Classifications

• • 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
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • 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
• • 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
  • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
• • 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
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
• • 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
• • 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
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to the field of integral membrane proteins which act as receptors or signal transducers. More specifically, it relates to the identification and preparation of specific antagonists of the function of such proteins.
• Receptors are the primary targets and mediators of hormone and drug actions.
• the cell surface receptors such as the G protein-coupled receptors (GPCRs), ion channel receptors, immunoglobulin receptors and tyrosine kinase receptors, belong to gene superfamilies based on sequence and structural similarities. Receptors belonging to these superfamilies are all integral membrane proteins predicted to exhibit extracellular, hydrophobic membrane- spanning and intracellular domains. Whereas tyrosine kinase receptors and most immunoglobulin receptors exhibit a single membrane spanning domain, G protein- coupled receptors are defined by seven putative
• the biogenic amine transporter proteins are also membrane-spanning proteins, with twelve transmembrane segments, that mediate the reuptake of released neurotransmitter.
• the ion channel receptors generally have separate subunits that associate together to form a functional receptor.
• the amino acid sequence of a receptor protein which is unique to each receptor, confers specific structure- related functions to the receptor, while conforming to the general structural determinants of the particular class of protein to which it belongs.
• TM transmembrane
• Figure 1 shows a two dimensional representation of the seven membrane-spanning domains of the D2 dopamine receptor.
• Figure 2 shows a two dimensional representation of the single membrane-spanning domain of the epidermal growth factor receptor.
• Figures 3A to 3G show immunoblots of isolated dopamine receptors after various treatments, monomers (M) and dimers (D) being indicated by arrows.
• Figure 3A shows the effect of the indicated
• D2-TM VII peptide aa407-4266
• D2 receptor aa407-4266
• concentrations of D2-TM VII peptide (aa407-426) on the D2 receptor (Lanes 1-7: 0, 0.3, 0.6, 1.0, 1.3, 1.6 and 2.5 mg/ml D2-TM VII peptide).
• Figure 3C shows the effect of incubation with the D2-TM VI and D2-TM VII peptides on the D2 receptor from human caudate nucleus.
• Lanes 1 and 3 buffer control; lane 2: D2-TM VI peptide; lane 4: D2-TM VII peptide.
• Figure 3D shows the effect of hydrophilic and hydrophobic receptor peptides on a D2 receptor
• D2 receptors were incubated m peptide buffer (lane 1); D2-C IIIA peptide (aa 244-263) (lane 2);
• D2-C IIIB peptide (aa 284-303) (lane 3); ⁇ 2-AR TM VI peptide (aa 276-296) (lane 4); D1-C IIIA peptide (aa 369- 383) (lane 5); and D1-C IIIB peptide (aa 416-431) (lane 6).
• Figure 3E shows the effect of D2-TM VII peptide on c-myc epitope-tagged human dopamine Dl receptor and c-myc epitope-tagged human serotonin 5-HT1B receptor.
• Dl receptors were incubated m peptide buffer without (lane 1) or with (lane 2) D2-TM VII peptide, and 5-HT1B
• Figure 3F shows the effect of temperature on the D2 dopamine receptor.
• the peptides tested were: D2-TM 7 (TWLGYVNSA) ( ⁇ ), D2-TM 5 (PAFWYSSIVSFYVPFIVTL)
• Figure 5 shows the dose-dependent competition displacement of agonist [ 3 H] qumpirole binding (shown as percentage of total [ 3 H] qumpirole binding) to membranes prepared from D2 receptor-expressing Sf9 cells by D2 receptor antagonist spiperone (o) and D2-TM 7 peptide,
• Figure 6 shows D2 receptor mediated dose-dependent dopamine activation of [ 35 S]GTP ⁇ S binding (left panel), and the dose-dependent inhibition of dopamine activation of [ 3b S]GTP ⁇ S binding by the D2 antagonists, spiperone (shaded bar) and D2-TM 7 peptide (TWLGYVNSA) (open bar) (right panel).
• D2 receptor mediated dopamine activation of [ 35 S]GTP ⁇ S binding (left panel) is shown as a percent of baseline activity, and the inhibition of dopamine activation of [ 35 S]GTP ⁇ S binding by the D2 antagonists, spiperone and D2-TM 7 peptide (TWLGYVNSA) (right panel) is shown as a percentage of the maximal response.
• Figure 7 shows D2 receptor mediated dose-dependent dopamine activation of [ 35 S]GTP ⁇ S binding (left panel), and the dose-dependent inhibition of dopamine activation of [ 35 S]GTP ⁇ S binding by the D2-TM 7 peptide (TWLGYVNSA) (open bar) and the GABA-TM peptide (GIFNLVYW) (solid bar) (right panel).
• D2 receptor mediated dopamine activation of [ 35 S]GTP ⁇ S binding (left panel) is shown as a percent of baseline activity, and the inhibition of dopamine activation of [ 35 S]GTP ⁇ S binding by the D2-TM 7 peptide and GABA-TM peptide (right panel) is shown as a
• Figure 8 shows the effect of co-expression of a D2- TM7 peptide and the full length D2 receptor on D2 receptor density in COS cells, estimated by spiperone binding. Co-expression of a D2-TM7 peptide and the D2 receptor (o) was compared with expression of the full length D2 receptor alone (•). A representative of two independent experiments is shown.
• Figure 9 shows the effect of coexpression of a D2- TM7 peptide with full length D2 receptors on D2 receptor function m COS cells (shaded bar) compared with
• Figures 10A to 10C show the duration (X axis) and extent (Y axis) of asymmetric body response of a rat after unilateral (left) intrastriatal injection of D2-TM
• Figure 11 shows the duration (X axis) and extent (Y axis) of asymmetric body response of a rat with bilateral cannulae after left intrastriatal injection of D2-TM VII peptide (TWLGYVNSA), 15 ng/3 ⁇ l (together with vehicle injection into the right striatum concurrently).
• Figure 12 shows immunoblots of an Ni-NTA resm- purified preparation from Sf9 cells co-expressing a c-myc epitope-tagged, ⁇ 2-adrenergic receptor (-c-myc- ⁇ 2AR+) and a histidine-tagged TMVII peptide of the ⁇ 2-adrenergic receptor ( 6xHis- ⁇ 2AR-TMVII) probed (left panel) with monoclonal antibody 9E10 against the c-myc epitope and (right panel) with a polyclonal antibody against the poly-histidine sequence of the TMVII ⁇ 2-adrenergic receptor peptide. Left lane of each panel shows
• Figure 13A shows the dose-dependent inhibition of [ 3 H] alprenolol binding to membranes prepared from ⁇ 2- adrenergic receptor-expressing Sf9 cells by various receptor subtype-specific peptides.
• the peptides tested were: ⁇ 2AR-TM I (LLIWGNVLVI) , ⁇ 1AR-TMVII (GYANSAFNP), CCR5-TMI (LYSLVFIFGFVGN), Fo c-TM ( E. Coll ATPase Fo c subunit: GQAIAFVLFVL) and Fo b-TM ( E. Coli ATPase Fo b subunit: LAAIGAAIGIGILG).
• Figure 13B shows the effect of the prototypical adrenergic receptor antagonist pindolol for comparison. Inhibition is represented as the
• Figures 14A to 14H show polygraph traces of blood pressure (Y axis: mm Hg) in a rat at indicated time intervals (X axis) after treatment with various agents:
• Figure 14B 1 mg/Kg isoproterenol ( ⁇ 1-AR agonist);
• Figure 14C 500 mg LYSAFTWLGYUNSAVNPIIY-NH2 ( ⁇ 1-AR TMVII peptide);
• Figure 14D 1 mg/Kg isoproterenol;
• Figure14F 1mg/Kgisoproterenol
• Figure 14G vehicle
• Figure 14H 1 mg/Kg isoproterenol.
• Figures 15A to 15K show polygraph traces of blood pressure (Y axis: mm Hg) in a rat at indicated time intervals (X axis) after treatment with various agents:
• Figure 15B 5 mg/Kg phenylephrine ( ⁇ 1A-AR agonist);
• Figure 15C 500 mg FFNWLGYANSAFNP-NH2 ( ⁇ 1A-AR TM7 peptide);
• Figure 15D 5 mg/Kg phenylephrine;
• Figure 15E saline;
• Figure 15F 5 mg/Kg phenylephrine;
• Figure 15G vehicle;
• Figure 15H baseline;
• Figure 151 5 mg/Kg phenylephrine;
• Figure 15J 1 mg/Kg prasozin ( ⁇ 1A-AR antagonist);
• Figure 15K 5 mg/Kg phenylephrine.
• Figure 16 shows urine output (Y axis) as a function of time (X axis) in a representative unilaterally nephrectomized rat under anesthesia after treatment with vehicle (10% DMSO) in water) or V2 receptor TM VII antagonist peptide, LMLLASLNSCTNPWIY, (V2 AT).
• FIGs 17A and 17B show the effect of a CCR5-TM I peptide antagonist, (CCR5-TMI: LYSLVFIFGFVGN) on HIV infection of PBMC cells as assessed by HIV reverse transcriptase activity ( Figure 17A) and by HIV P24 antigen production ( Figure 17B).
• CCR5-TMI CCR5-TMI peptide antagonist
• Figure 18 shows dose-dependent inhibition of EGF receptor tyrosine kinase activity m solubilized membrane preparations from cultured A431 cells by various
• LTVIAGLWIF a peptide derived from the TM domain of the EGF-erb3 receptor (EGF-tm:
• Figure 19 shows the antimicrobial effect of various transmembrane-based peptides on E. Coli as assessed by plating and counting growth of colonies on Luria Broth plates.
• Peptides examined were GIFNLVYW (GABA-TM),
• LAAIGAAIGIGILG Fo c-TM
• GQAIAFVLFVL Fo b-TM
• Figures 20 to 22 show the effect of intra-cerebral (IC) injection of the peptide ALGWIIATS on dopamine release (X axis) at various time intervals (Y axis) in caudate nucleus and nucleus accumbens measured by in vi vo microdialysis in an awake rat.
• IC intra-cerebral
• X axis dopamine release
• Y axis time intervals
• Figure 20 shows the release of dopamine in striatum as a function of time following cocaine administration (5 mg/Kg) with no pre-treatment (•) or with pre-treatment of rats with IC injection of DAT-TM 12 peptide (ALGWIIATS) 15 min before cocaine administration (o). Dopamine release is shown as a percentage of basal values.
• Figure 21 shows the effect of IC injection of the DAT-TM 12 peptide, ALGWIIATS, alone. Response is shown as percentage basal striatal release as a function of time.
• Figure 22 shows the release of dopamine in the nucleus accumbens as a function of time following cocaine administration (5 mg/Kg) with no pre-treatment (•) or with pre-treatment of rats with IC injection of DAT-TM 12 peptide, ALGWIIATS, 5 min before cocaine administration (o). Dopamine release is reported as a percentage of basal values.
• Figure 23 shows the effect of a CD4-TM peptide antagonist, LIVLGGVAGLLLF, on HIV infection of PBMC cells as assessed by HIV P24-ant ⁇ gen production.
• a peptide which has the amino acid sequence of a hydrophobic or transmembrane (TM) domain of an integral membrane protein, or of a portion of a transmembrane domain has a specific and selective antagonistic effect on the activity or function of the integral membrane protein from which it is
• the mechanisms and antagonist peptides of the invention may be utilised m advance of a complete understanding of their mechanism of action, it is hypothesised that the mechanism of the antagonism exerted by transmembrane domain peptides is the binding of such peptides to a transmembrane domain of the integral membrane protein, thereby interfering with intramolecular interactions which contribute to the proper three- dimensional conformation of the integral membrane protein monomer. It is hypothesised that the formation of a heterodimer of the antagonist peptide and the integral membrane protein monomer will interfere with binding of the integral membrane protein with its ligand and, for integral membrane proteins which are normally associated as dimers, will interfere with dimer formation.
• integral membrane protein shows specificity for that protein and does not interfere with the function of closely related integral membrane proteins.
• Integral membrane proteins comprise a great variety of proteins, including signal-transducing proteins such as G-protein coupled receptors and tyrosine kinase receptors, transporter proteins, membrane channel
• proteins proteins, lmmunoglobuin receptors and adhesins.
• Integral membrane proteins are found in the cell membranes of prokaryotic and eukaryotic cells and also within intracellular membranes m eukaryotic cells, for example the endoplasmic reticulum intracellular
• Antagonist peptides in accordance with the present invention can be used to control the function of integral membrane proteins found in all these various locations.
• fragments or analogues of the transmembrane amino acid sequences of an integral membrane protein which are effective to antagonise the function of that protein.
• a fragment or analogue of a transmembrane amino acid sequence of an integral membrane protein is effective if it is a functional equivalent of the transmembrane amino acid sequence.
• transmembrane or membrane-spanning domains of integral membrane proteins are believed to have a helical conformation and generally comprise a sequence of about 22 to 26 amino acids.
• integral membrane proteins are believed to have a helical conformation and generally comprise a sequence of about 22 to 26 amino acids.
• transmembrane domains adopt a barrel conformation.
• the antagonist peptide for a particular integral membrane protein may have the entire amino acid sequence of a transmembrane domain or may comprise a portion or fragment of the transmembrane amino acid sequence.
• Fragments of a transmembrane amino acid sequence may be selected by truncation of one or more amino acids from the amino terminus of the transmembrane amino acid sequence, by truncation of one or more amino acids from the carboxy terminus or by truncation of one or more amino acids from both amino and carboxy termini.
• transmembrane amino acid sequence within the total amino acid sequence of an integral membrane protein, there may be a variation of one or two amino acids in defining the termini of the transmembrane amino acid sequence, depending on the hydropathy analysis software used.
• the present invention provides antagonist peptides which correspond to the amino acid sequence of an
• integral membrane protein transmembrane domain fragments of such a TM amino acid sequence and peptides which include the amino acid sequence of an integral membrane protein transmembrane domain or fragments thereof.
• the present invention provides antagonist peptides comprising amino acid sequences corresponding to at least four, preferably ten and more preferably from fifteen to twenty consecutive amino acids of an integral membrane protein transmembrane domain.
• amino acid sequences of the transmembrane domains of integral membrane proteins are highly conserved in mammals.
• the function of an integral membrane protein from a first species may be antagonised by a peptide
• transmembrane domain amino acid sequence corresponding to the amino acid sequence of one of its own transmembrane domains or may be antagonised by a functionally equivalent transmembrane domain amino acid sequence from the corresponding region of the integral membrane protein of a second species.
• “functionally equivalent” means that the sequence of the transmembrane domain of the second species need not be identical to that of the first species but need only comprise a sequence which functions biologically and/or chemically as the equivalent of the transmembrane amino acid sequence of the first species.
• the present invention provides a generally
• a target integral membrane protein such as a receptor or transporter.
• antagonist peptides In addition to the antagonist peptides disclosed herein, one of ordinary skill in the art is enabled by the present invention to identify and prepare antagonist peptides specific for any selected integral membrane protein.
• Tables 1A to 1D show examples of the G-protein coupled receptors whose amino acid sequences can be accessed in public databases and Table 3 shows examples of other sequences available in databases.
• transmembrane amino acid sequence or sequences of a selected integral membrane protein identify a
• the present invention also enables the rational design of specific antagonist peptides or blockers active against the protein product of any gene predicted to encode an integral membrane protein.
• amino acid sequence of a new integral membrane protein is determined, for example by cloning and sequencing a gene or cDNA for the protein and
• the amino acid sequence can be subjected to hydropathic analysis, as described above, to identify the TM domains.
• the amino acid sequence of at least one transmembrane domain is then synthesised to provide a selective peptide
• suitable effective fragments or analogues of a transmembrane amino acid sequence may be selected and screened as described herein.
• the present invention enables novel specific pharmaceuticals for treatment of many disorders.
• receptor and transporter antagonists may be used to treat disorders associated with specific receptor overactivity such as schizophrenia which is associated with overactivity of the D2 dopamine receptor, or may be used to indirectly restore homeostasis m disorders which do not directly involve aberrant function of the particular receptor or transporter.
• disorders and antagonists in the latter category include: anti-Dl dopamine receptor for drug abuse, anti-histamine receptor for peptic ulcer disease, anti-angiotensm receptor for hypertension and anti- ⁇ adrenergic receptor for glaucoma.
• the inventors Using the D2 dopamine receptor as a model for other membrane spanning receptors, the inventors have shown a dopamine antagonist effect in vivo, in a rat model of rotational locomotion, by administering directly into the caudate nucleus of the brain a peptide comprising a fragment of one of the transmembrane amino acid sequences of the D2 dopamine receptor. Most importantly, the inventors have demonstrated specificity, with no
• a peptide comprising a portion of a transmembrane domain of the ⁇ 1-adrenergic receptor inhibited the function of that receptor
• a peptide comprising a portion of a transmembrane domain of the ⁇ 1A-adrenergic receptor inhibited the function of that receptor, as evidenced by the effect of these peptides on cardiac function and blood pressure.
• the models described herein are not, however, limited to GPCRS.
• the inventors have shown, for example, that specific antagonists can be prepared, in accordance with the invention, for tyrosine kinase receptors and immune receptors.
• Antagonist peptides in accordance with the invention have also been demonstrated to interfere with the
• Antagonist peptides m accordance with the invention may be prepared by any suitable peptide synthetic method.
• Chemical synthesis may be employed, for example standard solid phase peptide synthetic techniques may be used. In standard solid phase peptide synthesis,
• the solid support is generally based on a polystyrene resin, the resm acting both as a support for the growing peptide chain, and as a protective group for the carboxy terminus. Cleavage from the resin yields the free carboxylic acid.
• Peptides are purified by HPLC techniques, for example on a preparative C18 reverse phase column, using acetonitrile gradients in 0.1% trifluoroacetic acid, followed by vacuum drying.
• Antagonist peptides may also be produced by
• a DNA sequence encoding the desired peptide is prepared, for example by cloning the required fragment from the DNA sequence encoding the complete receptor, obtainable from genomic DNA or from commercially available genomic or cDNA libraries, and subcloning into an expression plasmid DNA.
• Suitable mammalian expression plasmids include pRC/CMV from
• the gene construct is expressed in a suitable cell line, such as a Cos or CHO cell line and the expressed peptide is extracted and purified by conventional methods. Suitable methods for recombinant synthesis of peptides are described in "Molecular
• Analogues of a transmembrane amino acid sequence of an integral membrane protein may be prepared by similar synthetic methods.
• the term "analogue” extends to any functional and/or chemical equivalent of a transmembrane amino acid sequence and includes peptides having one or more conservative amino acid substitutions, peptides incorporating unnatural amino acids and peptides having modified side chains.
• side chain modifications contemplated by the present invention include modification of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidation with methylacetimidate; acetylation with acetic
• anhydride carbamylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS) ; alkylation of amino groups with succinic anhydride and tetrahydrophthalic annydride; and pyridoxylation of lysine with pyridoxal- 5'-phosphate followed by reduction with NaBH 4 .
• TNBS trinitrobenzene sulfonic acid
• guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2, 3-butanedione,
• the carboxyl group may be modified by carbodiimide activation via -acylisourea formation followed by
• Sulfhydryl groups may be modified by methods such as carboxymethylation with lodoacetic acid or lodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercur ⁇ phenylsulfonic acid, phenylmercury chloride, 2-chloromercuric-4- nitrophenol and other mercurials; carbamylation with cyanate at alkaline pH.
• Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides.
• Tyrosine residues may be altered by nitration with tetranitromethane to form a 3- nitrotyrosine derivative.
• Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with lodacetic acid derivatives of N-carbethoxylation with
• Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid-, t-butylglycine, norvaline, phenylglycine,
• ornithine sarcosine
• 4-amino-3-hydroxy-6-methylheptanoic acid 2-thienyl alanine and/or D-isomers or amino acids.
• Group 1 F Y W
• Group 2 V L I
• Group 3 H K R
• Group 4 M S T P A G
• Group 5
• Fragments or analogues of the antagonist peptides of the invention may be conveniently screened for their effectiveness as receptor antagonists, for example by examining their ability to inhibit ligand-binding by the relevant receptor which has been pre-incubated with the peptide.
• Ligand-binding inhibition can be determined, for example, by a soluble receptor radioligand binding assay, as described herein.
• the antagonist peptides may also be screened for their effectiveness as receptor antagonists by examining their ability to impair receptor coupling to second messenger systems or their ability to impair some functional activity. For example, for a GPCR such as the D2 dopamine receptor, the ability of the antagonist to block D2 receptor mediated attenuation of adenylyl cyclase activity provides a convenient index of efficacy as described herein.
• tyrosine kinase receptor such as the EGF receptor
• the ability of antagonists to inhibit EGF receptor tyrosine phosphorylation, as described herein, provides an index of efficacy.
• peptide antagonists of the invention may be administered therapeutically by injection or by oral, nasal, buccal, rectal, vaginal, transdermal or ocular routes in a variety of formulations, as is known to those in the art.
• Stability can be improved if synthetic amino acids are used, such as peptoids or betidamino acids, or if
• Formulation may be, for example, in water/oil emulsion or in liposomes for improved stability.
• Oral administration of peptides may be accompanied by protease inhibitors such as aprotinin, soybean trypsin inhibitor or FK-448, to provide protection for the peptide.
• the nasal cavity provides a good site for absorption of both lipophilic and hydrophilic drugs, especially when coadministered with absorption enhancers.
• the nasal absorption of peptide-based drugs can be improved by using aminoboronic acid derivatives,
• the transdermal route provides good control of delivery and maintenance of the therapeutic level of drug over a prolonged period of time.
• a means of increasing skin permeability is desirable, to provide for systemic access of peptides.
• iontophoresis can be used as an active driving force for charged peptides or chemical enhancers such as the nonionic surfactant n- decylmethyl sulfoxide (NDMS) can be used.
• NDMS nonionic surfactant
• Peptides may also be conjugated with water soluble polymers such as polyethylene glycol, dextran or albumin or incorporated into drug delivery systems such as polymeric matrices to increase plasma half-life.
• water soluble polymers such as polyethylene glycol, dextran or albumin
• drug delivery systems such as polymeric matrices to increase plasma half-life.
• the peptide antagonists of the invention also provide a tool for the elucidation of the function of many important orphan receptors whose structures and locations are known but for which the endogenous ligand is unknown. Disruption of the function of an orphan receptor by a transmembrane peptide antagonist and observation of the resulting loss or disruption of function will assist in elucidating the role of the orphan receptor.
• the present invention also provides new methods of tissue imaging.
• An antagonist peptide derived from the transmembrane amino acid sequence of a membrane-spanning protein may be labelled with a suitable signalling moiety, such as an imaging radionuclide, and administered m vi vo .
• the labelled peptide binds stably to the receptor permitting visualisation and quantification of the receptor.
• Suitable radionuclides include
• Peptides may be labelled by conventional methods known to those skilled in the art.
• the specificity of the antagonist peptides of the invention for the receptor will provide improved accuracy and precision in the determination of receptor
• the invention provides new methods for gene therapy utilising a genetically
• transfection vector for introduction of the coding sequence into a selected cell or tissue, either ex vivo or in vi vo, in order to provide for in vi vo production of a selected integral membrane protein antagonist.
• adenovirus and vaccinia virus are employed as vectors for gene therapy. Gene therapy techniques are reviewed, for example, in (Hanania, E.G. (1995), Am. J. of Med. , v. 99, pp. 537-552).
• a recombinant nucleotide sequence encoding an antagonist peptide of the invention may be incorporated into a transfection vector under the control of a tissue-specific promoter which ensures expression of the nucleotide sequence only in the
• a viral vector may be employed incorporating a promoter which directs expression only in brain cells which have dopamine D2 receptors and a nucleotide sequence encoding an
• the viral preparation can be introduced directly into the brain, for example by mtra-cerebroventricular injection or infusion, where the virus is taken up by brain cells, but the peptide is produced only where required.
• Non-viral gene therapy methods are described, for example, in EP 289034.
• the invention provides transgenic animal models expressing transmembrane peptide antagonists which modulate endogenous integral membrane protein function. These animal models will provide a tool for testing the design, efficacy and toxicology of integral membrane protein antagonist peptides and will also provide models that mimic clinical diseases.
• Transgenic animal models in accordance with the invention can be created by introducing a DNA sequence encoding a selected peptide antagonist either into embryonic stem (ES) cells of a suitable animal, for example a mouse, by transfection or microinjection, or into a germ line or stem cell by a standard technique of oocyte microinjection.
• ES embryonic stem
• the ES cells are inserted into a young embryo and this embryo or an injected oocyte are implanted into a pseudo-pregnant foster mother to grow to term.
• the G-protein coupled receptors have a common pattern of seven hydrophobic membrane-spanning domains. These receptors are involved m a wide variety of pathways. Table 1 lists the various receptors which belong to this superfamily.
• dopamine receptor antagonist peptides and a method for regulating or inhibiting the activity of a selected dopamine receptor are provided.
• D1 to D5 Seeman, P. (1995). All belong to the family of G protein-coupled receptors (GPCRs) that have seven highly conserved membrane spanning regions which are linked by GPCRs.
• receptor and of peptide antagonists of that receptor provides a general illustration of the selection of a specific antagonist peptide to an integral membrane protein, in accordance with the invention. The same method may be applied by one of ordinary skill in the art to select an antagonist to any selected integral membrane protein.
• the dopamine D2 receptor is activated by the
• neurotransmitter dopamine, leading to the inhibition of intracellular adenylate cyclase.
• the D2 receptor gene encodes a long and a short form of the receptor, differing by a 29 amino acid segment in the third intracellular loop.
• the long and short forms have identical transmembrane domains.
• Figure 1 shows a two-dimensional representation of the D2 dopamine receptor spanning the cell membrane. The seven transmembrane domains and two cytoplasmic domains are identified, the transmembrane amino acid sequences being boxed.
• Table 2 shows the information available in the
• the transmembrane (TM) domains are identified by amino acid number, as follows:
• transmembrane domains can be determined from the complete amino acid sequence provided. Any one of these transmembrane amino acid sequences may be selected for use as a specific antagonist of the D2 dopamine receptor.
• a D2 dopamine receptor antagonist peptide may, therefore, be selected from the following transmembrane amino acid sequences:
• TM I ATLLTLLIAVIVFGNVLVCMAVS (Sequence ID NO: 1) TM II LIVSLAVADLLVATLVMPWWYLEW (Sequence ID NO: 2) TM III IVFTLDVMMCTASILNLCAISI (Sequence ID NO: 3) TM IV VTVMISIVWVLSFTISCPLLFGL (Sequence ID NO: 4) TM V PAFWYSSIVSFYVPFIVTLLVYI (Sequence ID NO: 5) TM VI MLAIVLGVFIICWLPFFITHILN (Sequence ID NO: 6) TM VII VLYSAFTWLGYVNSAVNPIIYTTF (Sequence ID NO: 7) or may be an effective fragment or analogue of any of these sequences.
• D2 receptors are found in brain, where the highest densities have been found in the striatum (Caudate- putamen, nucleus accumbens), olfactory tubercle, and substantia nigra and pituitary, whereas lower densities are present in the cortex, hippocampus and limbic brain regions (Bouthenet et al., 1987, 1991, Mansour et al., 1990).
• D2-l ⁇ ke receptors have been identified on synaptic nerve terminals, and there is evidence that D2 receptors are colocalized with D1 receptors in certain neuronal populations (Surmeier et al., 1992).
• mapping studies also indicate a presynaptic localization of D2-like receptors where they may function as autoreceptors regulating the synthesis and/or release of dopamine (Starke et al., 1989, Sokoloff et al., 1990).
• D2-TM I YATLLTLLIAVIVFGNVLVC (Sequence ID NO: 61);
• D2-TM II VSLAVADLLVATLVMPWWY (Sequence ID NO: 60); D2-TMIII.TLDVMMCTASILNLCAISID (Sequence ID NO: 59); D2-TM IV: RVTVMISIVWVLSFTISCPL (Sequence ID NO: 58); D2-TM V: PAFWYSSIVSFYVPFIVTL (Sequence ID NO: 57); D2-TM VI: LAIVLGVFIICWLPFFITHI (Sequence ID NO: 56); D2-TM VII: LYSAFTWLGYVNSAVNPIIY (Sequence ID NO: 55); D2-TM VII: TWLGYVNSA (Sequence ID NO:64).
• peptides of amino acid sequence corresponding to all or a portion of a D2 receptor TM domain are highly specific antagonists of D2 receptor binding and function in vi tro and in vivo .
• the dopamine D1 and D3 to D5 receptors are also receptors for the neurotransmitter dopamine and are found in brain as well as in other tissues.
• amino acid sequences of these receptors and identification of their transmembrane domains, can be obtained, for example, from SwissProt Database under the Accession Numbers listed in Table 1.
• an antagonist peptide may be selected from the following transmembrane amino acid sequences or may be an effective fragment or analogue of any of these sequences:
• TM 1 ILTACFLSLLILSTLLGNTLVCAAV (Sequence ID NO: 9);
• TM 2 FFVISLAVSDLLVAVLVMPWKAVAEIA (Sequence ID NO: 10);
• TM 3 NIWVAFDIMCSTASILNLCVISVD (Sequence ID NO: 11);
• TM 4 AAFILISVAWTLSVLISFIPVQLSW (Sequence ID NO:12);
• TM 5 TYAISSSVISFYIPVAIMIVTYTRI (Sequence ID NO: 13); TM 6: TLSVIMGVFVCCWLPFFILNCILPFC (Sequence ID NO: 14);
• TM 7 FDVFVWFGWANSSLNPIIYAFNAD (Sequence ID NO: 15).
• the D1 dopamine receptor has been associated with drug abuse and the D3 and D4 receptors have been
• Antagonists of these receptors in accordance with the invention provide specific therapeutic agents for use in these conditions.
• Antagonists of these receptors in accordance with the invention provide specific therapeutic agents for use in these conditions.
• adrenergic receptor antagonist peptides and a method for regulating or inhibiting the activity of a selected adrenergic receptor are provided.
• AR adrenergic receptors
• ⁇ 1- , ⁇ 2- , and ⁇ 3 AR are involved m the activation of adenylyl cyclase.
• activation of the platelet and ki ⁇ ney ⁇ 2 AR ⁇ 2 AR-C10 and ⁇ 2 AR-C4, respectively
• the ⁇ 1 AR receptors ( ⁇ 1A and ⁇ 1B ) have the ability to stimulate phospholipase C. Stimulation of this effector enzyme leads to membrane phospholipid hydrolysis and the subsequent mobilization of calcium from intracellular stores.
• the various AR, and the respective G proteins to which they couple, provide the means by which the two adrenergic agonists epinephrine and norepinephrine can elicit many different intracellular responses.
• an antagonist peptide may be selected from the following transmembrane amino acid sequences or may be an effective fragment or analogue of any of these sequences:
• TM I GMGLLMALIVLLIVAGNVLVIVAI (Sequence ID NO: 16); TM II: IMSLASADLVMGLLWPFGATIW (Sequence ID NO: 17); TM III:ELWTSVDVLCVTASIETLCVIALD (Sequence ID NO: 18); TM IV: RGLVCTVWAISALVSFLPILMHWW (Sequence ID NO: 19); TM V : RAYAIASSWSFYVPLCIMAFVYL (Sequence ID NO: 20); TM VI: LGIIMGVFTLCWLPFFLANWKAF (Sequence ID NO: 21); TM VII:RLFVFFNWLGYANSAFNPIIYCRS (Sequence ID NO: 22).
• an antagonist peptide may be selected from the following transmembrane amino acid sequences or may be an effective fragment or analogue of any of these sequences:
• an antagonist peptide may be selected from the following transmembrane amino acid sequences or may be an effective fragment or analogue of any of these
• TM I GVGVGFLAAFILMAVAGNLLVILSV (Sequence ID NO: 23);
• TM II FIVNLAVADLLLSATVLPFSATMEVL (Sequence ID NO: 24); TM III: DVWAAVDVLCCTASILSLCTISV (Sequence ID NO: 25); TM IV: AAILALLWWALWSVGPLLGWKEP (Sequence ID NO: 26); TM V: AGYAVFSSVCSFYLPMAVIWMYC (Sequence ID NO: 27); TM VI: LAIWGVFVLCWFPFFFVLPLGSL (Sequence ID NO: 28);
• TM VII EGVFKVIFWLGYFNSCVNPLIYPCS (Sequence ID NO: 29). The inventors have shown that the peptides
• FFNWLGYANSAFNP (Sequence ID NO: 30) and GYANSAFNP
• VFKVIFWLGYFNSCVN (Sequence ID NO: 31) and VFKVIFWLGYFNS (Sequence ID NO:73), both fragments of the TM VII domain of the human ⁇ 1A-adrenergic receptor, inhibited the function of that receptor in vi vo, as shown in Example 5.
• the adrenergic receptor antagonist peptides of the present invention provide new agents with previously unavailable specificity for use in treatment of hypertension.
• adenosine receptor antagonist peptides and a method for regulating or inhibiting the activity o f a selected adenosine receptor are provided.
• Adenosine is a neuromodulator which is released in response to increased activity or stress.
• Adenosine receptors are found in both central and peripheral neural locations. Four subtypes of adenosine receptors, designated A1, A2a, A2b and A3, have been identified.
• adenosine exerts a depressant action in the brain, heart and kidneys by activating adenosine receptors.
• the depressant action in the brain is
• adenosine is believed to confer neuroprotection. Moreover, centrally acting adenosine has been shown to be involved in pain, cognition, movement and sleep. Peripherally, adenosine is believed to have arrhythmic, hypotensive and
• caffeine are attributed to their action as adenosine receptor antagonists.
• Adenosine receptor antagonists have a role as therapeutics in the treatment of cardiovascular, renal and central nervous system disorders and are likely to be useful as anti-asthmatics, anti-depressants, anti- arrhythmics, anti-Parkinsonian therapeutics, cognitive enhancers and as renal protective agents.
• vasopressin type 2 receptor antagonist In accordance with a further embodiment of the invention, vasopressin type 2 receptor antagonist
• peptides and a method for regulating or inhibiting the activity of the receptor are provided.
• V2 vasopressin type 2 receptor of the kidney collecting tubules binds argmine vasopressin, leading to G protein-mediated activation of adenylate cyclase and decreased water permeability of the tubule cells
• V2 receptor Defects in the V2 receptor are a cause of congenital nephrogenic diabetes i nsipidus, characterized by excessive urine excretion (polyuria) and failure to concentrate urine in response to vasopressin.
• an antagonist peptide may be selected from the following transmembrane amino acid sequences or may be an effective fragment or analogue of any of these sequences:
• TMI AELALLSIVFVAVALSNGLVLAALA (Sequence ID NO: 74); TMII: IGHLCLADLAVALFQVLPQLAW (Sequence ID NO: 75); TMIII: AVKYLQMVGMYASSYMILAMTL (Sequence ID NO: 76); TMIV: VLVAWAFSLLLSLPQLFIFAQ (Sequence ID NO: 77); TMV: TYVTWIALMVFVAPTLGIA (Sequence ID NO: 78);
• TMVI MTLVIVWYVLCWAPFFLVQLW (Sequence ID NO: 79); TMVII: LLMLLASLNSCTNPWIYASF (Sequence ID NO: 80).
• Antagonist peptides based on the amino acid sequence of the TM domains of the V2 Receptor provide therapeutic agents which can reduce or prevent the function of that receptor
• LMLLASLNSCTNPWIY (Sequence ID NO: 53), a fragment of the TM VII domain of the V2 receptor, by inhibition of the V2 receptor, acted as a diuretic in the intact rat, as described in Example 6.
• chemokine receptor antagonist peptides and a method for regulating or inhibiting the activity of a selected chemokine receptor are provided.
• the cell surface receptors for chemokines belong to the family of G protein coupled receptors, and have oeen implicated in a number of physiological functions.
• chemokine receptors such as CCR5 and CXCR4, also called fusin
• the chemokine receptors, CCR5 and CXCR4 have been identified as the mam cofactors necessary for HIV entry into CD4-positive cells (Dragic et al., (1996); Weiss et al., (1996)).
• the phenotype of the HIV virus determines whether it
• CXCR4 or CCR5, or sometimes both receptors for entry into CD4-pos ⁇ t ⁇ ve cells.
• CCR2B and CCR3 are minor co-factors for HIV entry.
• an antagonist peptide may be selected from the transmembrane amino acid sequences of these receptors shown in Table 4 or may be an effective fragment or analogue of any of these sequences.
• an antagonist peptide based on the transmembrane domain of the CCR2B or CCR3 receptor can be used to block HIV entry, as these receptors may also act as coreceptors with the CD4 receptors for HIV virus entry.
• LYSLVFIFGFVGN (Sequence ID NO: 82), a fragment of the TM I domain of the CCR5 receptor, inhibited HIV infection of human PBMC cells, as described m Example 7.
• receptors in accordance with the invention, provides a therapeutic agent which can disrupt the function of the respective receptor, thereby preventing entry of the HIV virus into CD4-pos ⁇ t ⁇ ve cells containing that receptor.
• therapeutic agents may be used prophylactically or after exposure to the HIV virus.
• serotonin receptor antagonist peptides and a method for regulating or inhibiting the activity of a selected serotonin receptor are provided.
• serotonin neurotransmitter, serotonin, are mediated through a variety of serotonin receptors now numbering 15. All except the 5HT3 receptor belong to the superfamily of GPCRs.
• Serotonin has been associated with a number of neuropsychiatric disorders such as depression,
• an antagonist peptide may be selected from the transmembrane amino acid sequences of the receptor shown in Table 4 or may be an effective fragment or analogue of any of these sequences.
• An antagonist peptide based on the amino acid sequence of a transmembrane domain of any serotonin receptor provides a therapeutic agent which can disrupt the function of that receptor and hence can be used for specific directed therapy in neuropsychiatric disorders such as depression,
• opioid receptor antagonist peptides and a method for regulating or inhibiting the activity of a selected opioid receptor are provided.
• the diverse biological activities of the endogenous opioid peptides are mediated through a variety of opioid receptors including Mu, Delta, and Kappa.
• Opioid neuronal systems play important roles in a wide variety of physiological processes including pain, mood, learning, thermoregulation, mgestive behaviour, motor activity and the perception of reward, with
• an antagonist peptide may be selected from the transmembrane amino acid sequences of the receptor shown in Table 4 or may be an effective fragment or analogue of any of these sequences.
• An antagonist peptide based on the amino acid sequence of the transmembrane domains of the Mu-opioid receptor provides a therapeutic agent which can disrupt the function of that receptor and can be used m the treatment of disorders including substance abuse, obesity, eating disorders and bowel motility.
• angiotensin receptor antagonist peptides and a method for regulating or inhibiting the activity of a selected angiotensin receptor are provided.
• Angiotensin II (ANG II), a component in the
• renin-angiotensin system is an important factor in the pathogenesis of cardiovascular diseases including hypertension, cardiac left ventricular hypertrophy (LVH) and congestive heart failure.
• Angiotensin II also be considered as renin-angiotensin II.
• angiotensin II contributes to structural alterations of the vasculature such as medial hypertrophy, neointima formation and post-infarct remodeling of the heart.
• the biological activities of angiotensin II are mediated by the ANG II AT1 and AT2 receptor subtypes which display a
• ANG II physiological and cardiovascular actions of ANG II have been attributed to the AT1 receptor which is coupled to a G-protein, while stimulation of AT2 receptors, which are not G-protein-coupled, leads to an inhibition of cell proliferation and possibly induces cell differentiation. It is conceivable that under physiological conditions AT1 receptors facilitate, whereas AT2 receptors inhibit, angiogenesis. Under pathophysiological conditions, such as postmyocardial infarction or LVH, the AT2 receptor could be upregulated to control excessive growth mediated in part by the AT1 receptor.
• antagonist peptide may be selected from the transmembrane amino acid sequences of the receptor shown in Table 4 or may be an effective fragment or analogue of any of these sequences.
• Antagonist peptides based on the amino acid sequence of a TM domain of the AT1 receptor provide therapeutic agents which can be used in these same diseases.
• neuropeptide Y5 (NPY5) receptor antagonist peptides and a method for regulating or inhibiting the activity of that receptor are provided.
• Neuropeptide Y plays important roles in the central control of appetite and energy balance. These specific activities are mediated by the NPY5 receptor.
• the amino acid sequence deduced from rat Y5 cDNA shows only 30-33% identity to other NPY receptors, including Yl, Y2, and Y4/PP1.
• Pharmacological analysis shows that the Y5 receptors have high affinity for the peptides that elicit feeding (e.g. NPY, PYY, (2-36)NPY, and (LP)NPY) and low affinity for nonstimulating peptides (e.g.
• Antagonist peptides based on the amino acid sequence of the TM domains of the NPY5 receptor, provide
• therapeutic agents which can be used for the management of appetite regulation in obesity, and type 2 diabetes mellitus and related conditions.
• melanocyte stimulating hormone (MSH)
• MSH is a strong stimulator of pigment cells, modulating skin colour change in some animals. MSH has also been shown to act as a neurotransmitter in the central nervous system, as an endocrine stimulant and as a modulator of immune inflammatory responses. The hormone is considered a potential tool m the diagnosis and therapy of melanoma, as it has been used in
• Antagonist peptides based on an amino acid sequence of a TM domain of the MSH receptor, as shown in Table 4, provide therapeutic agents which can be used for the management of hyperpigmentation, melanoma and
• tyrosine kinase antagonist peptides and a method for regulating or inhibiting the activity of a selected tyrosine kinase receptor are provided.
• the tyrosine kinase receptors have an amino terminus involved in ligand binding, a single membrane-spanning domain and a homologous carboxyl tail catalytic domain with intrinsic tyrosine kinase activity (Kraus et al., 1989).
• tyrosine kinase receptors include receptor families for a number of growth factors, including epidermal growth factor (EGF), colony- stimulating factor 1/platelet derived growth factors and lnsulm/insulin-like growth factor , fibroblast growth factor, tumor necrosis factor, vascular endothelial growth factor.
• EGF epidermal growth factor
• Tyrosine kinase receptors are localized in a wide range of epithelial and fibroblastic cells.
• Tyrosine kinase receptors mediate a plethora of
• Binding of ligand to the extracellular portion of a tyrosine kinase receptor results m an association of two receptor molecules (dimerization) that leads to conformational changes resulting in the phosphorylation of the cytoplasmic domain of the receptor (Boni- Schnetzler et al., 1987).
• the epidermal growth factor (EGF) receptor has four subtypes, identified as erb1 to erb4.
• EGF receptors have been shown to act as oncogenes by mechanisms of overexpression, or mutations that
• EGF-erb3 receptor is overexpressed in a subset of human mammary tumors.
• the ability to inhibit or regulate activity of the EGF family of receptors by the antagonist peptides of the invention provides a new, specific tool to prevent the development of, or control, of neoplastic growth in psoriasis and cancer.
• an antagonist peptide may have the transmembrane domain amino acid sequence lATGMVGALLLLLVVALGIGLFM (Sequence ID NO: 32) or may be an effective fragment or analogue of that
• the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any combination sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be any one of the sequence, and for EGF-erb3, the antagonist may be
• MALTVIAGLVVIFMMLGGTFL (Sequence ID NO: 83) or an effective analogue or fragment thereof.
• LTVIAGLVVIF (Sequence ID NO:84), a fragment of the TM domain of the EGF-erb3 receptor, inhibited the tyrosine kinase function of that receptor in cultured A431 cells, as described in Example 8.
• Such an antagonist peptide provides a therapeutic agent for inhibition of cell proliferation, for use , for example, in cancer,
• Tyrosine kinase receptors such as Fibroblast Growth Factor receptor, FGFr (also belonging to the
• VEGFr also belonging to the
• Angiogenesis comprises the processes leading to the generation of new blood vessels through sprouting from already-existing blood vessels. Blood vessel growth is associated with wound healing, tissue growth and repair; abnormal angiogenesis occurs in pathologies such as cancer and diabetic retinopathy.
• Angiogenic inhibitors are of clinical significance because they can be used to influence directly the angiogenic processes involved, for example, in wound healing. Angiogenesis inhibitors will also be of value in treatment of diseases including pathogenic
• neovascularization such as Kaposi's sarcoma, diabetic retinopathy, and malignant tumor growth.
• the Fibroblast Growth Factor receptor (FGFr) has two forms, identified as FGFr1 and FGFr2.
• Antagonist peptides based on the amino acid sequence of the TM domain of the FGFr1 (IIIYCTGAFLISCMVGSVIVY: Sequence ID NO: 85) or FGFr2 (AIYCIGVFLIACMVVTVILC :
• Sequence ID NO: 86 receptors, provide therapeutic agents which can be used to regulate angiogenesis.
• vascular endothelial growth factor receptor 1 and 2 antagonist peptides and a method for regulating or inhibiting the activity of the selected receptor are provided.
• VEGFr Vascular Endothelial Growth Factor receptor
• TM domains ISYSFQVARGMEFLSSRKCIH Sequence ID NO: 87
• IIILVGTTVIAMFFWLLLVIILGTV Sequence ID NO:88
• Trk A is the receptor for nerve growth factor
• Trk B for brain derived neurotrpoc factor (BDNF) and neurotrophin-4
• trk C for the neurotrophin-3 receptor. Trk B and trk C are abundantly expressed in different parts of developing and adult brain. Like other members of receptor tyrosine kinase family, trk A
• Trk B and C are alternatively spliced into two different types of receptor lsoforms: the full-length, tyrosine kinase (TK) domain-containing form TK(+) and the truncated form TK(-). These isoforms are identical in their extracellular and transmembrane domains, but in place of the intracellular TK domain, TK(-) forms only contain short unique intracellular tail regions. Both receptor variants bind neurotrophins, but only TK(+) can activate intracellular signal
• an antagonist peptide may have the transmembrane domain amino acid sequence AVFACLFLSTLLLVI (Sequence ID NO: 89) or may be an effective fragment or analogue thereof.
• Antagonist peptides based on the amino acid sequence of the transmembrane domain of the trkA receptor provide therapeutic agents which can be used to reduce or inhibit nerve growth factor activity.
• a number of integral membrane proteins including the energy-dependent transporter pumps, form ion channels or ion channel receptors or are channel proteins.
• Bacterial Energy-dependent Transporter Antagonists In accordance with a further embodiment of the invention, bacterial energy-dependent transporter
• the integrity of bacterial membranes is maintained by a variety of membrane proteins, including bacterial ATPase transporter. Disrupting the function of the critical membrane protein may lead to loss of bacterial cell viability.
• the ion-translocating enzyme, F 1 F 0 ATPase synthesizes ATP using a proton gradient and is the enzyme responsible for oxidative phosphorylation.
• the energy of the proton gradient drives ATP synthesis, catalyzed by the F 1 F 0 ATPase.
• the E. coli unc operon which codes for the ATPase, contains nine genes coding for the F 0 and F 1 domains of the enzyme.
• the F 0 portion is membrane-intrinsic and has three sub-units, a, b and c.
• an antagonist peptide for the E. coli F 1 F 0 ATPase may have the Fo b sub- unit transmembrane sequence MAAAVMMGLAAIGAAIGIGILGG
• Sequence ID NO: 90 or the Fo c sub-unit TM sequence NATILGQAIAFVLFVLFCM (Sequence ID NO: 91) or may be an effective fragment or analogue of one of these sequences.
• peptide GQAIAFVLFVL (Sequence ID NO: 92), based on the amino acid sequence of the TM domain of the Gram negative ATPase Fo b subunit, and peptide LAAIGAAIGIGIL (Sequence ID NO:93), based on the Fo c subunit, used alone or in combination, could prevent the growth of E. coli , as described in Example 9.
• Antagonist peptides based on the amino acid sequence of a TM domain of the Gram negative ATPase Fo b subunit or Fo c subunit, provide therapeutic agents which can be used as anti-bacterials.
• mammalian energy-dependent transporter in accordance with a further embodiment of the invention, is provided.
• peptides and a method for regulating or inhibiting the activity of the selected transporters are provided.
• P-glycoprotein or MDR1 protein is an example of a mammalian energy-dependent transporter. It is an energy- -dependent efflux pump responsible for drug efflux and decreased drug accumulation in multi-drug resistant (MDR) cells.
• MDR multi-drug resistant
• the activation of the mdrl gene which encodes the protein can occur under various types of stimulation, including under the effect of anti-cancer drugs.
• P-glycoprotein is an ATPase transporter which is
• Antagonists of P-glycoprotein would be an important adjunct to treatment of cancer with
• an antagonist peptide for P-glycoprotein may be selected from the relevant TM domain amino acid sequences shown in Table 4 or may be an effective fragment or analogue of any of these sequences.
• Antagonist peptides based on the amino acid sequence of a TM domain of P-glycoprotein provide
• ion channel antagonist peptides and a method for regulating or inhibiting the activity of a selected ion channel antagonist are provided.
• GABA ⁇ -aminobutyric acid
• a receptor-chloride ion channel complex which belongs to the ligand-gated receptor superfamily, which also includes the 5HT3 serotonin receptor, the nicotmic acetylcholine receptor and the metabotropic glutamate receptor.
• the GABA-A receptor-chloride ion channel is believed to be a complex of five membrane-spanning protein subunits forming a heterooligomer.
• the subunits belong to ⁇ , ⁇ , ⁇ , ⁇ or p class.
• Each subunit has an N-terminus, four putative hydrophobic membrane-spanning domains and a C-terminus, linked by extracellular and intracellular loops (Schofield et al., (1987); Bernard, E.A., 1995).
• an antagonist peptide for the GABA-A receptor may be selected from the following transmembrane amino acid sequences of the human ⁇ l-subunit:
• GABA is the principal inhibitory neurotransmitter in the vertebrate brain which mediates its actions (neuronal inhibition) by binding to the integral membrane protein, the GABA-A receptor.
• GABA-A receptors form a fast-acting ligand-gated chloride ion-selective channel, that upon activation by agonist, results m the hyperpolarization of the neuron.
• GABA-A receptor channels mediate the major
• GABA-A receptor subtypes have been identified in hippocampus (Pyramidal and mterneurons), olfactory bulbs (Mitral and Granule cells), thalamus (relay neurons and Reticular nucleus), and in the cerebellum (Purkmje and Granule cells).
• a number of drugs which have their effect on the brain act by binding to the GABA agonist site or receptor channel; these include benzodiazepines which are
• GABA antagonist peptides as described herein may be used as therapeutics m similar disorders.
• the family of transporter proteins are glycoproteins with twelve putative membrane-spanning domains whicn mediate sodium- and chloride-dependent re-uptake of neurotransmitter.
• the neurotransmitter transporter proteins provide a re-uptake mechanism for
• an antagonist peptide may be selected from the following transmembrane amino acid sequences or may be an effective fragment or analogue of any of these sequences:
• T1 FLLSVIGFAVDLANVWRFPYL (Sequence ID NO: 37);
• T2 GAFLVPYLLMVIAGMPLFYM (Sequence ID NO: 38);
• T3 GVGFTVILISLYVGFFYNVII (Sequence ID NO: 39);
• T4 WQLTACLVLVIVLLYFSLW (Sequence ID NO: 40);
• VCFSLGVGFGVLIAFSSY (Sequence ID NO: 42)
• T7 IVTTSINSLTSFSSGFVVFSFL (Sequence ID NO: 43);
• T9 LFTLFIVLATFLLSLFCVT (Sequence ID NO: 45);
• GTSILFGVLIEAIGVAWFYGV (Sequence ID NO: 46);
• T11 LCWKLVSPCFLLFVVVVSIV (Sequence ID NO: 47);
• T12 LGWVIATSSMAMVPIYAAY (Sequence ID NO: 48).
• transporters The distribution of transporters is consistent with the distribution of neurotransmitters, suggesting that transporters might be expressed specifically for the neurotransmitter system. Transporter localization is chiefly in the presynaptic neuronal membrane.
• the dopamine transporter is also targeted by drugs of abuse such as cocaine and amphetamine.
• the antagonist peptides of the invention provide new, specific therapeutic agents useful in these dopamine transporter-related disorders as antidepressants and for the relief of drug craving and dependence.
• the CNS, vascular and immune systems share highly conserved specific, cell surface antigen receptors or immune receptors necessary for intercellular recognition.
• Members of this superfamily of receptors have a large amino terminus, typically involved in antigen
• cell surface antigen receptors include members of the immunoglobulin receptor
• CD4 superfamily such as CD4.
• immunoglobulin superfamily antagonist peptides and a method for regulating or inhibiting the activity of the selected antagonist are provided.
• CD4 is a T cell specific surface glycoprotein which shows homology to members of the immunoglobulin
• CD4 binds to nonpolymorphic regions of the major histocompatibility complex (MHC) class II molecule, thereby increasing the avidity of the T cell receptor for its ligand. CD4 interacts with at least two other T cell surface molecules known to be involved m T cell
• TCR T cell receptor
• CD4 receptor antagonists will provide new
• CD4+ T-cell mediated autoimmune diseases and allograft transplant rejection The CD4 receptor has also been identified as a necessary major coreceptor for HIV entry into cells.
• Antagonist peptides based on the amino acid sequence of the TM domain of the CD4 receptor,
• MALIVLGGVAGILLFIGLGIFF (Sequence ID NO: 94), provide therapeutic agents useful for the treatment of autoimmune disease, the control of allograft rejection and the prevention or reduction of HIV infection.
• peptide LIVLGGVAGLLLF (Sequence ID NO: 181) based on the amino acid sequence of the TM domain of the CD4 inhibited HIV infection of PBMC cells, as described in Example 11.
• Physiological cell death occurs when a cell within an organism dies by a mechanism orchestrated by proteins encoded by the organism's genome. The purpose of this process is to kill unwanted cells; apoptosis occurs in three situations, namely, during development and homeostasis, as a defence mechanism and in aging.
• Apoptosis inhibitors may be useful m treating ischemic conditions such as heart attacks, strokes or reperfusion injury, by blocking the apoptotic response of cells subjected to sublethal amounts of anoxia. They may also be useful in allograft rejection and m rheumatoid arthritis or other autoimmune disorders.
• Tumor Necrosis Factor receptors The Tumor Necrosis Factor receptors, TNFR1 and TNFR1
• TNFR2 belong to the TNF receptor superfamily which binds TNF- ⁇ as a mediator of apoptosis.
• an antagonist peptide may have the TM domain amino acid sequence VLLPLVIFFGLCLLSLLFIGLMY (Sequence ID NO: 95) or ALPVGLIVGTALGLLIIGVVNCIMTOV (Sequence ID NO: 96)
• CD94 is a type II membrane glycoprotem, and is a member of the C-type lectin superfamily. CD94 receptors have been implicated in the regulation of Natural Killer (NK) cell function.
• NK Natural Killer
• An unexpected feature of CD94 is the essential absence of a cytoplasmic domain, implying that association with other receptors may be necessary for the function of this molecule.
• CD95 is a member of the cytokine receptor
• CD95 is a mediator of apoptosis and binds to the cytokine ligand FASL.
• an antagonist peptide may have the transmembrane domain amino acid sequence LGWLCLLLLPIPLIVWV (Sequence ID NO: 97) or may be an effective fragment or analogue of any of this
• CD97 is a seven-transmembrane receptor belonging to the EGF TM7 superfamily. CD97 is expressed in leukocytes and leukocytes strongly positive for CD97 are
• CD97 may play a signal transduction role associated with the establishment or development of an inflammatory process.
• CD9 is an integral cell surface protein belonging to the TM4 superfamily of receptors. CD9 is involved in the aggregation of platelets and may participate also in cell-cell interactions critical for correct orientation and movement of maturing myeloid cells in bone marrow. 6. ANTIGEN RECEPTOR ANTAGONISTS
• a number of eukaryotic cell types have membrane- associated antigen receptors which are integral membrane proteins.
• the antigen receptor occurring on many mammalian cells which recognises the T cell antigen and provides the molecular basis for major histocompatability complex (MHC) antigen recognition.
• the receptor consists of two linked glyco-peptides, one of which, the ⁇ -glycopeptide, consists of a transmembrane domain and a cytoplasmic domain.
• the amino acid sequence of the human T cell antigen receptor ⁇ chain is disclosed in Yoshikai et al. (1985) and the TM domain has the amino acid sequence:
• DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (Sequence ID NO: 49). This sequence or an effective fragment or analogue thereof provides an antagonist which will interfere with the function of the T cell antigen
• Such an antagonist provides a therapeutic agent useful for control of autoimmune diseases or graft- versus-host reaction.
• rats were housed in the animal facility for acclimatisation for 1 week.
• Peptides were synthesized using standard solid state methodology by commercial suppliers. Peptides were stored dessicated under refrigeration. Peptides may be
• amide form is preferred for its improved stability.
• Most of the peptides described herein were prepared in amide form.
• peptides were prepared at a stock concentration of 5 mg peptide per 1 ml of peptide buffer. 5 mg of peptides were dissolved in 100 ml DMSO, then diluted to a volume of 1 ml with buffer (100 mM NaCl, 10 mM Tris-HCl, 2 mM EDTA), unless indicated otherwise.
• TM domain peptides prepared to provide antagonists of the indicated receptors:
• D2-TM I Human dopamine D2 receptor: D2-TM I:
• D2-TM II VSLAVADLLVATLVMPWVVY
• D2- TM III TLDVMMCTASILNLCAISID
• D2-TM IV TLDVMMCTASILNLCAISID
• RVTVMISIVWVLSFTISCPL D2-TM V: PAFVVYSSIVSFYVPFIVTL; D2- TM VI: LAIVLGVFIICWLPFFITHI; D2-TM VII:
• Rat ⁇ 1 adrenergic receptor ⁇ 1-AR TM VII: GYANSAFNP; ⁇ 1-AR TM VII: FFNWLGYANSAFNP;
• Human ⁇ 2 adrenergic receptor ⁇ 2-AR TM I: LIVVGNVLVI (Sequence ID NO: 98);
• CCR5-TM 1 LYSLVFIFGFVGN
• CCR5- TM 7 MQVTETLGMT (Sequence ID NO: 99);
• CXCR4 TM-1 PTIYSIIFLTGIV (Sequence ID NO: 100) ;
• S1A-TM VII PALLGAIIN
• SIB-TM VII FHLAIFDFFTWLG (Sequence ID NO: 102); SIB-TM VII: FHLAIFDFFTWLGYLNSLIN (Sequence ID NO:51); S1D-TM I: QALKISLAWLSV (Sequence ID NO:103);
• MOR-TM VII IPETTFQTVSWH (Sequence ID NO: 104);
• DTAMPITISIAY (Sequence ID NO: 105); AT1-TM VII:
• VDTAMPITICIAYFNN (Sequence ID NO: 52);
• V2-TM VII Human vasopressin 2 receptor: V2-TM VII:
• NPY5-TM I YFLIGLYTFVSL
• MSH- TM 1 ISDGLFLSLGLVS (Sequence ID NO:107).
• Human EGF-erb1 receptor EGFR1-TM: VGALLLLLVVALG (Sequence ID NO: 108);
• Human EGF-erb3 receptor EGFR3-TM: LTVIAGLVVIFMMLGG (Sequence ID NO:109); EGFR3-TM: LTVIAGLWIF;
• FGFr1-TM EIIIYCTGAFLIS (Sequence ID NO: 110); FGFr2-TM:
• WTVILCRMKNTT (Sequence ID NO:111);
• VEGFr1-TM Human Vascular Endothelial Growth Factor receptor: VEGFr1-TM: SYSFQVARGMEFL (Sequence ID NO:118);
• TrkA-TM FASLFLSTLLLVI
• P-glycoprotem VGTLAAIIHGAGL (Sequence ID NO: 114)
• GABA-A GIFNLVYW (Sequence ID NO: 115)
• DAT-TM XII ALGWIIATS
• DAT-TM XII PDWANALGWVIIATS (Sequence ID NO: 116);
• TNFR1-TM TVLLPLVIFFGLSL (Sequence ID NO:117); TNFR2-TM:
• PVGLIVGVTALGL (Sequence ID NO: 118)
• CD95-TM WLCLLLLPIPLIVW (Sequence ID NO: 119)
• baculovirus Recombinant baculovirus encoding the human D2L receptor was constructed using standard recombinant techniques. Briefly, a cDNA clone encoding the long form of the human D2 receptor (D2L) was extracted from the pZem 3 vector with Drain and Kpnl. The resulting fragment was blunt ended and isolated by electrophoresis on soft agarose. The transfer vector pJVETLZ New was digested with Nehl and blunt ended. The cDNA fragment coding for the D2L receptor was inserted into this vector by blunt-end ligation and the orientation verified by sequencing.
• D2L human D2 receptor
• Transfer of recombinant baculovirus encoding the D2L receptor into the AcNPV genome was achieved by co- transfection of plasmid and wild-type viral DNA m Sf9 cells using the calcium phosphate precipitation
• D2 receptor encoding pcDNA3 expression vector The pHD2 s -Zem plasmid containing the entire coding sequence of the human D2 S receptor was used as the template m the polymerase chain reaction (PCR) for the construction of pcDNA3 expression vectors encoding the full-length D2 receptor and truncation mutants.
• the D2/pcDNA3 expression vector was constructed as follows: The oligonucleotides
• TGGAGGATCTT3' and 5'GCAAGCTTGCCACCCAGTCGGTCCACCGC3') were used in the PCR reaction to generate a HmdIII/NotI fragment encoding the full-length D2 receptor which was isolated by agarose gel electrophoresis and inserted into pcDNA3 at Hindlll and Notl sites.
• the D2-TM VII/pcDNA3 expression vector was constructed as follows: The oligonucleotide encoding an initiator ATG codon, aa of TM VII of the D2 receptor, a stop TAG codon, and a Xbal site was chemically
• oligonucleotides which were digested with Xbal.
• the pcDNA3 vector was digested with HindiII and the ends filled with T4 DNA polymerase.
• the linearized pcDNA3 vector was digested with Xbal, and the double-stranded oligonucleotide subcloned into the pcDNA3 vector by blunt and sticky-end ligation.
• the sequence and insertion of the synthetic oligonucleotides into the vector at the appropriate site and orientation was verified by
• Sf9 Cell Culture Sf9 cells were grown in monolayer or suspension culture essentially as described by Summers and Smith (1987) in supplemented Grace's insect media at 27 °C. Pluronic F-68, a cell protective agent, was added to improve cell viability in suspension culture since Sf9 cells are very sensitive to mechanical shear. Cells at a density of 1-2 x 10 6 /ml were infected with the recombinant virus at a multiplicity of infection of 2-5 and harvested at 24 or 48 h post-infection. Typically, viral
• COS and CHO cell culture and DNA transfection COS-7 monkey kidney cells (American Type Culture Collection) were maintained as monolayer cultures at 37 C m Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and Penicillin and Streptomycin. CHO cells were cultured in Ham's F-12 media containing 10% fetal bovine serum. COS and CHO cells were transiently transfected with recombinant pcDNA3 expression vectors (8 ⁇ g) by calcium phosphate precipitation methodology as described by the manufacturer (BRL, Life Technologies Inc.).
• D2/pcDNA3 (8 ⁇ g) was co-transfected with D2 TM-VII/pcDNA3 (8 ⁇ g) or pcDNA vector (8 ⁇ g) into COS-7 cells as described above.
• Preparation of Cell Membrane Fractions The preparation of membranes was done at 4°C. Cells were centrifuged at 100xg for 7 min. and pelleted.
• buffer A washed once with buffer A, centrifuged again at high speed and resuspended in buffer A, and stored at -80 C or resuspended in buffer B: 75 mM Tris-HCl, 12.5 mM MgCl 2 , 2 mM EDTA, pH 7.4 and assayed immediately for adenylyl cyclase activity.
• Pelleted membranes P2 membranes were resuspended in buffer A and stored at -70° C or resuspended in the appropriate buffers for immediate use in various assays.
• Protein Determination Protein content was determined by the method of Bradford (BioRad). A standard protein concentration curve was made with bovine serum albumin (BSA). Protein concentration in the test sample was determined from the standard curve which was a plot of absorbance at 595 nm measured using a Hitachi model U- 2000 spectrophotometer against concentration.
• BSA bovine serum albumin
• Membranes were prepared by somcation m buffer A as described above. The pellet was resuspended and stirred at 4°C for 2 h in 2 ml of freshly prepared solubilization buffer consisting of 100 mM NaCl, 10 mM Tris-HCl pH 7.4, 2% digitonin, and 5 mM EDTA with protease inhibitors.
• the homogenate was centrifuged at 27000xg for 20 min and the solubilized fraction was washed and concentrated in Centriprep 30 four times with 10 ml cold buffer C: 100 mM NaCl, 10 mM Tris-HCl pH 7.4 with protease inhibitors.
• the washed fraction was precleared with 1/20 normal rabbit serum and protein A-Sepharose beads for 2 h on ice.
• Solubilized receptors were lmmunoprecipitated with the mouse monoclonal 9E10 antibody (D1 receptor) or rabbit polyclonal (D2 receptor) at a 1/37 dilution in buffer C for 2 h on ice, and agitated gently overnight at 4°C with 1/40 dilution of agarose fixed goat anti-primary IgG. The lmmunoprecipitate was washed 6 times with 5 volumes cold buffer C for 20 min, solubilized in SDS sample buffer, sonicated and electrophoresed on SDS-PAGE as described above.
• Soluble Receptor Binding The amount of solubilized and lmmunoprecipitated receptor was determined by radioligand binding. D2 receptor density was estimated by incubating m the presence of saturating concentrations ( ⁇ 2000 pM) of the benzamide antagonist [ 3 H] YM-01951-2 or
• Nonspecific binding was defined by binding not displaced by 1 mM (+)butaclamol.
• the binding preparation was loaded onto a Sephadex G-50 column (Pharmacia) and ligand bound receptor was separated from free ligand by elution. The eluate was counted in a scintillation counter for
• SDS-PAGE Electrophoresis Sodium dodecyl sulphate 10-12% polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli, 1970.
• lmmunoprecipitated membranes were solubilized in SDS sample buffer consisting of 50 mM Tris-HCl pH 6.5, 10% SDS, 10% glycerol, and 0.003% bromophenol blue with or without 10% 2-mercaptoethanol.
• Molecular mass (Da) of receptors was determined graphically by plotting the log molecular weight of known protein standards versus the RF (relative migration) of these proteins. The apparent molecular mass of proteins was estimated by determining the RF (from the centre of the band) and interpolating this value onto the standard curve.
• Human caudate tissue was obtained from the Canadian Bram Tissue Bank and cell membranes were isolated and receptors
• P2 membranes were prepared as described above. Saturation binding experiments were performed with ⁇ 25 ⁇ g P2 membrane protein with increasing concentrations of [ 3 H] spiperone (10-2000 pM, final concentration) in duplicate
• Nonspecific binding was defined as binding that was not displaced by 1 ⁇ M (+) -butaclamol.
• Bound ligand was isolated by rapid filtration and washing through a Brandel 48-well cell harvester using Whatman GF/C filters. Binding data were analysed by nonlinear least-squares regression using the computer-fitting program INPLOT version 3.0 GraphPad (San Diego).
• the assay mix contained 0.05 ml of crude membranes, 40 ml [ 35 S]GTP ⁇ S binding buffer (50 mM Tris-HCl, 5 mM MgCl 2 , 100 mM NaCl, 20 mM GDP, -280 pM [ 35 S] GTP ⁇ S ), 40 ml peptide antagonist (5 mg in 1 ml 10% DMSO buffer). This was preincubated for 15 min at 37°C and 20 ml agonist (1 mM, final concentration) added. Bound [ 35 S]GTP ⁇ S was
• In Vitro Receptor disruption 3 pmol of isolated and solubilised receptors, as determined by soluble binding assay, were prepared in buffer A (100 mM NaCl, 10 mM Tris-HCl pH 7.4, mM EDTA and 0.5% digitonin with 5 mg/ml leupeptin, 10 mg/ml benzamidine and 5 mg/ml soybean trypsin inhibitor), and incubated with 1.6 g/l (final concentration) of peptide (unless otherwise indicated). In these experiments, 5 mg of peptide were freshly dissolved in 100 ml DMSO and 100 ml Digitonin (5% w/v solution) and 800 ml buffer A.
• SDS buffer 50 mM Tris- HCl, pH 6.5/12% SDS/10% 2-mercaptoethanol/10% (vol/vol) glycerol/0.003% bromophenol blue
• SDS buffer 50 mM Tris- HCl, pH 6.5/12% SDS/10% 2-mercaptoethanol/10% (vol/vol) glycerol/0.003% bromophenol blue
• Receptors were prepared in buffer A and incubated at 23, 37, 65, and 90°C for 30 min with SDS buffer at a final volume of 30 ml, and subjected to SDS- PAGE and immunoblot analysis.
• Receptor pH-stability experiments were performed with 1.5 pmol of immunoprecipitated D2 receptors prepared in buffer A. Receptors were treated with H 2 O, or 0.1 N tartaric acid (final concentration), or 0.1 N HCL (final concentration), or 0.1% glacial acetic acid (final concentration). Samples were then incubated at 37°C for 30 min with SDS buffer at a final volume of 30 ml, and subjected to SDS-PAGE and immunoblot analysis.
• D2 dimers Fig. 3A, lane 1
• the dissociation of the D2 dimer to monomer was accomplished in a dose-dependent manner upon addition of the hydrophobic peptide LAIVLGVFIICWLPFFITHI, aa 375-394 of the D2 receptor, within the TM VI domain (Fig. 3B) or the peptide LYSAFTWLGYVNSAVNPIIY, aa 407-426 of the D2 receptor, within the TM VII domain(Fig. 4B). Both of these peptides, containing no strongly polar residues, had similar ability to dissociate D2 dimers
• LSSTSPPERTRYSPIPPSHH (Sequence ID NO: 63), aa 284-303) of the D2 receptor C IIIB domain or third cytoplasmic loop of the D2 receptor, or a hydrophobic peptide (aa 276-296) corresponding to a portion of the TM-VI region of the ⁇ 2- adrenergic receptor ( ⁇ 2-AR), or two peptides derived from the carboxyl tail of the D1 receptor (aa 369-383 and aa 416-431) (Fig. 3D).
• Fig. 3E no dissociation of immunoprecipitated human dopamine D1 and serotonin 5-HT1B receptor dimers was observed with the D2-TM VII peptide fragment.
• synthetic peptides derived from hydrophobic putative transmembrane domains of receptor proteins can interact specifically to disrupt receptors as noncompetitive antagonists.
• TM peptide disruption of D2 receptor ligand binding Peptides derived from the TM 5 (PAFWYSSIVSFYVPFIVTL) and TM 7 (TWLGYVNSA) domains of the D2 receptor inhibited [ 3 H] -spiperone binding to P2 membranes prepared from D2 receptor-expressing Sf9 cells in a dose-dependent manner (Fig. 4).
• the inhibition was receptor subtype-specific, since TM peptides from the vasopressin V2 receptor-TM7 (LMLLASLNSCTNPWIY), TM-12 of the dopamine transporter (ALGWIIATS) and TM-1 of the GABA receptor A subunit (GIFNLVYW) had a much smaller effect on D2 receptor binding (Figure 4).
• TM peptide inhibition of D2 receptor stimulated GTP- ⁇ S binding In control P2 membranes, dopamine mediated a dose-dependent stimulation of [ 35 S]-GTP- ⁇ S binding
• Adenylyl Cyclase Activity Adenylyl Cyclase activity was conducted essentially as described (Salomon et al., 1974). The assay mix contained 0.02 ml of membrane suspension (10-25 ⁇ g of protein), 0.012 mM ATP, 0.1 mM cAMP, 0.053 mM GTP, 2.7 mM phosphoenolpyruvate, 0.2 units of pyruvate kinase, 1 unit of myokmase and 0.13 ⁇ Ci of [ 32 P]ATP in a final volume of 0.05 ml.
• Enzyme activities were determined in duplicate or triplicate assay tubes containing 10 -3 - 10 -9 M dopamine or 100 uM forskolin or 10 mM sodium fluoride and incubated at 37° C for 30 mins.
• inhibitory receptors such as the D2L receptor
• adenylyl cyclase activity mix contained 100 uM forskolin.
• Inhibition of adenylyl cyclase assays was determine following incubation at 27° C for 20 mins. Reactions were stopped by the addition of 1 ml of an ice-cold solution containing 0.4 mM ATP, 0.3 mM cAMP and [ 3 H]cAMP (25000 cpm). Antagonist inhibition of dopamine stimulated cyclase was performed with increasing concentrations of peptide in the presence of 10 uM dopamine. cAMP was isolated by sequential column chromatography using Dowex cation exchange resm and aluminum oxide. Data were analysed by computer fitted nonlinear least-squares regression.
• Stereotaxic surgery Male Wistar rats ( ⁇ 300-350 g) were anaesthetized with ketamine (66 mg/kg i.p.), acepromazine (3 mg/kg i.p.) and pentobarbital (22 mg/kg i.p.) for chronic stereotaxic implantation.
• a unilateral stainless steel guide cannula (G22) was placed into the centre of the left caudate putamen (Ant. +1.5, Lat. -2.2, Vert. - 5.0, Paxinos and Watson, 1982) to allow drug or vehicle injection.
• bilateral stainless steel guide cannulas (G22) were stereotaxically placed into the centre of left and right caudate putamens for both drug and vehicle injections respectively.
• the guide cannula was kept patent by stylets (Plastic Products Company, Roanoke, VA) terminating 0.5 mm below the guide tips which were located 2 mm above the point of injection.
• the rats were allowed a week postoperative recovery before experimental use.
• Intracerebral injection technique The stylets were withdrawn and injections (drug or vehicle) made into conscious rats using a 28 gauge internal cannula
• Rats with bilateral cannulas were administered drug into the left striatum and vehicle into the right striatum, followed 15 min after by a subcutaneous injection of 0.25 mg/kg
• Asymmetry (ipsilateral to the side of peptide or vehicle injections) was scored on the 0-3 system, 15 min after subcutaneous challenge with apomorphine (0.25 mg/kg). Animals showing an ability to move in right and left directions were not categorized as circling. However, an ability to circle in one direction (asymmetric body posture) was scored on a 0-3 response according to observations in the open field and to the lifting of the tail. The criteria which met the 0-3 scores were:
• D2 receptor transmembrane peptides were studied using the accepted animal model described by Costall et al. (1983), for screening dopamine receptor antagonists. Effect of unilateral intrastriatal injection of receptor peptides on motor behavior. Following unilateral injection of peptides (D2-TM VII or ⁇ 2-AR-TM VI) or vehicle into the left striatum, animals were observed for 1 h. All treatments failed to induce circling responses.
• baculoviruses Recombinant baculovirus encoding the c- myc epitope tagged b 2 -adrenergic receptor was constructed using standard recombinant techniques, as described above. Recombinant baculovirus encoding a histidine- tagged TM-7 domain of the ⁇ 2 -adrenergic receptor was constructed using the Bac-to-Bac system according to manufacturers' instructions. Briefly a complementary oligonucleotide encoding the TM-7 domain of the ⁇ 2 - adrenergic receptor was ligated and subcloned into the multiple cloning site of the pFASTBAC His expression vector by standard recombinant techniques. The
• Ni-NTA purification of histidine-tagged proteins Sf9 cells co-expressing histidine-tagged ⁇ 2-AR Tm7 peptide and full-length c-myc tagged ⁇ 2-AR were disrupted under native conditions. The cell lysate was passed by gravity-flow over a Ni-NTA resin to purify histidine- tagged proteins. Histidine-tagged proteins generated in Sf9 cells were eluted from the resin according to the BAC-to-BAC kit instructions (BRL, Life Technologies).
• Soluble ⁇ 2-adrenerg ⁇ c receptor binding was estimated by incubating solubilized membranes from ⁇ 2-adrenergic receptor expressing Sf9 cells (75 ml) in the presence of saturating
• Non-specific binding was defined as binding not displaced by 1 mM pindolol.
• Bound receptor was separated from free ligand by elution from Sephadex G-50 columns and binding activity determined by scintillation counting.
• histidine-tagged TM-7 peptide of the ⁇ 2 -adrenergic receptor was co-expressed with a full-length c-myc- epitope tagged ⁇ 2 -adrenergic receptor in Sf9 cells.
• a histidine-tagged ⁇ 2-TM 7 peptide - c-myc epitope tagged ⁇ 2 receptor heterodimer should be detectable on immunoblots of Ni-NTA purified receptors.
• An antibody against the His tag of the TM-7 peptide revealed on immunoblot a species at the expected molecular mass ( ⁇ 50 kDa) of a heterodimer ( Figure 12, right panel). In the identical preparation, the same species was detected as an
• GQAIAFVLFVL E. Coli ATPase Fo c subunit
• LAAIGAAIGIGILG LAAIGAAIGIGILG
• LYSLVFIFGFVGN a peptide derived from the TM 1 domain of the CCR5 receptor
• transmembrane domain of the receptor but not by peptides derived from the transmembrane domain of the closely related ⁇ 1 adrenergic receptor.
• Example 5 Inhibition of ⁇ adrenergic and ⁇ adrenergic receptor activity in vi vo
• rats were housed in the animal facility for acclimatisation for 1 week.
• the rats Prior to the experiments, the rats were fasted overnight but provided with water ad libi tum. Under halothane anesthesia, the left femoral or jugular vein was exposed, cleaned, clamped and cannulated with polyethylene tubing (Tygon; PE 10-20) for intravenous injections of drugs. The right carotid artery was then exposed adjacent to the trachea and cannulated for blood pressure recording (PE 10-20 tubing). The cannulae were tunneled subcutaneously to the midback of the animal where they were brought out onto the skin surface and capped with rubber injection ports. All catheters were filled with a solution of heparin (10 units/ml) and were flushed periodically with the same solution to prevent clotting.
• PE 10-20 tubing polyethylene tubing
• the cannulae were tunneled subcutaneously to the midback of the animal where they were brought out onto the skin surface and capped with rubber injection ports. All catheters were filled with a solution of he
• adrenergic receptor antagonist peptides The effect of adrenergic receptor antagonist peptides was examined using an accepted animal model for assessing cardiovascular drugs.
• the rat was given 1 mg/Kg of the ⁇ 1-adrenergic receptor agonist
• Figures 14E to 14H show the results of a control experiment.
• Baseline cardiac parameters were 240
• VFKVIFWLGYFNSCVN a fragment of the ⁇ 1A-adrenergic receptor TM VII peptide was administered intravenously.
• heart rate was reduced to 240 beats/min before recovery to 420 beats/min, during which time there was a significant, transient drop in blood pressure to 130-120/60.
• Figures 15E-15G show the results of a control experiment in the same rat. Administration of saline resulted in an unexpected transient increase in blood pressure from 150/100 to 180/100, with no change in heart rate, before stabilising to baseline values (Figure 15E).
• Rats and surgery Sprague Dawley rats were obtained from Charles River (220-230 g). Rats were acclimizatized for 48 h prior to experiments. Rats were anesthesized with halothane and nephrectomized (right kidney) and placed back into the vivarium for seven days. Following this post-operative recovery time, rats weighed 250-270 g.
• Rats were then anesthesized with sodium pentobarbital at a dose of 36 mg/ kg (i.p.) and maintained under
• a catheter was placed in the right carotid artery (PE 50) for measuring blood pressure, one in the left jugular vein (PE 60) for injection of vehicle, peptide, or drugs and one in the left ureter (PE50) for collecting urine.
• PE 50 right carotid artery
• PE 60 left jugular vein
• PE50 left ureter
• MAP mean arterial pressure
• V2 antagonist Peptide V2 receptor peptide antagonism and its effects on MAP and urine output was assessed by single bolus dose of 1 mg peptide (V2-TM VII:
• LMLLASLNSCTNPWIY in 200 ml vehicle (water with 10% DMSO and protease inhibitors).
• peptides act as antagonists of V2 receptors.
• Example 7 Inhibition of HIV infectivity by antagonists of the CCR5 receptor
• CCR5 Peptide Antagonist Treatments 10 mg of peptide (CCR5-TM 1: LYSLVFIFGFVGN-NH2) was dissolved in 500 ml of DMSO, and 50 ml was used to pretreat 1 ml of cultured cells (4 X 10x6 cells/well) for 4 h.
• CCR5-TM 1 LYSLVFIFGFVGN-NH2
• concentration at this stage was 0.5 mg/ml. Peptide concentration was then diluted in half upon addition of virus for 2 h, in a total assay volume of 2 ml.
• HIV-1 Infectivity Assay As Assessed By HIV-RT Activity: HIV-1 Infectivity Assay was performed according to methods described by Mark A. Wamberg (Soudeyns et al., Antimicrobial Agents and Chemotherapy 35, 1386-1390, 1991).
• HIV-IIIB isolate of HIV-1 was employed because of its high infectiousness. Briefly, human PBMC cells were infected at a multiplicity of infection of 0.1. To determine the levels of inhibition of HIV-1 adsorption, by CCR5-TM1, PBMC cells were pretreated for 1/2 h with the peptide, exposed to the virus for 2 h and viral infection was assessed 7 days post-infection by
• CCR5-TM 1 LYSLVFIFGFVGN-NH2
• CCR5-TM 1 LYSLVFIFGFVGN-NH2
• C Peptide inhibition of HIV cellular adsorption and uptake as assessed by P24-ant ⁇ gen production: In PBMC cells pretreated with CCR5-TM1, the HIV P24 level was 4730 pg/ml compared to 20960 pg/ml for non-peptide treated cells. These data indicate 77% inhibition of HIV infection by the CCR5 peptide antagonist (Fig. 17B).
• Example 8 Inhibition of EGF-mediated tyrosine kinase activity
• the human A431 cell line (ATCC CRL 1555) was established from an epidermoid carcinoma of a 85 year old female patient. It expresses an extremely high number of EGF receptors on its cell surface (3 x10E6/cell), due, at least in part, to the amplification of EGF receptor DNA sequences (30-fold).
• EGF receptor tyrosine kinase enzyme assay The Biotrak* assay system and kit by Amersham Life Science was used. In brief, the system is designed to detect epidermal growth factor receptor tyrosine kinase enzyme activity in solubilized tissues and cells. Enzyme present in the samples will catalyze the transfer of the g-phosphate of adenosine-5 t -tr ⁇ phosphate to the tyrosine group on a peptide which is specific for EGFr tyrosine kinase domain. Specific detection of the enzyme is further assured by using epidermal growth factor to activate the EGF receptor tyrosine kinase enzyme activity.
• EGF dependent tyrosine kinase activity may be determined from the difference between the enzyme activity in the presence or absence of added epidermal growth factor.
• the assay is performed at pH 7 .4 in Hepes buffer with MgCl 2 as the essential metal ion.
• the assay will give linear incorporation of phosphorus-32 into substrate peptide corresponding to at least 20% of ATP
• Phosphorylated peptide is separated on binding paper. After washing the paper, the extent of phosphorylation may be detected by scintillation counting.
• a solubilized membrane preparation was prepared from A431 cells maintained in culture as a monolayer, and premcubated for 30 min at 30 °C with various
• EGFR3-TM EGF-erb3 receptor
• D2- TM VII-TWLGYVNSA GABA-A: GIFNLVYW
• tyrosine kinase assay concentrations of a peptide derived from the TM domain of the EGF-erb3 receptor (EGFR3-TM: LTVIAGLWIF) or with peptides derived from TM domains of other receptors (D2- TM VII-TWLGYVNSA; GABA-A: GIFNLVYW) prior to tyrosine kinase assay.
• EGF-erb3 receptor TM peptide inhibited EGF mediated tyrosine kinase activity.
• the EGFr peptide antagonist (LTVIAGLVVIF) inhibited EGF mediated tyrosine kinase activity in a dose-dependent manner, in comparison to a peptide derived from GABA TM domain (GIFNLVYW) (Fig. 18).
• Peptide D2-TM VII (TWLGYVNSA) had a similar lack of effect on EGF mediated tyrosine kinase activity (data not shown).
• a peptide derived from E. Coli ATPase Fo b subunit, GQAIAFVLFVL and the peptide derived from E. Coli ATPase Fo c subunit, LAAIGAAIGIGILG were tested for antimicrobial activity in comparison to a peptide derived from the GABA-A ion channel (GIFNLVYW), and a peptide derived from the D2 dopamine receptor (D2-TM VII- TWLGYVNSA) .
• 5 mg of each peptide was dissolved in DMSO and diluted with water (DMSO concentration was 10% of the final volume). When tested alone, 50 ml of peptide solution was used for each 500 ml culture. When tested in combination, 25 ml of each peptide solution was used for each 500 ml culture.
• E. coli strain LE392 was grown in LB medium to a density of O.D.600 between .8-1.0. A 10:1 dilution was made for a final volume of 500 ml. 50 ml of test peptide (5 mg/ml stock concentration) was added to the culture (0.5 ug/ul, final peptide concentration) which was then grown at 37 C for 2 h. 50 ml of the culture was then plated on to LB plates at 10 -6 dilution. Plates were then incubated overnight and colonies counted the next morning. The remaining culture was stored at 4 °C overnight. 50 ml of this culture was then plated the following day on to LB plates at 10 -6 dilution. Plates were incubated overnight and colonies counted next morning.
• mtracerebral guide cannulae (Plastic Products Company, Roanoke, VA, U.S.A.) for the microdialysis probes
• ketamine 66 mg/kg
• pentobarbital 22 mg/kg
• the cannula was inserted into the medial nucleus accumbens (coordinates relative to bregma: A +1.5, L +1.3, V +7.6, Paxinos and Watson, 1982) or caudate nucleus
• the steel insert from the guide cannula was replaced by the dialysis probe (2 mm, CMA/12, Carnegie Medicin, Sweden) and perfused with artificial CSF (NaCl 145 mM, KCl 2.7 mM, CaCl 2 .2H 2 O 1.2 mM, MgCl 2 1.0 mM, Na 2 HPO 4 2 mM, ascorbic acid 0.2 mM, pH 7.4) at a rate of 1 ml/min. Perfusion in the awake, unrestrained and mobile animals was continued for 3-4 hours until the basal efflux of dopamine and its
• Dialysate was collected over 30 min periods and injected directly into a HPLC system equipped with a Biophase ODS 5 mm, 4.6 x 250 mm column.
• the mobile phase consisted of 50 mM sodium phosphate monobasic, 0.5 mM EDTA, 1.8 mM sodium octyl sulfonate, 14 % methanol, with pH adjusted to 3.50 with phosphoric acid. Sensitivity of dopamine detection was 2 pg. The percentage recovery of dopamine through the dialysis cannula was calculated each time.
• An antagonist peptide for the dopamine transporter was made based on the amino acid sequence of TM-12 (DAT-TM XII :ALGWIIATS).
• DAT-TM XII :ALGWIIATS An antagonist peptide for the dopamine transporter
• Fig. 20 solid circles.
• Pretreatment with the antagonist peptide for the dopamine transporter 70 mg given intracerebroventricularly by slow infusion in 7 ml of buffered saline 12 min before injection of cocaine
• Intracerebroventricular injection of the dopamine transporter antagonist peptide alone in doses of 70 mg or 98mg had no effect on dopamine release in striatum (Fig. 21).
• the HIV P24 level was 14970 pg/ml compared to 20960 pg/ml for non-peptide treated cells.

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