 to 90, more particularly 62 to 90.
  • the region 60-85 of the ⁇ -1 domain, more particularly 62-85 or 72-82 are found to be of particular interest.
  • One MHC sequence of particular interest is ERETQIAKGNEQSFRVDLRTLLR, (SEQ ID NO:l; U.S. Patent No. 5,385,888).
  • ERETQIAKGNEQSFRVDLRTLLR SEQ ID NO:l; U.S. Patent No. 5,385,888
  • Suitable cell surface receptors include, but are not limited to, insulin receptor, insulin-like growth factor receptor, growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), transferrin receptor, epidermal growth factor receptor, low density hpoprotein receptor, high density hpoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGF receptor, PDGF receptor, EPO receptor, TPO receptor, ciliary neurotrophic factor receptor, prolactin receptor, and T-cell receptors.
  • SwissProt ML1B rat melatonin receptor type IB
  • SwissProt SCRC human secretin receptor precursor
  • SwissProt NY1R Xenopus nueropeptide Y receptor
  • SwissProt PAFR rat platelet activating factor receptor
  • SwissProt BLR1 Bokitt's Lymphoma receptor for human, mouse and rat. Algorithms for sequence analysis are known in the art, and include, but are not limited to, the Best Fit sequence program described by Devereux et al. , Nucl. Acid
  • the amino acid sequence of the receptor region of interest will have at least about 10% sequence identity, and frequently at least about 15-20% sequence identity.
  • sequence similarity will be at least about 30%, with at least about 35% being preferred, and frequently at least about 45-50%.
  • the examples provide the results of exemplary similarity searches.
  • Exemplary activation amino acid sequences of interest include, but are not limited to:
  • TWLGRQGPEGPSSIPPGTLTTLW (SEQ ID NO:2) from human glucose transporter, GLUT4, which is 13% identical and 39% similar to SEQ ID NO:l;
  • KTDSQILKELEESSFRKTFEDYLH (SEQ ID NO:3) from human insulin receptor, which is 35% identical and 56% similar to SEQ ID NO: 1 ;
  • EAEAAVATQETSTVRLKVSSTAVRT (SEQ ID NO:4) from human LDL receptor, which is 16% identical and 88% similar to SEQ ID NO:l;
  • KTEAEKQAEKEEAEYRKVFENFLH (SEQ ID NO:5) from human insulin like growth factor receptor, which is 32% identical and 54% similar to SEQ ID NO:l; 5.
  • KKENKIVPSKEIVWWMNLAEKIP (SEQ ID NO:6) from human leptin receptor, which is 17% identical and 43% similar to SEQ ID NO:l ;
  • EKKPVPWESHNSSETCGLPTLVQTY (SEQ ID NO:7) from human GCSF receptor;
  • GPHCVKTCPAGVMGENNTLVWKY (SEQ ID NO:8) from human epidermal growth factor receptor, which is 17% identical and 43% similar to SEQ ID NO:l;
  • EYELQYKEVNETKWKMMDPILTTSVPVY (SEQ ID NO:9) from human growth factor receptor, which is 32% identical and 48% similar to SEQ ID NO:l;
  • ARGGTLELRPRSRYRLQLRARLN (SEQ ID NO: 10) from human thrombopoietin receptor, which is 22% identical and 43% similar to SEQ ID NO:l; 10. QRVEILEGRTECVLSNLRGRTRY (SEQ ID NO: 11) from human erythropoietm receptor, which is 26% identical and 43% similar to SEQ ID NO:l;
  • EMQSPMQPVDQASLPGHCREPPPW (SEQ ID NO: 12) from interleukin-2 (IL- 2) receptor alpha chain, which is 12% identical and 37% similar to SEQ ID NO:l;
  • DPDEGVAGAPTGSSPQPLQPL (SESQ IDNO.13) from IL-2 receptor beta chain, which is 19% identical and 38% similar to SEQ ID NO: 1 ;
  • EPSLRIAASTLKSQISYRARVRAWAQCY SEQ ID NO: 15
  • IL-4 interleukin-4 receptor
  • DYETRITESKCVTILHKGFSASVRTILQ SEQ IDNO:16
  • IL-5 which is 30% identical and 52% similar to SEQ ID NO:l; 16.
  • PAQEVARGVLTSLPGDSVTL SEQ ID NO: 17
  • interleukin-6 IL-6 receptor
  • GKSNICVKVGEKSLTCKKIDLTTIVK (SESQ IDNO: 18) from interleukin-7 (IL-7), which is 26% identical and 61% similar to SEQ ID NO:l; 18.
  • EDMGNNTANWRMLLRILPQSF (SEQ ID NO:19) from interleukin-8 (IL-8) receptor-B, which is 24% identical and 43% similar to SEQ ID NO:l ;
  • EVLGNDTAKWRMVLRILPHTF (SEQ ID NO:20) from interleukin-8 (IL-8) receptor- A, which is 19% identical and 52% similar to SEQ ID NO:l;
  • ELDPGFIHEARLRVQMATL (SEQ ID NO:21) from interleukin-9 (IL-9) receptor, which is 32% identical and 63% similar to SEQ ID NO: 1 ;
  • EVITDAVAGLPHAVRVSARDFL (SEQ ID NO:22) from interleukin-11, which is 32% identical and 45% similar to SEQ ID NO:l ;
  • EQPTQLELPEGCQGLAPGTEVTYRLQLHML (SEQ ID NO:23) from interleukin- 12, which is 43% identical and 67% similar to SEQ ID NO:l; 23. EWSDKQCWEGEDLSKKTLLRFW (SEQ ID NO:24) from interleukin- 13, which is 27% identical and 59% similar to SEQ ID NO:l;
  • KQDKKIAPETRRSIEVPLNERI (SESQ ID NO:25) from interleukin- 13 (a second sequence), which is 23% identical and 41% similar to SEQ ID NO:l;
  • EITTDVEKIQEIRYRSKLKLI (SEQ ID NO:27) from human platelet derived growth factor (PDGF) receptor;
  • EARCDFCSNNEESFILDADSNM (SEQ ID NO:28) from human vascular endothehal growth factor (VEGF) receptor, which is 27% identical and 50% similar to SEQ ID NO:l;
  • TWQTPSTWPDPESFPLKFFLRY (SEQ ID NO:29) from human ciliary neurotrophic factor receptor-alpha, which is 27% identical and 59% similar to SEQ ID NO:l;
  • DSQTNVSQSKDSDVYITDKTVL (SEQ ID NO:30) from T-cell receptor alpha chain, which is 14% identical and 50% similar to SEQ ID NO: 1 ;
  • EWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:31) from T-cell receptor beta chain, which is 17% identical and 37% similar to SEQ ID NO: 1 ;
  • SQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRH (SEQ ID NO:32) from T-cell receptor gamma chain, which is 29% identical and 50% similar to SEQ ID NO:l;
  • VHTEKVNMMSLTVLGLRMLF (SEQ ID NO:33) from T-cell receptor delta chain, which is 37% identical and 58% similar to SEQ ID NO: 1 ;
  • EKTDRFVMKKLNDRVMRVEYHFLSPY (SEQ ID NO:34) from human transferrin receptor, which is 29% identical and 54% similar to SEQ ID NO:l;
  • EWEIHFAGQQTEFKILSLHPGQKYL (SEQ ID NO:35) from human prolactin receptor, which is 20% identical and 48% similar to SEQ ID NO:l.
  • ARRKAKAERKLRLRPSDLRSFLTMF (SEQ ID NO:38) from rat melatonin receptor type IB.
  • KLRTQETRGNEVSHYKRLARSTLLLIP (SEQ ID NO:39) from human secretin receptor.
  • GKYVCLEDFPEDKRFLSYTTLLFIL (SEQ ID NO:40) from Xenopus neuropeptide Y receptor type 1.
  • SFRVDSEFRYT (SEQ ID NO:41) from rat platelet activating factor receptor.
  • CLNPMLYTFAGVKFRSDLSRLLTKL (SEQ ID NO:42) from human Burkitt's lymphoma receptor.
  • CLNPMLYTFAGVKRFSDLSRLLTKL (SEQ ID NO:43) from mouse Burkitt's lymphoma receptor.
  • CLNPMLYTFAGVKRFSDLSRLLTKL (SEQ ID NO:44) from rat Burkitt's lymphoma receptor.
  • the receptor sequence of interest i.e. the activation sequence
  • oligopeptides are made, either synthetically or through recombinant means, which correspond to the activation sequence of the extracellular domain of the cell surface receptor.
  • oligopeptide is identical to all or part of the activation sequence, or that the oligopeptide has substantial homology to the activation sequence; that is, as described below, the oligopeptide may have amino acid substitutions, insertions or deletions as compared to the activation sequence.
  • the activation sequences may be modified, either as modified oligopeptides or as modified receptors, where the receptors are made with modified activation sequences.
  • the activation sequences of the regulatory oligopeptides are altered.
  • any modifications do not substantially alter the biological activity, i.e. they do not inhibit internalization or aggregation, or prevent activation, of the activation sequence for the corresponding receptor. This is easily tested using the binding assays described herein. For example, amino acid substitutions, insertions and deletions may be made.
  • amino acid substitutions are made.
  • residues critical for biological activity are either not altered or conservatively altered.
  • Critical residues may be elucidated using known mutagenesis techniques followed by activity or binding assays; for example, using scanning mutagenesis techniques, wherein single amino acid residues within the activation sequence are modified by substitution with an aliphatic amino acid, e.g. serine, alanine, glycine, valine, etc.
  • substitutions deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the oligopeptide are desired, substitutions are generally made in accordance with the following chart:
  • substitutions that are less conservative than those shown in Chart I, although these generally are not preferred.
  • substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
  • the substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • leucyl isoleucyl, phenylalanyl, valyl or alanyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
  • an electronegative residue e.g. glutamyl or aspartyl
  • a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
  • not more than about three substitutions or deletions will be made, and that the change will not be more than about 20 number %, usually not more than about 10 number %, of the number of amino acids in the active motif, although in some instances higher numbers of alterations may be made.
  • the present invention provides oligopeptides that have at least about 60% identical homology to the activation sequence of each receptor described herein, with at least about 75% being preferred and at least about 80% being especially preferred; in some instances the identity will be as high as 90 to 95 or 98%.
  • non-critical residues are altered, this may be higher.
  • the biological function of the activation sequence is to be decreased, the amount of changes may also be greater.
  • conservative substitutions as known in the art, including substitutions within the large hydrophobic group: isoleucine, leucine, valine and phenylalanine; between serine and threonine; glycine and alanine; asparagine and glutamine; aspartic acid and glutamic acid; or lysine, arginine and histidine.
  • non-conservative alterations are done.
  • the oligopeptides may contain additional sequences, as will be appreciated by those in the art.
  • the oligopeptides may be extended to: 1) provide convenient linking sites, e.g. cysteine or lysine; 2) to enhance stability; 3) to bind to particular receptors; 4) to provide for site-directed action; 5) to provide for ease of purification (for example, epitope or purification (His 6 ) tags); 6) to alter the physical characteristics (e.g. solubility, charge, etc.); or 7) to stabilize the conformation; etc.
  • the oligopeptides may be joined to non-wild-type flanking regions as fused proteins, joined either by linking groups or covalently linked through cysteine (disulfide) or peptide linkages.
  • the oligopeptide may be linked through a variety of bifunctional agents, such as maleimidobenzoic acid, methyidithioacetic acid, mercaptobenzoic acid, S-pyridyl dithiopropionate, etc.
  • the oligopeptides may be joined to a single amino acid at the N- or C-terminus of a chain of amino acids, or may be internally joined.
  • the subject peptides may be covalently linked to an immunogenic protein, such as keyhole limpet hemocyanin, ovalbumin, etc. to facilitate antibody production to the subject oligopeptides.
  • the oligopeptides may be shorter than those depicted herein; that is, residues from either the N- or C-terminus of the oligopeptide may be deleted with the retention of biological activity, preferably full biological activity. In some cases, internal residues may be removed from the oligopeptide. Generally, this will be done by sequentially removing residues and assaying for the ability to bind to the activation sequence of a receptor; once binding has been established, activation may be evaluated.
  • the subject oligopeptides may be expressed in conjunction with other peptides or proteins, so as to be a portion of the chain, either internal, or at the N- or C- terminus.
  • Various post-expression modifications may be achieved.
  • one may provide farnesylation or prenylation, such that the subject peptide will be bound to a lipid group at one terminus, and will be able to be inserted into a lipid membrane, such as a liposome.
  • the subject oligopeptides may be modified by the addition of chemical moieties or groups.
  • the oligopeptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.
  • the subject oligopeptides may also be combined with other proteins, such as the Fc of an
  • IgG isotype to enhance complement binding, or with a toxin, such as ricin, abrin, diphtheria toxin, or the like, particularly the A chain.
  • the oligopeptides may be linked to antibodies for site directed action.
  • conjugation techniques see, for example, U.S. Patent Nos. 3,817,837; 3,853,914; 3,850,752; 3,905,654; 4,156,081; 4,069,105; and 4,043,989, which are inco ⁇ orated herein by reference.
  • the oligopeptides may be labelled as well.
  • Oligomers of the regulatory oligopeptides of the invention may also be made.
  • oligopeptides of interest for drug screening include, but are not limited to: 1) an oligopeptide having at least substantially the sequence of the receptor region of interest; 2) MHC/receptor oligopeptide heterodimers having the sequence of the receptor region of interest and the amino acid sequence of bioactive oligopeptides of the major histocompatibility locus class I antigens; and 3) receptor derived oligopeptide homodimers, generally as a head to tail dimer, where a spacer of from 1 to 3 small neutral amino acids may be present between the two active peptide sequences, as is generally described in WO US96/15426, specifically incorporated herein by reference.
  • the oligopeptides comprising the activation sequences may be prepared in accordance with conventional techniques, such as synthesis (for example, use of a Beckman Model 990 peptide synthesizer or other commercial synthesizer).
  • Peptides may be produced directly by recombinant methods (see Sambrook et al.
  • fusion protein for example to a protein that is one of a specific binding pair, allowing purification of the fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at a site engineered to yield the desired peptide (see for example Driscoll et al. (1993) J. Mol. Bio. 232:342-350).
  • the activation sequence contained within the receptor is altered, to form a modified receptor.
  • the sequence corresponding to the regulatory peptide i.e. the activation sequence
  • the modification may include a deletion or substitution of the complete oligopeptide sequence, or a portion thereof. Substitutions of interest also include scanning mutations as outlined above.
  • the modification is performed using recombinant DNA technology.
  • the DNA sequence encoding the desired receptor may be obtained from various sources, or may be obtained from a cDNA library using probes derived from publically available sequence information. Techniques for in vitro mutagenesis of cloned genes are known; methods for site specific mutagenesis can be found in Sambrook, et al. supra. pp 15.3-15.108; Weiner et al. (1993) Gene 126:3541; Sayers et al. (1992) Biotechniques 13:592-6; Jones and Winistorfer (1992) Biotechniques 12:528-30; Barton et al. (1990) Nucleic Acids Res. 18:7349-55; Marotti and Tomich (1989) Gene Anal. Tech.
  • primers are devised that span the region. On hybridization, the region to be deleted forms a single stranded loop. The loop may be excised by nuclease digestion, or a suitable polymerase may be used to extend out from the primer.
  • the DNA sequences are inserted into an appropriate expression vector, where the native transcriptional initiation region may be employed or an exogenous transcriptional initiation region, i.e., a promoter other than the promoter which is associated with the gene in the normally occurring chromosome.
  • the promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome.
  • transcriptional initiation regions are known for a wide variety of expression hosts. Generally a selectable marker operative in the expression host will be present.
  • the promoter may be operably linked to the coding sequence of the genes of interest so as to produce a translatable mRNA transcript.
  • Expression vectors have convenient restriction sites located near the promoter sequence so as to provide for the insertion of nucleic acid sequences encoding heterologous proteins.
  • the promoters in suitable expression vectors may be either constitutive or inducible.
  • the expression vectors are transformed into a host cell.
  • the expression hosts may involve prokaryotes or eukaryotes, particularly E. coli; B. sublilis; yeast cells; mammalian cells; e.g., COS and CHO cells, HeLa cells, L(tk-), primary cultures; insect cells; Xenopus laevis oocytes; and the like.
  • Particularly preferred host cells are mammalian cells.
  • the oligopeptides are used in methods for inhibiting the internalization of a cell surface receptor response of a mammalian cell.
  • the methods comprises adding oligopeptides as defined herein to mammalian cells expressing the cell surface receptor.
  • the oligopeptide inhibits the receptor internalization.
  • the ligand need not be added to alter receptor internalization.
  • the oligopeptides are used in methods of activating receptors. As discussed above, for some receptors, the addition of the oligopeptide can replace or augment the requirement for ligand binding to effect receptor activation. As outlined briefly above, cell-surface receptors appear to fall into two general classes: type 1 and type 2 receptors. Type 1 receptors have generally two identical subunits associated together, either covalently or otherwise, in the absence of bound ligand; they are essentially preformed dimers, even in the absence of ligand. The type 1 receptors include the insulin receptor and the IGF receptor.
  • the type-2 receptors generally are in a monomeric form, and rely on either binding of one ligand to each of two monomers, or, as shown in the present invention, the binding of one or more oligopeptides that result in multimer formation, to achieve receptor activation.
  • Type-2 receptors include the growth hormone receptor, the leptin receptor, the LDL receptor, the GCSF receptor, the interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, IL-17, etc., receptors, EGF receptor, EPO receptor, TPO receptor, VEGF receptor, PDGF receptor, T-cell receptor, transferrin receptor, prolactin receptor, and the ciliary neurotrophic factor receptor.
  • the present invention provides receptors with exogeneous compounds bound to their activation sequences, and specifically with bound oligopeptides as defined herein.
  • each individual receptor monomer can bind a ligand molecule, and thus will not be brought together into a dimer form; that is, each monomer has a bound ligand, rather than two monomers sharing a ligand.
  • a receptor may be classified as either type 1 (ligand dependent) or type 2 (ligand independent) on the basis of several tests. As noted above, type 2 receptors will exhibit a decrease in receptor activation at high levels of ligand. Type 2 receptors may also be classified as such using the present invention, since the addition of the internalization oligopeptides of the invention will result in receptor activation even in the absence of ligand.
  • oligopeptides based on the activation sequences of type 2 cell- surface receptors result in activation of the receptor even in the absence of ligand. It is important to note in this case that the ligand binding site is distinct from the activation or activation sequence.
  • the present invention provides methods for activating a type 2 cell surface receptor by binding an exogeneous compound to the activation sequence of the receptor. As a result of this binding, two monomeric receptors dimerize and activate the receptor.
  • activation may be prevented by binding an antagonistic exogeneous agent to the activation sequence to prevent activation of the receptor; that is, certain agents will prevent activation of the receptor, even in the presence of ligand.
  • exogeneous compound herein is meant a compound not produced endogeneously by the cell or organism; that is, it is artificially introduced to the cell or organism.
  • Exogeneous compounds include, but are not limited to, bioactive agents as defined below such as chemical and small organic moieties; and the oligopeptides described herein. As will be appreciated by those in the art and described below, having determined the location of the binding, and the mechanism of receptor activation by binding, it will be routine to screen for other molecules that will accomplish the same thing as the oligopeptides of the invention.
  • a preferred embodiment utilizes the oligopeptides of the invention as the exogeneous compounds.
  • receptor activation or grammatical equivalents herein is meant the biological function associated with ligand binding to a cell-surface receptor. As will be appreciated by those in the art, this will vary widely depending on the identity of the receptor. For example, cell-surface receptors frequently cause phosphorylation as a result of ligand binding, that generally, although not always, is due to a conformational change in the receptor as a result of ligand binding. Thus “activation” may comprise a conforaiational change either within the receptor, or as a result of monomeric receptors becoming multimeric, which allows the receptor do facilitate signalling. For example, this conformational change may allow the receptor to become phosphorylated, or allow the receptor to associate with a third molecule which then results in phosphorylation of either the receptor, the third molecule, or yet another molecule. In this way signalling is accomplished.
  • the exogeneous compound is added to the cell in the absence of exogeneous ligand.
  • exogeneous ligand herein is meant ligand which is not produced endogeneously by the cell or organism; i.e. exogeneous ligand is introduced to the cell or organism. It will be appreciated that the composition of the ligand is generally the same, whether produced endogeneously or introduced exogeneously; the designation goes to the source of the ligand, not its composition. As will be appreciated by those in the art, there may or may not be endogeneous ligand present.
  • the exogeneous compound acts as a ligand replacement, although it does not bind to the ligand binding site as a competitor; rather, as outlined herein, it binds to the activation sequence of the receptor.
  • the presence or absence of binding of the exogeneous compound may be tested in a variety of ways, as will be appreciated by those in the art, including labeling the exogeneous compound and detecting labelled receptors, competitive assays with known binding agents, such as the oligopeptides of the invention, or by utilizing modified receptors which do not contain activation sequences.
  • the exogeneous compound is added to the receptor in the presence of the ligand which normally activates the receptor.
  • the ligand which normally activates the receptor.
  • the level of receptor activation is greater with a combination of the ligand and the exogeneous compound as compared with the same amount of ligand alone.
  • the effect of adding ligand and exogeneous compound in this case, an oligopeptide of the invention
  • is greater than either ligand alone or exogeneous compound alone as is generally shown in the examples and Figures.
  • the oligopeptides are administered therapeutically.
  • the subject oligopeptides act to enhance the cellular response to hormones that bind to the surface membrane receptor corresponding to the oligopeptide, e.g. insulin response is enhanced by the oligopeptide SEQ ID NO:3, glucose transport is enhanced by the oligopeptide SEQ ID NO:2, etc.
  • Hormones including insulin, insulin-like growth factor, human growth hormone, glucose transporters, transferrin, epidermal growth factor, EPO, TPO, low density hpoprotein, human growth hormone and interleukins are herein referred to as "therapeutic hormones" or "ligands".
  • Enhancement of the cellular response to therapeutic hormones by the subject oligopeptides provides a means of improving the response of patients that are either unresponsive, e.g. resistant, to the action of such hormones, or, in the case of the type 2 receptors, the oligopeptides can actually serve as ligand replacements. This is particularly desirable in some situations where the administration of the hormone ligand has undesirable side-effects, for example in the case of growth hormone; the use of the oligopeptides or other exogeneous compounds may allow receptor activation without significant side-effects.
  • the subject oligopeptides may be administered to patients requiring enhancement of the response to naturally occurring levels of the therapeutic hormone.
  • the oligopeptides may be administered to patients in conjunction with a therapeutic hormone.
  • a therapeutic hormone Of particular interest is the treatment of insulin resistance, which may be associated with defects in glucose transport, or in the cellular response to insulin.
  • Administration of the subject oligopeptides improves the response to insulin therapy.
  • enhancement of the effect of human growth hormone is also of particular interest. Human growth hormone is current given in a number of clinical situations as an injectible drug; alternative therapies may include augmenting the response of endogeneous hormone.
  • exogeneous compounds including the oligopeptides of the invention, may be administered to patients either as ligand replacements, or to augment the response of a given amount of ligand hormone, as is outlined above.
  • the present invention provides for methods of treating patients with disorders associated with a ligand that normally activates a type 2 cell surface receptor.
  • patients with either insufficient amounts of growth hormone, no growth hormone, or insufficient response to a normal level of growth hormone may all be treated by the administration of an exogeneous compound which binds to the activation sequence of the growth hormone receptor and causes both receptor activation and retarded receptor internalization.
  • other disorders such as obesity or weight problems associated with leptin may be treated.
  • diseases associated with both not enough ligand present in the patient, and too much ligand present in the patient may be treated using the exogeneous compounds of the invention.
  • the addition of the exogeneous compounds, for example the oligopeptides of the invention can allow dimerization of the receptor and activation can actually cause the body to make less ligand.
  • the administration can be simultaneous or sequential, as will be appreciated by those in the art.
  • the oligopeptides may be administered topically or parenterally, e.g. by injection at a particular site, for example, subcutaneously, intraperitoneally, intravascularly, intranasally, transdermally or the like.
  • Formulations for injection will comprise a physiologically-acceptable medium, such as water, saline, PBS, aqueous ethanol, aqueous ethylene glycols, or the like.
  • Water soluble preservatives which may be employed include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol and phenylethanol. These agents may be present in individual amounts of from about 0.001 to about 5% by weight and preferably about 0.01 to about 2%.
  • Suitable water soluble buffering agents that may be employed are alkali or alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate.
  • Additives such as carboxymethylcellulose may be used as a carrier in amounts of from about 0.01 to about 5% by weight.
  • the formulation will vary depending upon the purpose of the formulation, the particular mode employed for modulating the receptor activity, the intended treatment, and the like.
  • the formulation may involve patches, capsules, liposomes, time delayed coatings, pills, or may be formulated in pumps for continuous administration.
  • the specific dosage can be determined empirically in accordance with known ways. See, for example Harrison's, Principles of Internal Medicine, 11th ed. Braunwald et al. ed, McGraw Hill Book Co., New York, 1987.
  • a therapeutically effective dose of the subject oligopeptides will be in the range of about 0.005-10, more usually from about 0.01-1 mg/kg of host weight, and preferably from about 0.1 to about 1 mg/kg; for example, from about 0.3 to about 0.9 is preferred for the leptin receptor oligopeptide.
  • Such a dose will be sufficient to enhance the action of the therapeutic hormone, usually by at least as much as 50%.
  • Administration may be as often as daily; usually not more than one or more times daily, or as infrequent as weekly, depending upon the level of drug which is administered.
  • the oligopeptides may be administered alone, or in combination with the therapeutic hormone.
  • the hormone may be administered at a normally therapeutically effective dose, or the dose may be decreased by as much as 50%, usually by as much as 25%, to compensate for the oligopeptide enhancement.
  • the host may be any mammal including domestic animals, pets, laboratory animals and primates, particularly humans. The amount will generally be adjusted depending upon the half life of the peptide, where dosages in the lower portion of the range may be employed where the peptide has an enhanced half life or is provided as a depot, such as a slow release composition comprising particles, introduced in a matrix which maintains the peptide over an extended period of time, e.g., a collagen matrix, use of a pump which continuously infuses the peptide over an extended period of time over a substantially continuous rate, or the like.
  • the oligopeptides, modified receptors and cells containing the modified receptors are used in screening assays. Identification of the amino acid sequence in this region of receptors permits the design of drug screening assays for compounds that modulate receptor internalization or serve as ligand replacements for type 2 receptors.
  • Drug screening assays utilize the subject sequence information and peptide compositions, e.g., proteins, oligopeptides and synthetic derivatives thereof, to identify agents that modulate the internalization of cell surface receptors, or, in the case of type 2 receptors, allow the identification of agents that serve as ligand replacements.
  • Drug candidates capable of modulating surface receptor internalization are identified by first screening the drug candidates for the ability to compete with a bioactive oligopeptide for association with the intact receptor or that interfere with the binding of an oligopeptide to the subject receptor derived oligopeptides.
  • the methods comprise combining a cell surface receptor which contains an activation sequence and a candidate bioactive agent, and determining the binding of the candidate agent to the activation sequence.
  • cell surface receptor herein is meant any of number of cell surface receptors which are usually internalized upon ligand binding. Suitable cell surface receptors are as outlined above. Preferred embodiments utilize the human cell surface receptors, although other mammalian receptors may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep, pigs, horses, etc.) and primates. This latter embodiments may be preferred in the development of animal models of human disease.
  • cell surface receptors include amino acid substitions, insertions, or deletions of the naturally occuring sequence. Preferably, these do not alter the biological activity of the receptors, although as outlined herein, in some instances it may be desirable to modify the biological activity of the receptors.
  • cell surface receptors included within the definition of cell surface receptors; that is, either the full-length receptor may be used, or functional portions thereof.
  • the functional domain comprises at least a ligand binding domain and an activation sequence, such that the conformational change that occurs upon ligand binding to the receptor will still occur.
  • the function domain for a type 2 receptor may comprise only the activation sequence.
  • the cell surface receptor is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.).
  • the insoluble supports may be made of any composition to which peptide or receptor can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape.
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflonTM, etc.
  • Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
  • the particular manner of binding of the peptide or other protein is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the peptide and is nondiffusable.
  • Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the receptor is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the receptor on the surface, etc. Following binding of the peptide or receptor, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BS A), casein or other innocuous protein.
  • BS A bovine serum albumin
  • a ligand or analog bound by the cell surface receptor will also be added to the assay. That is, when insulin receptors are used, the ligand is insulin; when human growth hormone receptors are used, the ligand is human growth hormone; etc. As will be appreciated by those in the art, ligand analogs may also be used. In a preferred embodiment, this is not required, for example when assaying type-2 receptors.
  • Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
  • agent or "exogeneous compound” as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., with the capability of directly or indirectly altering cell surface receptor internalization and/or activation, which can be in response to ligand binding or in the absence of ligand binding.
  • assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs. The determination of the binding of the candidate bioactive agent to the receptor may be done in a number of ways.
  • the candidate bioactive agent is labelled, and binding determined directly. For example, this may be done by attaching all or a portion of the cell-surface receptor to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
  • a labelled candidate agent for example a fluorescent label
  • washing off excess reagent for example a fluorescent label
  • determining whether the label is present on the solid support Various blocking and washing steps may be utilized as is known in the art.
  • label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal.
  • the oligopeptides may be labeled at tyrosine positions using 125 I (for example, the activation sequences of the human GLUT4, insulin, IGF-1, G-CSF, IL-2 and hGH receptors all contain tyrosine residues), or with fluorophores.
  • 125 I for example, the activation sequences of the human GLUT4, insulin, IGF-1, G-CSF, IL-2 and hGH receptors all contain tyrosine residues
  • fluorophores for example, more than one component may be labeled with different labels; using 125 I for the oligopeptides, for example, and a fluorophor for the candidate agents.
  • the binding of the candidate bioactive agent is determined through the use of competitive binding assays.
  • the competitor is an oligopeptide as described herein.
  • the candidate bioactive agent is labeled.
  • Either the candidate bioactive agent, or the oligopeptide, is added first to the receptor for a time sufficient to allow binding, if present.
  • Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
  • the oligopeptide is added first, followed by the candidate bioactive agent.
  • Displacement of the oligopeptide is an indication that the candidate bioactive agent is binding to the activation sequence and thus is capable of modulating the internalization of the receptor.
  • either component can be labeled.
  • the oligopeptide is labeled, the presence of label in the wash solution indicates displacement by the agent.
  • the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
  • the candidate bioactive agent is added first, with incubation and washing, followed by the oligopeptide.
  • the absence of binding by the oligopeptide may indicate that the bioactive agent is bound to the receptor with a higher affinity.
  • the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of oligopeptide binding, may indicate that the candidate agent is capable of binding to the activation sequence and modulating the internalization of the receptor.
  • the methods comprise combining a cell surface receptor, a ligand bound by the receptor (if required), and an oligopeptide as described herein, to form a test mixture.
  • the candidate bioactive agent is added to the test mixture, and the binding of the candidate bioactive agent to the activation sequence of the receptor is determined.
  • either or both of the oligopeptide or the candidate bioactive agent is labeled, with preferred embodiments utilizing labeled oligopeptides, such that displacement of the label indicates binding by the candidate bioactive agent.
  • the methods comprise differential screening to identity bioactive agents that are capable of modulating the internalization or activating receptors.
  • the methods comprise combining a cell surface receptor, a ligand (if required), and an oligopeptide in a first sample.
  • a second sample comprises a candidate bioactive agent, a cell surface receptor, a ligand (if required), and an oligopeptide.
  • the binding of the oligopeptide is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding the activation sequence and potentially modulating the internalization of the receptor. That is, if the binding of the oligopeptide is different in the second sample relative to the first sample, the agent is capable of binding the activation sequence.
  • a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native receptor, but cannot bind to modified receptors, for example those that have the activation sequences deleted.
  • the structure of the receptor sequence of interest may be modeled, and used in rational drug design to synthesize agents that interact with that site.
  • Drug candidates that affect receptor internalization or receptor activation are also identified by screening drugs for the ability to either enhance or reduce the effect of the subject receptor derived oligopeptides on the internalization or activation of a selected surface receptor.
  • Screening may be performed to find agents that interfere with the association of a bioactive MHC-derived oligopeptide with the subject oligopeptides, where the agents will be capable of modulating the internalization or activating the receptors from which the subject oligopeptides are derived. This is done using the methods described above, but replacing the oligopeptides with a sequence from a ⁇ l -domain of an MHC Class I antigen, such as SEQ ID NO:l.
  • the drug candidate and varying concentrations of the subject receptor-derived oligopeptides are added to each of the sample receiving areas containing support-bound peptide.
  • the oligopeptide added is of substantially the same amino acid sequence as the oligopeptide bound to the support and is labeled.
  • Positive controls for binding of active peptide and competitive binding of active peptide may include samples containing labeled active peptide alone and a mixture of labeled active peptide and unlabeled active peptide, respectively. Samples containing labeled active peptide and unlabeled inactive peptide that does not aggregate with the bound peptide may serve as a negative control for competitive binding with peptide.
  • control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the labeled active peptide to the support-bound peptide. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, labeled peptide determined. For example, where a radiolabel is employed in labeling the peptide, the samples may be counted in a scintillation counter to determine the amount of bound, labeled peptide.
  • test samples containing the drug candidate if the amount of labeled active peptide bound to the support-bound peptide or receptor is in the range of values of the positive control samples for competitive binding and is significantly less than the negative control samples for competitive binding, then the drug candidate in the test sample is able to successfully competitively bind the support-bound peptide.
  • Drug candidates capable of such competitive binding may mediate modulation of cell surface expression of a receptor.
  • reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used.
  • the mixture of components may be added in any order that provides for the requisite binding.
  • agents that activate type 2 receptors are done in a manner similar to those outlined above.
  • it may be desirable to prevent activation of the type 2 receptors using the same basic assays described herein, but looking for agents that interfere with activation and/or internalization, i.e. an antagonist.
  • agents which bind to the activation sequences but do not allow multimerization of the receptors could be antagonists.
  • antagonists may be linked together, for example via covalent bonds, to provide for a "dimer" of the antagonist that now may act as an activator, since the dimer will now bind at least two receptors. This can be done using linkers as will be appreciated by those in the art.
  • candidate bioactive agent as defined above are added to a type 2 cell surface receptor with an activation sequence.
  • the receptor may be on the surface of a cell, or may be done in vitro.
  • the system is then assayed to determine whether the candidate bioactive agent binds to the activation sequence and activates the receptor.
  • a preferred embodiment provides methods of screening comprising adding a candidate bioactive agent to a mammalian cell which has a type 2 cell surface receptor with an activation sequence. The system is then assayed to determine whether the candidate bioactive agent binds to the activation sequence and activates the receptor.
  • Binding to the site on the receptor corresponding to the subject oligopeptides is indicative of such activity, as is the ability to interfere with the binding of the subject oligopeptides to the cognate receptor.
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described.
  • the agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds.
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
  • oligopeptides may also serve as ligand replacements.
  • the oligopeptides of the invention may be used to elucidate the function of unknown receptors, so called "orphan receptors". That is, for receptors for which no known function is associated, it is possible to add oligopeptides to the receptors, either in vivo or in vitro, and look for effects, as will be appreciated by those in the art.
  • EXPERIMENTAL Example 1 In order to determine the region on the external domain of a cell surface receptor that is involved in receptor internalization, a sequence similarity comparison was performed. The comparisons were performed with the commercially available Wisconsin Package, version 8.0-openVMS, Genetics Computer Group. The complete receptor sequences were obtained from public databases, as previously described in the
  • the similarity is based on the evolutionary distance between amino acids, as measured by Dayhoff and normalized by Gribskov and Burgess (1986) Nucl. Acids Res. 14:6745-6763.
  • the "local homology” algorithm of Smith and Waterman (1981) Advances in Applied Mathematics 2:482-289 finds the best segments of similarity between the two sequences.
  • Insulin Receptor modification and expression The human insulin receptor gene, as described in the database references and in Ebina et al. (1985) Cell 40:747-758) with a pCR3 expression vector (Invitrogen, catalog no. K3000-01) was transfected by electroporation into HeLa cells. Methods of electroporation are described in Boggs et al. (1986) Ex. Hematol. 149:988-994. In the transfected cells the receptors show insulin dependent internalization.
  • a mutated form of the insulin receptor was created by deleting residues 713 to 740 (SEQ ID NO:36; PKTDSQILKELEESSFRKTFEDYLHNV) using amplification primers that spanned the region to be deleted.
  • the deletion mutant, mIR was transfected into HeLa cells and internalization of the mIR was then tested.
  • Receptor internalization was performed essentially as described in Stagsted et al. (1990) CeU 62:297-307. Briefly, 50 ⁇ l of the transfected cells at 106 cells/ml were incubated in a shaking water bath at 37°C with 625 pM 1251 -labeled insulin in the absence or presence of 10 ⁇ M of peptide as shown in Table 1, and the final volume brought to 1OO ⁇ l. The cells were then diluted with 50 ⁇ l of KRHB ( ⁇ H7.2) (no acid wash) or 50 ⁇ l of KRHB (pH 2.0) (acid wash) and incubated on ice for 5 min. The cells were finally harvested by centrifugation on top of silicone oil, and both free and cell-associated radioactivity was measured.
  • Glucose Transport in Adipose Cells The biological activity of the peptides were measured by their effect on glucose uptake in rat adipose cells as described (Stagsted et al. (1991) J. Biol. Chem. 266:12844-12847). Briefly, rat adipose cells were obtained from epididymal fat pads and suspended in Krebs-Ringer HEPES buffer (KRH) with 5% bovine serum albumin at a lipocrit of 10% (final). The peptide effect was measured in cells maximally stimulated with insulin (10 nM). After equilibration at 37°C for 30 min the cells were incubated for 30 min at 37°C with buffer (basal), 10 nM insulin plus peptide.
  • KRH Krebs-Ringer HEPES buffer
  • bovine serum albumin bovine serum albumin
  • the peptides were assembled stepwise either on a phenylacetamidomethyl (PAM) resin using the t-Boc NMP/HOBt protocol of an Applied Biosystems Model 430A peptide synthesizer, or on a p-alkoxy benzyl alcohol (Wang) resin using a modified Fmoc/BOP protocol of a Milligen Biosearch Model 9600 synthesizer.
  • PAM phenylacetamidomethyl
  • Wang p-alkoxy benzyl alcohol
  • the desired peptides were confirmed by sequence analysis, amino acid composition, and fast atom bombardment mass spectrometry.
  • the peptides were activated by incubation of 1 mM stock solution at 37°C in 0.1 M NaCI overnight (Stagsted et al. (1991) J. Biol. Chem. 266:12844-12847).
  • GNEQSFRVDLRTLLRYAGGGNEQSFRVDLRTLLRYA (DS-A85). The data are shown in Table 2, where the numbers indicate percent internalized receptor.
  • the data show that the mutated insulin receptor mIR does not internalize upon insulin binding, whereas more than 50% of the wild type IR is internalized within 30 minutes.
  • the pepIR peptide inhibits receptor internalization to the same extent as DS-A85.
  • the GLUT4pep (SEQ ID NO:2), at a concentration of 10 ⁇ M, does not affect insulin receptor internalization by the transfected cells. In the presence of peptide the per cent internalized receptor is 6 ⁇ 9, in the absence of peptide it is 64 ⁇ 7. The peptide does inhibit the internalization of GLUT4, as shown by the effect on glucose uptake at maximal insulin stimulation. In the presence of 10 nM insulin, the enhancement of glucose uptake was 12 ⁇ 4 fold. The enhancement was increased to 24 ⁇ 2 fold with the addition of the GLUT4pep. The peptide therefore seems to inhibit internalization of GLUT4, but not insulin receptor.
  • oligopeptides having the sequence of the extracellular domain of a cell surface receptor, and having sequence identity with a region of an MHC class I antigen are effective in inhibiting the internalization of the corresponding receptor.
  • the peptides are therapeutically useful in enhancing the cellular response to hormones such as insulin.
  • Appropriate cell lines have the following attributes: they are responsive to the appropriate hormone, they expresses the cognate receptor, they possess molecules required for down stream signaling of hormone induced response.
  • the growth medium is as recommended for cell line.
  • Antibodies Immunoprecipitation: 2-5 ⁇ g of Anti PY-99 (Santa Cruz Biotechnology), Anti-Receptor or signaling molecule specific antibody (e.g., Jak, STAT); Western blot: Anti Receptor specific antibody, Signaling protein specific antibody (Santa Cruz
  • Immunoprecipitation Protein G beads (Pharmacia); 20-40 ⁇ l of slurry.
  • Protease inhibitor mix (lOOx) in water 1 mg/ml Aprotinin, 0.1 mg/ml Pepsatin A, 0.1 mg/ml Leupeptin, 0.1 mg/ml Chymostatin, 0.1 mg/ml AEBSF.
  • Cell wash buffer PBS with 1 mM ortho-vanadate, 50 mM NaF, 20 mM ⁇ - glycerophosphate and 2 mM sodium phyrophosphate.
  • Lysis buffer 50 mM HEPES, 150 mM NaCl, 1% Triton X-l 00, IX protease inhibitor mix, 50 mM NaF, 5 mM EDTA, and 2 mM sodium phyrophosphate.
  • Blocking buffer Blotto: TST (0.01 M Tris pH 7.4, 0.15 M NaCl, 0.075% Tween 20,
  • the cells were grown cells to an approximate density of 1 x 10 6 cells, centrifuged down and resuspended in recommended media for growth.
  • the cells were starved for 14 - 18 hours at 37°C (5% CO 2 ) in a media without serum, spun down and resuspended cells at a density of 1 x 10 6 cells/ml in a media without serum.
  • 10 ml of cell suspension per PI 00 tissue culture dish was used.
  • the cell suspension was treated with 0.01 - 30 ⁇ M peptide, 15 - 30 min at 37°C (5% CO 2 ); (thaw peptide very shortly before the assay).
  • 10 ml of ice cold cell wash buffer was added per dish, transfered to a Falcon tube and centrifuged down quickly at 4°C and 3000 rpm. The media was carefully aspirated and the wash was repeated at 4°C.
  • Immunoprecipitation The protein G beads were washed with 0.5 x lysis buffer in Eppendorf tubes, 1 - 4 ⁇ g of Anti-PY-99 antibody was added, with receptor specific or signaling molecule (JAK2, STAT5) specific antibody per IP. The tubes were incubated with end-over-end rotation for 2 h at RT. The lysate was added, the tubes incubated with end-over-end rotation over night at 4°C. The beads were washed 3 times with 1 x lysis buffer, 40 ⁇ l of SDS-sample buffer was added, and the samples were boiled for 3 min.
  • Protocol for EPO-R activation (phosphorylation) TF-1 cells from ATCC were used, with the growth medium being RPMI 1640 with 1 x Penicillin/Streptomycin, 2 mM glutamin, 10% fetal bovine serum (Hyclone), and 1 ng/ml GM-CSF.
  • Cells were grown to an approximate density of 1 x 10 6 cells, centrifuged down and resuspended in media with 5% serum and no GM-CSF. The cells were starved for 14 - 18 hours at 37°C (5% CO 2 ), spun down and resuspended cells at a density of 1 x 10 6 cells/ml in a media without serum and GM-CSF. 10 ml of cell suspension per PI 00 tissue culture dish was used. The cell suspension was treated with 1 - 30 ⁇ M peptide, 15 - 30 min at 37°C (5% CO 2 ); (thaw peptide very shortly before the assay).

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Abstract

La présente invention concerne des oligopeptides dont la séquence d'acides aminés correspond à un domaine extracellulaire de récepteur et qui présentent une analogie de séquence avec des peptides régulateurs d'antigènes MHC de classe I. Ces peptides favorisent ou remplacent la réponse physiologique d'un ligand se liant avec le récepteur correspondant. Ces oligopeptides sont utilisés dans le diagnostic et le traitement de maladies en rapport avec une réponse insuffisante ou inappropriée des récepteurs, ainsi que pour le criblage de médicaments candidats qui ont une incidence sur l'expression de surface des récepteurs. Une molécule réceptrice modifiée, dont la séquence correspondant au peptide régulateur a été modifiée ou supprimée, convient également pour le criblage de médicaments.
EP99908383A 1998-02-24 1999-02-23 Peptides derives de recepteurs agissant en tant que modulateurs de l'activite de ces recepteurs Withdrawn EP1064015A2 (fr)

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EP2316922B1 (fr) 2002-05-24 2013-05-22 Merck Sharp & Dohme Corp. Anticorps anti-IGFR humain neutralisant
US7396913B2 (en) 2002-10-14 2008-07-08 Abbott Laboratories Erythropoietin receptor binding antibodies
EP1694850B1 (fr) 2003-11-12 2011-06-29 Schering Corporation Systeme de plasmide pour l'expression multigenique
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AU2010239125B2 (en) * 2009-04-22 2015-05-14 The Walter And Eliza Hall Institute Of Medical Research Structure of the C-terminal region of the insulin receptor alpha-chain and of the insulin-like growth factor receptor alpha-chain
CN110251668A (zh) 2010-11-30 2019-09-20 霍夫曼-拉罗奇有限公司 低亲和力血脑屏障受体抗体及其用途
CN111732635B (zh) * 2020-06-22 2022-03-22 中国医学科学院基础医学研究所 与cd123蛋白特异性结合的多肽、多肽复合物、共递送系统及其制备方法与应用

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US5753625A (en) * 1995-05-12 1998-05-19 Sangstat Medical Corporation Treatment for inhibiting the progression of autoimmune disease
US6346390B1 (en) * 1996-03-08 2002-02-12 Receptron, Inc. Receptor derived peptides involved in modulation of response to ligand binding

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