WO1998000538A2 - Peptides and peptidomimetic compounds affecting the activity of g-protein-coupled receptors by altering receptor oligomerization - Google Patents
Peptides and peptidomimetic compounds affecting the activity of g-protein-coupled receptors by altering receptor oligomerization Download PDFInfo
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- WO1998000538A2 WO1998000538A2 PCT/IB1997/000814 IB9700814W WO9800538A2 WO 1998000538 A2 WO1998000538 A2 WO 1998000538A2 IB 9700814 W IB9700814 W IB 9700814W WO 9800538 A2 WO9800538 A2 WO 9800538A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to novel peptides and/or peptidomimetic compounds, methods for generating such compounds, and the use of such compounds to affect receptor oligomerization of G protein-coupled receptors that form multimeric associations for activity.
- GPCRs G protein-linked receptors
- G proteins The class of receptors known as G protein-linked receptors (GPCRs) are typically characterized by a 7-helix organization, whereby the receptor protein is believed to traverse the membrane seven times. GPCRs share a common signalling mechanism, whereby signal transduction across the membrane involves intracellular transducer elements known as G proteins.
- GDP guanosine diphosphate
- GTP guanosine triphosphate
- GTP When GTP is bound to its surface, the G protein regulates the activity of an effector.
- effectors include enzymes such as adenylyl cyclase and phospholipase C, channels that are specific for calcium ions (Ca 2+ ), potassium ions (K + ), or sodium ions (Na + ) and certain transport proteins.
- activation of GPCRs by transmitters will induce one or another of the following effector responses: activation of adenylyl cyclase, inhibition of adenylyl cyclase or stimulation of phospholipase C activity.
- activation of adenylyl cyclase activation of adenylyl cyclase
- inhibition of adenylyl cyclase or stimulation of phospholipase C activity.
- phospholipase C activity When the effector adenylyl cyclase is either activated or inhibited it produces changes in the concentration of the molecule cyclic adenosine monophosphate
- IP 3 inositol triphosphate
- DAG diacylglycerol
- IP 3 then causes calcium ions (Ca 2+ ) to be released into the cytoplasm.
- Alterations in cellular levels of cAMP and Ca + are two of the most important intracellular messages that in turn act to alter the behaviour of other target proteins in the cell.
- GPCRs may be classified according to the type of signalling pathway they activate in cells. This occurs at the level of the G proteins, which detect and direct signals from diverse receptors to the appropriate effector-response pathway.
- the three main groups of G proteins are: Gs-like, which mediate adenylyl cyclase activation; Gi-like, which mediate inhibition of adenylyl cyclase; and Gq-like, which mediate activation of phosphoplipase C. Since one receptor can activate many G proteins, the signal can be greatly amplified through this signal transduction pathway.
- GPCRs A wide variety of chemical messengers involved in regulating key functions in the body act through GPCRs. These include neuro transmitters such as dopamine, acetylcholine and serotonin, hormones of the endocrine system such as so atostatin, glucagon and adrenocorticotropin, lipid mediators such as prostaglandins and leukotrienes and immunomodulatory proteins such as interleukin-8 and monocyte-chemoattractant polypeptide.
- the family of GPCRs also includes the receptors for light (rhodopsin), for odors (olfactory receptors) and for taste (gustatory receptors). Over one hundred different G protein-coupled receptors have been identified in humans, and many more are expected to be discovered. All or most of these receptors are believed to utilize one of the three principal
- G protein-effector signalling pathways stimulation or inhibition of adenylyl cyclase or activation of phospholipase C.
- 5-HT 1A 5-HT. is 5-HT 4 5-HT 2 Serotonin
- GPCRs are believed to function as monomeric units. Diagrams and text describe the majority of members within this class of receptors as singular units, generally spanning the membrane seven membrane times (For eg. see: descriptions of the muscarinic receptor in Basic N euro chemistry, fifth edition, eds: Siegel et al; Raven Press: N.Y.;254-259, 1994). However, it must be noted that there are some GPCRs that are not members of the seven membrane spanning family. For example, the receptor for insulin-like growth factor II that directly activates G ; . 2 , has only a single membrane spanning domain.
- the human ⁇ 2 adrenergic receptor has been used as a model to illustrate the common structural features shared by members of the GPCR family (Kobilka, G., Annu Rev. Neurosci, 15:87-114, 1992).
- Evidence from biochemical Dohlman et al., J. Biol. Chem., 262:14282-14288, 1987
- immunologic studies of the topology of the ⁇ , adrenergic receptor Wang et al. J. Biol. Chem, 264: 14424-14431, 1989
- most GPCRs comprise seven membrane spanning domains.
- Such drugs may be classified into two types: 1) agonists, which mimic the action of natural transmitter by provoking activation of G protein-effector signalling pathways when they bind to the transmitter site; and 2) competitive antagonists, which block the binding of the transmitter by occupying the transmitter binding site but do not themselves activate G protein-effector pathways.
- a useful analogy is that of a lock and key, whereby agonists are different keys which are able to open the same receptor lock, whereas antagonists will block the key-hole but will not open the lock.
- compounds which can bind to a specific region of the receptor are called ligands: agonists and antagonists are ligands which bind to the transmitter recognition site on the receptor.
- Ligands that block or otherwise interfere with the interaction of agonists with the receptor, and thereby prevent agonists from activating the receptor are known as competitive antagonists. These compounds are generally thought to act by binding to the transmitter site, but to possess no intrinsic activity themselves (i.e. they do not turn on the signalling function of the receptor.) Studies have shown that competitive antagonists can be further categorized into two closes, 'neutral antagonists' which block agonist binding but have no effect on signalling, and 'inverse agonists' (also known as negative antagonists) which can inhibit the 'background' or basal level of signalling displayed by receptors in the absence of agonists.
- the present invention resides in the discovery that novel peptide compounds modelled on the transmembrane region of GPCRs can selectively modulate the function of such receptors by affecting the ratio of receptor monomer to multimeric forms (homo-meric or hetero-meric).
- the present invention relates to novel short peptides of a preferred length of up to about 15 - 20 amino acid residues, or peptidomimetic compounds, modeled on transmembrane domains of GPCRs which form oiigomers (eg. dimers) for activity that can be used to selectively affect activities of GPCRs.
- a working example is provided, based on residues 276 - 296 of the ⁇ 2 -adrenergic receptor, wherein the peptide inhibits agonist promoted stimulation of adenylyl cyclase activity.
- the present invention also provides for novel peptides modelled on the transmembrane region of GPCRs that can selectively modulate the function of such receptors by affecting the ratio of receptor monomer to multimeric forms (homo-meric or hetero-meric) that can be greater than 20 amino acid residues but less than 50 amino acid residues.
- the present invention also provides one skilled in the art with the ability to model a u-ansmembrane domain of GPCRs which form multimers for activity in order to generate novel peptides or peptidomimetic compounds that selectively affect receptor oligomerization (eg. dimerization), thereby selectively affecting receptor function.
- novel peptides or peptidomimetic compounds of this invention may be utilized in compositions and methods for specifically controlling certain GPCR activities.
- the invention also involves a process for affecting GPCR activity in mammals which comprises: administering to a subject an effective amount of the novel compound to affect GPCR activity.
- a further embodiment involves a pharmaceutical preparation for treating disease and psychoses which comprises administering a pharmaceutically effective amount of the novel peptide or peptidomimetic compound, with a suitable pharmaceutical carrier, sufficient to affect GPCR.
- Another aspect of this invention involves generating peptides and peptidomimetic compounds that are useful for in vitro and in vivo studies of GPCRs.
- the compounds of the present invention may be prepared by chemical synthesis techniques, commercially feasible amounts may be produced inexpensively. Moreover, because the compounds of the present invention are relatively small and may be peptidergic in nature, they are less likely to stimulate an undesirable immune response in patients treated with them.
- Figure 1 shows immuloblotting of human ⁇ 2 AR expressed in Sf9 cells.
- Crude membrane preparations (lane l), digitonin-solubilized membrane proteins (lane 2) and affinity purified receptors (lane 3) derived from Sf9 cells expressing either c-mvc tagged (lane 3) or HA-tagged (lanes 1 and 2) ⁇ 2 AR were immunoblotted following SDS-PAGE using the appropriate antibody (9E10 and 12CA5, respectively).
- the blots reveal immunoreactive bands coixesponding to the expected monomeric form (43-50kDa) as well as a higher moleculai * weight species (85-95 kDa).
- the right panel illustrates immunoblots of crude membrane preparations derived from Sf9 cells expressing HA-tagged ⁇ : AR treated (lane 5) or not (lane 4) with the membrane-permeant photoactivatible crosslinker BASED. Position of receptor bands are denoted by arrows and molecular weight markets are as shown.
- Figure 2 shows effects of various peptides and ⁇ 2 AR ligands on receptor dimerization.
- Lanes 1 and 2 c-myc (lane 1) or anti-HA (lane 2) mAbs.
- the two immunoprecipitates were then immunoblotted with the anti- HA mAb.
- the occurrence of dimerization between the HA- and c-myc-tagged receptors is revealed by the fact that the HA-tagged ⁇ 2 AR is co-immunoprecipitated with the c-myc tagged receptor by the anti-c-myc mAb (lane 1).
- Lanes 3 and 4 c-myc tagged ⁇ 2 AR was expressed in Sf9 cells and immunoprecipitated with anti-c-myc mAb. The immunoprecipitates were then immunoblotted with either anti-HA (lane 3) or anti-c-myc or anti-c-myc (lane 4) mAbs. Lanes 5 and 6: HA-tagged ⁇ 2 AR was expressed in Sf cells, immunoprecipitated with anti-HA mAb and then immunoblotted with either anti-c-myc (lane 5) or anti-HA (lane 6) mAbs. These controls demonstrate the specificity of each antibody towards their respective targets.
- Lane 7 and 8 HA-tagged ⁇ ,AR and c-myc tagged M2 muscarinic receptors were co-expressed in Sf9 cells, immunoprecipitated with either anti-HA (lane 7) or anti-c-myc (lane 8) mAbs. Immunoblotting with the anti-c-myc mAb did not reveal the presence of a ⁇ 2 AR/M2 muscarinic receptor heterodimer (lane 8). Results shown are representative of three separate experiments.
- FIG. 3 demonstrates Immunoblotting of V2-vasopressin receptors (V2-R) expressed in COS-7 cells.
- V2-R V2-vasopressin receptors
- Crude membrane preparations from COS-7 cells transiently transfected with c-myc tagged V2-R (lane 1) or c-myc tagged V2-R truncation mutant ol 1 (lane 2) were immunoblotted with the anti-c-myc mAb.
- the molecular weight markets are as shown. Square brackets highlight the dimeric species of both ildtype and 0-11 V2 vasopressin receptors while asterisks denote the monomeric species. Data are representative of three independent experiments.
- Figure 4 shows effects of various peptides on receptor dimerization.
- TM VI peptide [residues 276-296: NH 2 - GUMGTFTLCWLPFFIVNIVH-COOH] at a concentration of 0.15 ⁇ g/ ⁇ L for 0 (lane 1), 15 (lane 2), 20 (lane 3) or 30 minutes (lane 4). Membranes were then subjected to SDS-PAGE, transferred to nitrocellulose and immunoblotted with the anti-c-myc antibody. A representative immunoblot is shown.
- Figure 5 demonstrates, in A, effects of increasing concentrations of TM VI peptide on the amount of ⁇ 2 AR dimer.
- Increasing concentrations (0-6.3 mM) of the peptide were added to purified c-myc tagged ⁇ ,AR and the amount of dimer assessed by immunoblotting using the a i c-myc mAb (lanes 1 - 8).
- lanes 1 - 8 purified ⁇ 2 AR was treated (lane 10) or not (lane 9) with the D2 TM VII peptide.
- the data shown are representative of three distinct experiments.
- control peptides used to determine the selectivity of the effect observed with the TM VI peptide included one derived from the C-terminal tail of the ⁇ 2 AR [residues 347-358 NH 2 -LKAYGNGYSSNG-COOH] or an additional control peptide unrelated to the ⁇ 2 -AR but of similar size as the TM VI peptide [NH 2 - SIQHLSTGHDHDDVDVGEQQ-COOH] were also found to be without effect on the amount of dimer (data not shown).
- B Densitometric analyses of three experiments similar to that shown in B. The relative intensity of the dimer is expressed as percent of total receptor (monomer + dimer) immunoreactivity.
- FIG. 1 shows superimposed densitometric scans of immunoblotted receptors which were previously treated with increasing concentrations of the TM VI peptide.
- the monomer is denoted by M while the dimeric species is marked by D.
- the concentration of peptide added for the curves shown was: none ( ), 0.07 mM (—...- — ), 0.05 mM ( — — ), and 1.25 mM ( ).
- Figure 6 demonstrates effects of TM VI peptide on ⁇ 2 AR stimulated adenylyl cyclase activity in Sf cells.
- Membrane preparations derived from ⁇ 2 AR expressing Sf9 cells were either not treated (open circles), or treated with TM VI peptide (closed squares), control peptide TM VI Ala (closed circles), or second control peptide from TM VII of the D2 dopamine receptor (open triangles).
- Isoproterenol stimulated adenylyl cyclase activity was then assessed for these membranes. Data are expressed relative to the maximal stimulation obtained with the untreated membranes and represent mean +/- SEM for 8 independent experiments. Peptides were used at a concentration of 0.15 ⁇ g/ ⁇ l.
- Figure 7 shows effects of ⁇ 2 AR ligands on receptor dimerization.
- Figure 8 depicts effects of TM VI peptide on ⁇ 2 AR expressed in mammalian cells.
- Figure 9 demonsu-ates sequence data collated from numerous published articles oriented to compare the peptide sequences of the putative TM VI regions of twenty-seven GPCRs. While hydropathy analysis may yield results of uncertain reliability when identifying the TM VI and TM VII domains of particular groups of GPCR, sequence analysis can identify the "GGL motif," corresponding to TM VI, with greater certainty.
- IP inositol phosphate
- the abbreviation BASED is bis [ ⁇ -(4 azidosalicylamindo) ethyl] disulphide.
- 5-HT is 5-hydroxytryptamine.
- DOI 2,5-dimethoxy-4-iodoamphetamine hydrobromide.
- PBS phosphate buffered saline.
- ⁇ 2 AR is ⁇ 2 -adrenergic receptor.
- GPCR G protein-coupled receptor
- GpA glycophorin A
- HA influenza hemagglutinin.
- TM VI transmembrane domain 6.
- NDI nephrogenic diabetes insipidus.
- any amino acid as used herein includes the L-isomers and D-isomers of the naturally occurring amino acids, as well as other "non-protein" ⁇ -amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogues of naturally occurring peptides.
- the naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, Y-carboxyglutamic acid, arginine, ornithine and lysine.
- non- protein -amino acids include norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Hyp), isonipecotic acid (Inp), homoserine, cyclohexylglycine (Chg), -amino-n-buty ⁇ * ic acid (Aba), cyclohexylalanine (Cha), aminophenylgutyric acid (Pba), phenylalanines substituted at the oitho, meta, or para position of the phenyl moiety with one or two of the following: a (C,-C 4 ) alkyl, a (C r ) alkoxy, halogen or nitro groups or substitute with a methylenedioxy group, ⁇ -2- and 3-thienylalanine, ⁇ -2- and 3-furanylalanine, ⁇ -2-, and 3- and 4-pyridylalanine
- amino acid containing an aryl side chain means any amino acid having an aromatic group.
- Tyrosine, phenylalanine, tryptophan, O-sulfate esters of tyrosine and 5-nitrotyrosine exemplify such amino acids.
- polar amino acid means any amino acid having an uncharged side chain which is relatively soluble in water. Examples include glutamine, asparagine, glycine, serine, hydroxyproline and homoserine.
- hydrophobic amino acid means any amino acid having an uncharged side chain which is relatively insoluble in water. This group includes leucine, valine, tryptophan, norleucine, norvaline, alloisoleucine, thioproline, de hydro pro line, cyclohexylalanine and cyclohexylglycine.
- patient refers to any mammal, especially humans.
- backbone chain refers to the portion of a chemical structure that defines the smallest number of consecutive bonds that can be traced from one end of that chemical structure to the other.
- the atomic components that make up a backbone chain may comprise any atoms that are capable of forming bonds with at least two other atoms.
- peptide-lead refers to the undervitized peptide that is modelled on one of the transmembrane domains in the first step of the design of the compounds of this invention.
- Example III Examples of peptide-leads are listed in Example III.
- the te ⁇ n "parent GPCR” as used herein, refers to the GPCR from which the peptide-lead is derived.
- Molecules of this invention ai'e designed using the peptide-lead as the model for GPCR-peptides that ai'e selectively inhibitory towards the parent GPCR or closely related receptors.
- GPCR-peptide refers to peptides, modified or unmodified, which have been designed and synthesized according to the methods of this invention, that can be used to selectively prevent or disrupt functional aggregation of GPCR's which form multimers (eg. dimers) for activity.
- oligomeric GPCR refers to G protein-coupled receptors that form oligomers (eg. dimers) and that such interactions have functional consequences. Oligomers can be homo-multimeric or hetero - multimeric .
- selective inhibition and “selectively inhibits” as used herein, refers to the ability of the peptide to inhibit the actions of a species of GPCR and/or closely related receptors without affecting the activity of other non-related species of receptors to any significant extent.
- Significant extent means there must be at least a 10-fold magnitude difference in inhibitory activities between the parent receptor and other types of receptors.
- the present invention relates to novel peptides and/or peptidomimetic compounds that are modeled on the transmembrane region of GPCRs.
- the peptides and peptidomimetic molecules of this invention selectively bind to the GPCR from which they were designed. This property allows the molecules of this invention to affect those activities of the GPCR that are mediated through this receptor.
- the GPCR-peptides of the present invention are characterized by complimentarity to one of the transmembrane domains of the parent receptor. Preferably, that region is modelled on one of the transmembrane domains.
- the GPCR-peptides of this invention are further characterized by: (1) the ability to affect (positively or negatively) the activity of the parent GPCR and possibly closely related receptors, and (2) the inability to significantly affect other types of receptors (i.e. other receptors can be inhibited if there is a 10-fold magnitude difference in activities)
- TMS Candidate Transmembrane Sequences
- Each transmembrane sequence provides a potential model for a peptide-lead which will be used to design peptides or peptidiomimetic compounds that could function to disrupt or prevent functional aggregation of the same GPCR (parent-GPCR) and possibly closely related receptors.
- the first step is to identify the transmembrane domain of the receptor of interest.
- techniques well known in the art available for determining transmembrane regions of the GPCR. These include hydropathy plots to identify the hydrophobic segments.
- the secondary structure can also be analyzed to identify alpha-helix structures.
- GPCRs of the family A/rhodopsin related subfamily share a number of features common to all members of its class.
- One of these features is the presence of recurring patterns in their amino acid sequence.
- Each transmembrane domain (TM) can be characterized by a recurring pattern that is unique for that TM.
- the alignment of the TMs is therefore based on recurring patterns rather than on homology alone.
- the patterns used were:
- TM2 I O XDXXXXXXXP or LXXXDXXXXXXP TM3 : SXXXLXXD XDR or SXXXLXXI XXHR
- TM4 WXXXXXXXP or WXXXXXXXP
- TM5 FXXPXXXXXXY
- TM6 FXXCXXP
- TM7 LXXXXXXDPXXY or LXXXXXNPXXY
- a "sequence identifier” is assigned to each position in the alignment.
- the "extended notation” convention is applied. This reads as follows:
- transmembrane (TM) sequences it may not be desirable to use the hydropathy methods for determining transmembrane (TM) sequences.
- TM VI and TM VII, respectively the hydrophobic sequence patterns are not as clear.
- other methods such as comparisons of a unique amino-acid motif found within one successful peptide-lead may be useful.
- a unique amino acid motif of the GPCR of interest such as the GGL motif found within the TM VI sequence of the ⁇ 2 AR
- one skilled in the art could predict with which groups of receptors within the GPCR family a polypeptide of a particular amino acid sequence might interact more or less strongly, and with which ones that same polypeptide would be unlikely to interact.
- one skilled in the art might also be able to select a sequence structure for a peptide or peptidomimetic compound in order to select the breadth or nai'rowness of interactions amongst related GPCRs, as might be desired.
- hydropathy analysis may yield results of uncertain reliability when identifying the
- TM VI and TM VII domains of particular groups of GPCR sequence analysis identifies a "GGL motif, corresponding to TM VI, with greater certainty.
- Data presented in Figure 9 demonstrates that the reproducibility of the GGL motif between GPCRs is grouped into subgroups of receptors with similar functions.
- the GGL motif is located in a transmembrane region of the GPCR that is not known to be involved in any of the domains recognized as participating in protein-protein interactions within the signal transduction complex. A possible relationship exists between the GGL sequence and GPCR specificity which could be compatible with a self-recognition role for this domain.
- GPCR might be predictable. This in turn provides a possible method of predicting the selectivity (or otherwise) of the action of any particular TM VI peptide.
- the molecule In designing a GPCR-peptide according to this invention, two important considerations must be taken into account. First, the molecule must be able to physically associate with the parent-GPCR. The present theory of peptide binding suggests that the initial step in binding requires, at a minimum, an ionic interaction between the receptor and the peptide. It is also probable that other molecular interactions, such as hydrogen bonding and hydrophobic interactions, ai'e important for this association. Therefore, the identification and maintenance of these interactions are critical in designing a potent GPCR antagonist.
- the second consideration in designing the GPCR-peptides of this invention is secondary and tertiary structure. While certain portions of the GPCR-peptide will not directly participate in molecular interactions with the receptor, they may play a role in the overall conformation of the GPCR-peptide. This, in turn, can have a dramatic effect on potency. If the GPCR-peptide cannot assume the proper conformation, the molecular- interactions required for association with the receptor cannot be achieved, even if the components capable of forming such interactions are present in the molecule.
- GPCR-peptides of this invention must be designed so that they assume a conformation which allows them to associate with the receptor.
- Conformational requirements may be in the nature of overall three-dimensional structure and orientation of the GPCR-peptide, or merely the spacing between two sites on the GPCR-peptide which directly interact with the receptor.
- Those alanine-substituted peptides which retain an ability to prevent or disrupt functional aggregation of GPCRs which form multimers for activity indicate portions of the peptide-lead that do not directly interact with the receptor and which do not have side chains which play a critical role in the folding of the GPCR-peptide.
- Such peptides are preferred peptide-leads and GPCR-peptides of the present invention.
- those peptides which lack or have greatly reduced disruptive activity point out areas of the peptide-lead and GPCR-peptide that are important for activity. These latter peptides suggest the nature of an important interior intramolecular interaction based upon the amino acid substituted for.
- an arginine-to-alanine substitution which resulted in reduced activity suggests the location of an important positive charge - either an ionic interaction with the receptor or an intramolecular ionic interaction within the peptide - which is required to maintain optimal conformation.
- a serine-to-alanine substitution which had a negative effect on activity indicates the location of an important hydrogen bond.
- the hydrogen bond may be between the peptide-lead and the GPCR, or it may be an intramolecular hydrogen bond that plays an important role in the conformation of the peptide-lead.
- a single position deletion analysis is performed.
- a series of peptides containing single deletions at positions which do not affect inhibitor activity are synthesized and assayed for activity.
- the peptides from this series that retain significant activity indicate areas of the peptide-lead and GPCR-peptide that are not essential for proper conformation.
- Such peptides are also included within the scope of this invention.
- Deletion peptides from this series which have significantly lower attenuating activity indicate the location of components which provide critical spacing in the peptide-lead or GPCR-peptide. This may be verified by replacing the deleted amino acid with a different, yet analogous structure. For example, substitution of any conformationally important amino acid with a three carbon alkyl chain without a significant loss of activity confirms that spacing is critical at that part of the molecule.
- Additional information about important structural and conformational features necessary for designing a potent GPCR-peptide of this invention may be obtained through 3-dimensional X-ray crystailographic procedures coupled with computer modelling. Specifically, one of ordina ⁇ * y skill in the ait may analyze a GPCR/peptide-lead using such a method. Alternatively, one of average skill in the ait could employ multiple alanine substitutions or multiple deletions to identify important intramolecular interactions in the antagonist itself. It will also be apparent that each new GPCR- peptide designed and tested will, itself, provide additional information about structural features important for inhibition of GPCR activity.
- GPCR- peptides of this invention may be designed and synthesized. This is achieved by substituting the identified key residues of the peptide-lead with other components having similar features. These substitutions will initially be conservative, i.e., the replacement component will have approximately the same size, shape, hydrophobicity and charge as the key residue. Those of ordinary skill in the art ai'e well aware of appropriate replacements for a given amino acid [Dayhoff et al., in Atlas of Protein Sequence and Structure No. 5, 1978 and Argos et al., EMBO J., 8, pp. 779-85 (1989)].
- Typical conservative substitutions for an amino acid are other amino acids with similar charges, for example, aspartic acid for glutamic acid, arginine for lysine, asparagine for glutamine, hydroxyproline for proline and vice versa. Substitutions with non-natural amino acids may also be performed to reduce the peptidic nature of the peptide-lead. Some examples are cyclohexylalanine for tyrosine, sarcosine for glycine. statine for threonine and homoarginine for arginine. These modifications may increase the biological stability of the antagonist, in addition to increasing its potency.
- the molecule containing the substitute component is shown to be a compound effective for selectively preventing or disrupting the aggregation of GPCRs, less conservative replacements may be made at the same position.
- substitutions typically involve the introduction of non-amino acid components which contain the important feature imparted by the amino acid at that position.
- Such substitutes are well-known in the art.
- the sequence Leu-Val-Arg (corresponding to amino acids 65-67 of thrombin) can be replaced by p-guanidinobenzoic acid. This substitution maintains the hydrophobicity of Leu-Val, as well as the guanidinium functionality of Arg.
- GPCR-peptides according to this invention may be designed by insertions at various sites along the peptide-lead. To determine areas of the peptide-lead where a component may potentially be inserted, a series of peptides having a single alanine insertion at various sites is synthesized. Those peptides from this series which retain activity for selectively preventing or disrupting functional aggregation of GPCRs which form multimers for activity indicate potential insertion sites.
- a component to be inserted In choosing a component to be inserted, one should be guided by the same considerations set forth above in selecting a substitute component. Specifically, one must keep in mind how the insertions may potentially affect the moleculai" interactions between the GPCR-peptide and the GPCR and how they affect conformation of the GPCR-peptide. For example, the insertion of an anionic component adjacent to a critical cationic amino acid in the peptide-lead could interfere with an important ionic interaction and should therefore be avoided. Similarly, the insertion of a component which is known to cause structural perturbations, e.g., a proline, should also be avoided.
- Cyclization may allow the peptide to assume a more favorable conformation for association with the GPCR. Cyclization may be achieved by methods well-known to those in the art. One method is the formation of a disulfide bond between two non-adjacent cysteine residues (D- or
- the most preferred peptides of the present invention are modelled after the peptide-lead which comprises the formula: SEQ ID NO: 1: NH 2 -GIIMGTFTLCWLPFHVNIVH-COOH.
- SEQ ID NO: 1 NH 2 -GIIMGTFTLCWLPFHVNIVH-COOH.
- the GPCR peptide is entirely peptidic and is synthesized by solid-phase peptide synthesis techniques, solution-phase peptide synthesis techniques or a combination thereof which constitute the most cost- efficient procedures for producing commercial quantities of these peptides.
- non-protein amino acids When “non-protein” amino acids are contained in the GPCR peptide, they may be either added directly to the growing chain during peptide synthesis or prepai'ed by chemical modification of the complete synthesized peptide, depending on the nature of the desired "non-protein” amino acid. Those of skill in the chemical synthesis art are well aware of which "non-protein” amino acids may be added directly and which must be synthesized by chemically modifying the complete peptide chain following peptide synthesis.
- GPCR peptides of this invention which contain both non-amino acid and peptidic portions is preferably achieved by a mixed heterologous/solid phase technique.
- This technique involves the solid-phase synthesis of all or most of the peptide portion of the molecule.
- the peptide chain can be prepai'ed by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence.
- various n-protecting groups e.g., the carbobenzyloxy group or the t- butyloxycarbonyl group (BOC)
- various coupling reagents e.g., dicyclohexylcarbod ⁇ mide or carbonyldimidazole
- various active esters e.g., esters of N-hydroxypthalimide or N-hydroxy- succinimide
- vaiious cleavage reagents e.g., trifluoracetic acid (TFA), HCl in dioxane, boron tris-(trifluoracetate) and cyanogen bromide
- reaction in solution with isolation and purification of intermediates is well-known classical peptide methodology.
- a preferred peptide synthesis method follows conventional Merrifield solid-phase procedures. See
- peptidomimetic There are a number of methods for designing peptidomimetic compounds that are known in the art.
- the starting point for designing a peptidomimetic compound is the sequence and/or conformation of a particular oligopeptide or peptide of interest.
- a particular oligopeptide or peptide of interest For example, see, Spatola, A.F. Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins (Weistein, B, Ed.), Vol. 7, pp. 267-357, Marcel Dekker, New York (1983), which describes the use of methylenethio bioisostere [CH,S] as an amide replacement in enkephalin analogues; and Szelke et al., In Peptides: Structure and Function, Proceedings of the Eighth American Peptide Symposium, (Hruby and Rich,
- the method involves computer simulation of the peptide in a manner that simulates a real-size primary structure in an aqueous environment, shrinking the size of the polypeptide isobarically and isothermally, and expanding the simulated polypeptide to its real size in selected time periods.
- a useful set of tools, terms Balaji plots, energy conformational maps and probability maps, assist in identifying those portions of the predicted peptide structure that are most flexible or most rigid.
- the ability of the peptides and peptidomimetic compounds of this invention to selectively affect oligomerization of GPCRs which form multimers for activity can be measured by choosing from the many techniques available in the art. In general, the goal of these types of assays would be to measure the ratio of monomeric receptor to multimeric receptors (dimers, trimers, etc.) The change in ratio of the relative amounts of monomer to multimer will reflect conversion of monomers to multimers or vice versa.
- results of these assays are correlated with measurements of the activity of the GPCR (using techniques described below), one skilled in the art will be able to ascertain whether the peptide-lead, GPCR-peptide or peptidomimetic compound will interfere with the functional aggregation of receptor subunits which form multimeric associations for functional activity.
- Those compounds which promote oligomerization would be predicted to have one activity (eg. agonist or positive efficacy) while those which promote dissociation of oligomers would be predicted to demonstrate opposite activity (eg. inverse agonists or negative efficacy).
- the magnitude of change in ratio and/or rate of change effected by the compound would provide a measure of the compound's efficacy and/or potency in modulating receptor activity.
- Measuring the ratio of monomeric receptor to multimeric receptor there ai ' e many different techniques available for determining the relative amount of monomer to multimer (eg. dimer) formed in the presence and absence of the peptide-lead, GPCR-peptide, or peptidomimetic compound.
- different assay systems can be designed to measure the ability of compounds to modify the ratio of monomers/multimers.
- any procedure that permits measurement of the relative amounts of monomer and oligomer in receptor preparations eg. membranes, solubilized receptor preparations, purified receptors, etc) can be used.
- a sample containing the compound to be tested or a control sample lacking the compound would be added to a suspension or solution of receptor preparation. After an incubation period, the receptor preparation would be analyzed to determine the relative amounts of monomeric and oligomeric species such that changes in the ratio produced by the test compound could be used to predict the activity and efficacy of the compound in regulating receptor function.
- Immunological methods can be used to measure compound efficacy.
- differential epitope tagging can be used in combination with differential co-immunoprecipitation to demonstrate the formation or absence of multimeric subunit aggregation.
- immunological techniques can be used to purify and identify the presence of each subunit in a multimer. If the complex is made up of two or more identical subunits (eg. homodimer or homotrim ⁇ r), each subunit is treated as if it is unique, such that the subunits bear tags in proportion to the number of units in the multimer.
- the complex is a homodimer
- one-half of the cDNA will be tagged with tag A and the other-half will be tagged with tag B.
- the resulting dimers will form between A- A, AB, and BB subunits, but will be observable by their migration in the SDS-PAGE gel, relative to the individual units. These will be visualized by immunoblotting with either or both types of anti-A MAbs or anti-B MAbs.
- each set comprises cDNA encoding one subunit of a receptor and one unique immunologic tag, one set for each subunit;
- An immunological method for measuring monomer/oligomer ratio entails separating monomers and oligomers based on size and measurement of relative amounts of each using reporter systems.
- the following steps would be followed: 1) receptor cDNA would be tagged with epitope for monoclonal antibody and expressed in a heterologous system (eg. baculovirus-insect cell system);
- membranes or pure receptor can be solubilized in SDS sample buffer and components separated by size on SDS-polacrylamide gels;
- monomeric and oligomeric receptor species would be identified by size and relative amounts of each species determined by densitometric scanning; 6) the ratio of monomer/oligomer species would be compared for different concentrations of the test compound.
- alternate means of separating monomeric and oligomeric receptor species by size can be used: eg. gel filtration, ultracentrifugation or others followed by antibody detection of different size forms and determination of ratio of monomeric to oligomeric species.
- Alternate means of labelling the receptor could entail labelling the receptor with some reporter permitting specific detection of the receptor (eg. fluorescent label specifically incorporated into the receptor protein which can be quantitated following size separation of monomeric and oligomeric species.
- the association of monomers in ohgomeric receptor complexes can be measured directly using Fluorescence Resonance Energy Transfer, involving use of two different fluorophores with distinct excitation and emission spectra, where the emission spectrum of the first fluor overlaps with excitation spectrum of the second fluor.
- Two separate preparations of receptor would be labelled with one or the other tluor and labelled receptor preparations would be reconstituted together in solution or in phospholipid vesicles. The mixture would then be irradiated at the excitation wavelength of the first and second fiuors. Monomers would show major emission and emission wavelength for the first fluor.
- Oligomers would show increased emission at the emission wavelength of the second fluor due to close proximity of the two fiuors and energy transfer from the first to the second fluor.
- the ratio of emission intensities at emission wavelengths for the first and second fiuors would provide a measure of the relative amounts of monomeric (no energy transfer) and the oligomeric receptor species.
- Compounds which modify the ratio of monomeric and oligomeric species of the receptor will also modify the ratio of emission intensities at the two emission wavelengths and permit prediction of activity and efficacy of the compound in regulating receptor activity.
- Modifications to this Fluorescence Resonance Energy Transfer method can be made by using receptors tagged with different epitopes and two corresponding monoclonal antibodies labelled with first and second fiuors.
- two receptor populations tag 1 and tag 2 in the same preparation (by co-expression of two receptors in insect cells or mammalian lines; or by separate expression and reconstitution into single preparation) are incubated with anti-tag 1 labelled with fluor 1, and anti-tag 2 labelled with fluor 2.
- Monomers will not show energy transfer between fiuors 1 and 2 on different receptor monomers, whereas oligomers will bring two receptor-bound antibodies into proximity and permit energy transfer, measured as an increase in emission intensity at the emission wavelength of fluor-2.
- Reverse-phase HPLC can also be used as a method for measuring subunit recombination.
- peptide-assessment assays would thus involve the following steps: Adding aqueous solution containing peptide, derivative, or peptidio mimetic compound to be tested to solution containing a GPCR preparation (tissue, cell or extract); adding agonist to the same solution; measuring the response to agonist by means of assay as described above; comparing the magnitude of the response to agonist in presence of the peptide or peptidio mimetic compound to that in absence of test molecule under otherwise identical conditions. Decrease in agonist-induced response in the presence of peptide or peptidiomimetic compound indicates antagonist activity.
- Activity of GPCR-peptide can be further characterized by testing: varying concentrations of peptide with fixed concentration of peptide or peptidiomimetic compound with fixed concentration agonist
- Activity of the GPCR-peptide or peptidiomimetic compounds can also be assessed by measuring the compound's affect on spontaneous receptor activity (i.e., basal activity in absence of added agonist). In this case, the same assay systems can be used without agonist and look for decrease in receptor activity in presence of compound. All assays described here are familial- to those versed in the ait, and described in detail in numerous scientific publications and methods manuals.
- the present invention also provides a method for treatment of G protein-coupled receptor mediated disease in patients, such as mammals, including humans, which comprises the step of administering to the patient a pharmaceutically effective amount of a compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described.
- compositions which comprise a pharmaceutically effective amount of the peptides or peptidomimetic compounds of this invention, or pharmaceutically acceptable salts thereof, and, preferably, a pharmaceutically acceptable carrier or adjuvant.
- Therapeutic methods of this invention comprise the step of treating patients in a pharmaceutically acceptable manner with those compounds or compositions.
- Such compositions may be in the form of tablets, capsules, caplets, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
- the therapeutic agents of the present invention may be administered alone or in combination with pharmaceutically acceptable carriers.
- the proportion of each carrier is determined by the solubility and chemical nature of the compound, the route of administration, and standard pharmaceutical practice.
- a composition of the invention is in the form of a unit dose.
- the unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional excipients.
- binding agents such as acacia, gelatin, sorbitol, or polyvinylpyrolidone
- fillers such as lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine
- tabletting lubricants such as magnesium stearate
- disintegrants such as starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose
- pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.
- the compounds may be injected parenterally; this being intramuscularly, intravenously, or subcutaneously.
- the compound may be used in the form of sterile solutions containing other solutes, for example, sufficient saline or glucose to make the solution isotonic.
- the compounds may be administered orally in the form of tablets, capsules, or granules containing suitable excipients such as starch, lactose, white sugar and the like.
- the compounds may be administered orally in the form of solutions which may contain colouring and/or flavouring agents.
- the compounds may also be administered sublingually in the form of traches or lozenges in which each active ingredient is mixed with sugar or corn syrups, flavouring agents and dyes, and then dehydrated sufficiently to make the mixture suitable for pressing into solid form.
- the solid oral compositions may be prepared by conventional methods of blending, filling, tabletting, or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are, of course, conventional in the art.
- the tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
- Oral liquid preparations may be in the form of emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
- Such liquid preparations may or may not contain conventional additives.
- suspending agents such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, or hydrogenated edible fats
- emulsifying agents such as sorbitan monooleate or acacia
- non-aqueous vehicles which may include edible oils), such as almond oil, fractionated coconut oil, oily esters selected from the group consisting of lycerine, propylene glycol, ethylene glycol, and ethyl alcohol
- preservatives for instance methyl para-hydroxybenzoate, ethyl para-hydroxybenzoate, n- propyl parahydroxybenzoate, or n-butyl pai'ahydroxybenzoate of sorb
- fluid unit dosage forms may be prepared by utilizing the peptide and a sterile vehicle, and, depending on the concentration employed, may be either suspended or dissolved in the vehicle.
- the compound Once in solution, the compound may be injected and filter sterilized before filling a suitable vial or ampoule and subsequently sealing the carrier or storage package.
- Adjuvants such as a local anaesthetic, a preservative or a buffering agent, may be dissolved in the vehicle prior to use. Stability of the pharmaceutical composition may be enhanced by freezing the composition after filling the vial and removing the water under vacuum, (e.g., freeze drying the composition).
- Parenteral suspensions may be prepared in substantially the same manner, except that the peptide should be suspended in the vehicle rather than being dissolved, and, further, sterilization is not achievable by filtration.
- the compound may be sterilized, however, by exposing it to ethylene oxide before suspending it in the sterile vehicle.
- a surfactant or wetting solution may be advantageously included in the composition to facilitate uniform distribution of the compound.
- the phai'maceutical compositions of this invention comprise a phai'maceutically effective amount of a compound of this invention and a pharmaceutically acceptable carrier. Typically, they contain from about 0.1% to about 99% by weight, preferably from about 10% to about 60% by weight, of a compound of this invention, depending on which method of administration is employed.
- Dosages may vary with the mode of administration and the particular peptide or peptidomimtic compound chosen.
- the dosage may vary with the particular patient under treatment.
- the dosage of the compound used in the treatment will vary, depending on the seriousness of the disorder, the weight of the patient, the relative efficacy of the compound and the judgment of the treating physician.
- Such therapy may extend for several weeks, in an intermittent or uninterrupted manner, until the patient ' s symptoms are eliminated.
- the compounds of the present invention can be modified by one skilled in the ait in such a manner as to prevent access into the central nervous system such that they can function in peripheral tissues to affect peripheral G protein coupled receptor mediated events.
- the transmembrane regions are believed to form a right-handed coiled coil where non-covalent helix packing (hydrophobic) interactions dominate.
- a dimerization motif 7;i LIXXGVXXG 8 - VXXT
- Gl 3 was found to be essential for dimerization as substitution with either hydrophobic or larger polar residues prevented dimer formation (Lemmon, M.A., et al., supra, 1992).
- Additional glycine and leucine residues shown in bold were also found to be important determinants of GpA dimerization.
- Analysis of ⁇ 2 AR transmembrane sequences revealed that leucine and glycine residues positioned with a similar spacing exist in the cytoplasmic end of the sixth
- TM VI U'ansmembrane domain
- Peptides were solubilized in the following buffer: 100 mM NaCl, 10 mM Tris-HCl pH 7.4, 2 mM EDTA (plus a protease inhibitor cocktail consisting of 5 mg/ml leupeptin, 10 mg/ml benzamidine and 5mg/ml soybean trypsin inhibitor), 0.05% digitonin and 10% DMSO. Peptide sequences were confirmed either by mass spectrometry or amino acid analysis.
- Peptides used were as follows: l) ⁇ 2 AR TM VI peptide consisting of residues 276-296; NH 2 - GIIMGTFTLCWLPFFIVNIVH-COOH. 2) a second peptide with Ala residues substituted at positions 276. 280, and 284 NH 2 -A ⁇ MATFTACWLPFFIVNIVH-COOH, 3) a peptide derived from residues 407-426 of the D2 dopamine receptor TM VII NH 2 -YIIPNVASNVYGLWTFASYL-
- membrane preparations from mammalian or Sf9 cells infected with recombinant bacculovirus expressing human ⁇ 2 AR were treated with increasing concentrations of the different peptides at room temperatures and for various times as indicated below.
- membrane preparations from mammalian or Sf9 cells or affinity purified receptors derived from Sf9 cells expressing c-myc tagged ⁇ 2 AR were treated at increasing concentrations of the different peptides at room temperature for various times as indicated (see results). Samples were then run on SDS-PAGE and then transferred to niUOcellulose.
- membrane preparations were also treated with either 10 ⁇ M timolol or 1 ⁇ M isoproterenol instead of, or in addition to the different peptides.
- Peptide antagonist activity was assessed by assaying adenylyl cyclase activity. In these assays, membranes were also used to determine the effect of vaiious peptides on the ability of the ⁇ 2 AR to stimulate adenylyl cyclase activity described below.
- the recombinant baculoviruses encoding the c-myc or hemaglutinin (HA) tagged wildtype human ⁇ ,AR, the c-myc tagged human M2 muscarinic receptor and c- yc tagged Dl dopamine receptor (c- myc ⁇ 2 AR and HA- ⁇ 2 AR, c-/;;yc M2-R, and c- yc Dl-R respectively) were constructed as described (Mouillac, B., et al., J. Biol. Cem., 267:21733-21737, 1992).
- HA Tetyr-Pro-Tyr-Asp-Val- Pro-Asp-Tyi'-Ala
- c-myc Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu
- St9 cells were maintained at 27°C in serum-supplemented [10% fetal bovine serum (FBS) v/v] Grace's insect medium (Gibco-BRL) with gentamycin and ftmgizone.
- FBS fetal bovine serum
- Gibco-BRL Grace's insect medium
- Cells were grown either as monolayers in T flasks or in suspension in spinner bottles supplemented with pluronic acid to prevent cell taring due to agitation.
- Cells were infected at log phase at a density of 1 x 10 6 cells per ml for 48 h.
- CHW and LTK cell lines with and without stably transfected ⁇ 2 AR were maintained as described (34).
- Cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with L- glutamate, 10% FBS, gentamycin and fungizone.
- DMEM Dulbecco's modified eagle medium
- Transfected CHW cells expressed -5 pmol receptor/mg protein while transfected LTK cells expressed 200 fmol receptor/mg protein.
- Stably transfected cell lines were grown in the presence of 150 ug/ml G418.
- V2 vasopressin receptors For uaroient expression of V2 vasopressin receptors the following procedures were followed. COS- 7 cells were maintained in supplemented DMEM as described above. Genomic DNA for the V2 vasopressin receptor was isolated from nepl rogenic diabetes insipidus (NDI) patients or unaffected individuals, subcloned into a construct containing a c-myc epitope tag and ligated into a mammalian expression vector, pBC12BI (Cullen, B.R, Meth. Enzymol., 152:684-704, 1987).
- NDI nepl rogenic diabetes insipidus
- COS-7 cells were ttansiently transfected with the expression vector encoding either wildtype V2 vasopressin receptor, a truncation mutant O- 11 or with vector alone for 48 hours.
- Membranes were prepai'ed as follows and washed. Sf9 or mammalian cells were washed twice with ice-cold PBS. The cells were then disrupted by homogenization with a polytron in 10 ml of ice-cold buffer containing 5 mM Tiis-HCI, pH 7.4. 2 mM EDTA (plus a protease inhibitor cocktail consisting of 5 mg/ml leupeptin, J O mg/ml benzamidine and 5 mg/ml soybean trypsin inhibitor). Lysates were centrifuged at 500 x g for 5 minutes at 4°C, the pellets homogenized as before, spun again and the supernatants were pooled.
- receptors were then solubilized in 2% digitonin or 0.3% N-dodecyl- ⁇ -D-maltoside and purified by affinity chromatography on alprenolol- sepharose as or by immuno precipitation as described below.
- Solubilized receptors were affinity purified by alprenolol-separose chromatography as described (Mouillac, B., et al., J. Biol. Cem., 267:21733-21737, 1992; Shorr, R.G.L., et al, J. Biol. Chem., 256:5820-5826, 1981).
- the affinity purified preparations were concentrated using Centriprep and Centricon cartridges (Amicon) and the amount of ⁇ 2 AR in each sample was determined in soluble [ 125 I]CYP radioligand binding assays as described (Mouillac, et al., 1981, supra). Purified receptors were desalted on Sephadex G-50 columns prior to SDS-PAGE.
- Tagged ⁇ 2 ARs were immunoprecipitated with either a mouse anti-c-wyc monoclonal antibody (9E10; Evan, G.I., et al., Mol. Cell. Biol., 5:3610-3616, 1985) or a mouse anti-hemagglutinin monoclonal antibody (12CA5; Nimar, H.L., et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953, 1983) as described previously (Mouillac, et al., 1981, supra).
- a mouse anti-c-wyc monoclonal antibody (9E10; Evan, G.I., et al., Mol. Cell. Biol., 5:3610-3616, 1985
- a mouse anti-hemagglutinin monoclonal antibody (12CA5; Nimar, H.L., et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953, 1983
- Removal of digitonin and concentration of the solubilized receptor was performed by dialysis using Centriprep cartridges (Amicon) against an ice- cold solution (Buffer A) containing 100 mM NaCl, 10 mM Tris-HCl pH 7.4, 2 mM EDTA (plus protease inhibitors described above) until the digitonin concentration was reduced below 0.05%.
- Purified 9E10 or 12CA5 antibody (1: 1000 dilution) was added to the concentrate and gently agitated for 2 hours at 4°C.
- Anti-mouse IgG agarose Sigma; at an 11 :1 secondary to primary Ab molar ratio
- protease inhibitor cocktail were then added.
- the reaction was allowed to proceed overnight at 4°C with gentle agitation.
- the immunoprecipitate was centrifuged at 12,000 ipm in a microcentrifuge for 10 minutes at 4°C.
- the pellet was washed three times in buffer A and finally resuspended in 200 ⁇ L of non-reducing SDS PAGE loading buffer for 30 minutes, sonicated and centrifuged at 12,000 rpm.
- the supernatant was then subjected to SDS PAGE and Western blotting as described below.
- Membrane preparations from Sf9 or mammalian cells or in some cases affinity-purified or immunoprecipitated ⁇ 2 AR were prepai'ed for non-reducing SDS-PAGE on 1 % slab gels as described previously (Laemmli, U.K., Nature, 227:680-686, 1970). In the case of the V2 vasopressin receptors reducing SDS-PAGE was performed.
- Immunoblots against the anti-c-wvc or anti-HA antibodies were revealed using a goat anti-mouse alkaline phosphatase-coupled second antibody (GIBCO-BRL) or a chemiluminescent substrate for a horseradish peroxidase coupled second antibody (Renaissance, NEN Dupont).
- GBCO-BRL goat anti-mouse alkaline phosphatase-coupled second antibody
- Renaissance, NEN Dupont a chemiluminescent substrate for a horseradish peroxidase coupled second antibody
- AR western blots were developed using a chemiluminescent substrate for goat anti-rabbit coupled horseradish peroxidase antisera (Sigma).
- blots were scanned by laser densitometry (Pharmacia-LKB Ultrascan).
- Receptor number was calculated from saturation binding experiments using [ l2S l] cyanopindolol (CYP) as the radioligand (Bouvier et al., Mol. Pharmacol., 267:7- 19. 1994). Briefly, 10 ⁇ L of a membrane preparation in a total volume of 0.5 mL was labelled with 250 pmol of [ 125 I]-CYP which is at a near saturating concentration. Non-specific binding was defined using 10 ⁇ L alprenolol.
- Adenylyl cyclase activity was assayed by the method of Salomon et al., (Anal. Biochem., 58:541-548, 1974). Membranes were prepai'ed and washed as described above. Again 10 uL of membranes (3-5 ug of protein) were used in a total volume of 50 uL. In some experiments, the peptides or the buffer used to soiubilize them were added to the enzyme assay mix. Enzyme activities were determined in the presence of nM to 100 uM isoproterenol, 100 uM forskolin or 10 mM NaF. Data were calculated as pmoles cAMP produced/min/mg protein and were analyzed by least squares regression using SigmaPlot 4.17 (Jandel Scientific).
- the dimer which was readily observed in membrane prepai'ations, was also detected in digitonin-solubi ⁇ zed receptors (lane 2) and following affinity purification of receptors on alprenolol-sepharose (lane 3).
- the dimer to monomer ratio as assessed by immunoblotting was increased by two-fold. This suggests that the dimer is already present before cell fractionation and that crosslinking stabilizes this form of the receptor, therefore, the dimeric species does not represent an artifact of membrane preparation or solubili .ation.
- Identical results were obtained when membranes were solubilized with 0.3% N-dodecyl- ⁇ -D-maltoside instead of digitonin (data not shown).
- the anti-HA mAb was used to blot receptors immunoprecipitated with either the anti-HA mAb or the anti-c- vc mAb.
- blotting of the anti-HA immunoprecipitate revealed both the 45 kDa and the 90 kDa forms of the receptor.
- the ⁇ 2 AR could also be detected by the anti- HA mAb in the c-myc immunoprecipitate of co-expressed receptors but the dimer then represented the predominant form (lane 1).
- V2 vasopressin receptors are also dimeric
- the vasopressin receptor is critical for regulation of water retention in the kidney. Recently, several mutations of this receptor have been linked to congenital nephrogenic diabetes insipidus (NDI. Bichet, D.G., et al., Am J. Hum. Genet., 55:278-286, 1994).
- NDI. Bichet D.G., et al., Am J. Hum. Genet., 55:278-286, 1994.
- transient expression of both wildtype and a truncated form of the V2 vasopressin receptor in COS-7 cells was studied. Both monomeric (appx. 64-69 kDa) and dimeric (appx. 120-135 kDa) forms of the wildtype human V2 vasopressin receptor were detected when expressed in COS-7 cells ( Figure 3, lane 1).
- a mutant form of the V2 receptor truncated in the C- terminal tail at residue 33y was also capable of forming dimers when expressed in
- VI peptide on receptor-stimulated adenylyl cyclase activity As shown in Figure 6a, the addition of TM VI peptide to membrane prepai'ations at a concentration of 0.15 ⁇ g/ ⁇ l significantly reduced isoproterenol-stimulated adenylyl cyclase activity (p ⁇ 0.05). In contrast, neither the peptide solubilization buffer (data not shown) nor control peptides (TM VI-Ala or TM VII of the D2 dopamine receptor) had significant effects on isoproterenol-stimulated adenylyl cyclase activity.
- TM VI peptide was without effect on basal cyclase activity in Sf9 cells which were infected with the wildtype baculovirus (data not shown). Also consistent with a receptor-specific action of the peptide is the observation that Dl dopamine receptor-stimulated adenylyl cyclase activity was not significantly affected by the TM VI peptide ( Figure 6c). As was the case for the inhibition of dimerization, the inhibitory action of the TM VI peptide on receptor-mediated adenylyl cyclase activity was dose-dependent ( Figure 6c).
- peptide IC 50 values for the inhibition of dimer formation are very similar (2.14 ⁇ 0.05 ⁇ M and 3.2 ⁇ 0.04 ⁇ M, respectively) thus suggesting that receptor dimerization may be an important step in ⁇ 2 AR-mediated signalling.
- our data suggest a role for dimerization in receptor activity, one cannot exclude the possibility that the effect of the TM VI peptide is not directly due to an effect on the monome ⁇ dimer equilibrium. Still, these results clearly show that this domain of the receptor is important in modulating ⁇ 2 AR signal transduction.
- the peptide represents a novel pharmacological tool for the study of receptor activity.
- the relative amount of dimer can be altered by a peptide derived from TM VI and by receptor ligands suggesting that under basal conditions there appears to be a dynamic equilibrium between monomeric and dimeric species of receptors.
- the data also suggest that shifting the equilibrium away from the dimeric form of the receptor interferes with the ability of the ⁇ 2 AR to productively interact with its signalling pathway.
- GPCR-peptides are receptor specific
- Receptor specificity is illustrated in the present examples by the observation that the M2 muscarinic receptor forms homodimers (see Debburmann, et al., supra, 1995 and data not shown) yet does not form heterodimers with the ⁇ 2 AR ( Figure 2). Similarly, the ⁇ 2 AR TM VI peptide had little effect on Dl dopamine receptor-stimulated adenylyl cyclase activity ( Figure 6c) or on D2 dopamine receptor dimer formation.
- V2 vasopressin receptor see discussion above - this study, Figure 3
- platelet activating factor receptor metabotropic glutamate receptor, substance P receptor, neurokinin-2 receptor, the C5a anaphylaxo toxin receptor, glucagon receptor, the dopamine Dl receptor, D2 receptor, the 5HT 1D receptor, the M2 muscarinic receptor and the M3 muscai'inic receptor
- GPCR-peptides and peptidomimetic compounds could be designed for these receptors that would function to as demonstrated in these examples to selectively prevent or disrupt the functional aggregation of these receptors, thereby attenuating receptor activity.
- GPCRs of the family A rhodopsin related subfamily share a number of features common to all members of its class.
- One of these features is the presence of recurring patterns in their amino acid sequence.
- Each transmembrane domain (TM) can be characterized by a recurring pattern that is unique for that TM.
- the alignment of the TMs is therefore based on recurring patterns rather than on homology alone.
- the patterns used were:
- a "sequence identifier” is assigned to each position in the alignment.
- the "extended notation” convention is applied. This reads as follows:
- TM2 138 VVYMLHLATADVLFVSVLPFKJSYYFSG 165 TM3 176 RFVTAAFYCNMYASILLMTVISIDR 200 TM4 215 TLGRASFTCLAIWALAIAGVVPLVLKE 241 TM5 268 AYYFSAFSAVFFFVPLIISTVCYVSIIRC 296 TM6 313 FLSAAVFCIFIICFGPTNVLLIAHYSFL 340
- TM 118 KITITVVLAVLILITVAGNVVVCLAVGLNRR 48 TM2 54 NCFIVSLAITDLLLGLLVLPFSAIYQLS 81 TM3 92 NIYTSLDVMLCTASILNLFMISLDR 116 TM4 131 TPVRVAISLVLIWVIS1TLSFLSIHLG 157 TM5 180 EVYGLVDGLVTFYLPLLIMCITYYRIFKV 208
- Dopamine Receptor subtype 2 (long form) M29066
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Abstract
Description
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU37050/97A AU3705097A (en) | 1996-07-01 | 1997-07-01 | Peptides and peptidomimetic compounds affecting the activity of g-protein-coupled receptors by altering receptor oligomerization |
EP97933814A EP0910640A2 (en) | 1996-07-01 | 1997-07-01 | Peptides and peptidomimetic compounds affecting the activity of g-protein-coupled receptors by altering receptor oligomerization |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US2103196P | 1996-07-01 | 1996-07-01 | |
US60/021,031 | 1996-07-01 |
Publications (2)
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WO1998000538A2 true WO1998000538A2 (en) | 1998-01-08 |
WO1998000538A3 WO1998000538A3 (en) | 1998-05-07 |
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PCT/IB1997/000814 WO1998000538A2 (en) | 1996-07-01 | 1997-07-01 | Peptides and peptidomimetic compounds affecting the activity of g-protein-coupled receptors by altering receptor oligomerization |
Country Status (4)
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---|---|
EP (1) | EP0910640A2 (en) |
AU (1) | AU3705097A (en) |
CA (2) | CA2259132A1 (en) |
WO (1) | WO1998000538A2 (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998000715A2 (en) * | 1996-07-01 | 1998-01-08 | Biosignal Inc. | Method of assaying compounds which affect the activity of g protein-coupled receptors based on measurement of receptor oligomerization |
WO1999043711A1 (en) * | 1998-02-27 | 1999-09-02 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | G protein-coupled receptor antagonists |
WO2001016182A2 (en) * | 1999-08-27 | 2001-03-08 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | POLYPEPTIDES THAT BIND HIV gp120 AND RELATED NUCLEIC ACIDS, ANTIBODIES, COMPOSITIONS, AND METHODS OF USE |
WO2001066723A2 (en) * | 2000-03-10 | 2001-09-13 | Mcgill University | Hetero-oligomeric g protein-coupled receptors as drug target |
WO2001081408A3 (en) * | 2000-04-21 | 2002-07-18 | New England Medical Ct | G-protein coupled receptor (gpcr) agonists and antagonists and methods of activating and inhibiting gpcr using the same |
US6555522B1 (en) | 1998-02-05 | 2003-04-29 | Mount Sinai School Of Medicine Of The City Of New York | Peptides and other small molecules derived from regions of interacting proteins and uses thereof |
US7105488B1 (en) | 1998-02-27 | 2006-09-12 | The United States Of America As Represented By The Department Of Health And Human Services | G protein-coupled receptor antagonists |
US7304127B2 (en) | 1999-08-27 | 2007-12-04 | United States Of America As Represented By The Secretary, Department Of Health And Human Services | Polypeptides that bind HIV gp120 and related nucleic acids, antibodies, compositions, and methods of use |
US7696168B2 (en) | 2000-04-21 | 2010-04-13 | Tufts Medical Center, Inc. | G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same |
US8440627B2 (en) | 2004-11-04 | 2013-05-14 | Tufts Medical Center, Inc. | G protein coupled receptor agonists and antagonists and methods of use |
US10709763B2 (en) | 2017-12-19 | 2020-07-14 | Gpcr Therapeutics, Inc. | GPCR heteromer inhibitors and uses thereof |
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WO1997040148A2 (en) * | 1996-04-22 | 1997-10-30 | L'universite De Montreal | Peptide antagonists derived from the transmembrane domains of g protein-coupled receptors |
-
1997
- 1997-07-01 CA CA 2259132 patent/CA2259132A1/en not_active Abandoned
- 1997-07-01 EP EP97933814A patent/EP0910640A2/en not_active Withdrawn
- 1997-07-01 AU AU37050/97A patent/AU3705097A/en not_active Abandoned
- 1997-07-01 WO PCT/IB1997/000814 patent/WO1998000538A2/en not_active Application Discontinuation
- 1997-07-02 CA CA 2205543 patent/CA2205543A1/en not_active Withdrawn
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WO1998000715A2 (en) * | 1996-07-01 | 1998-01-08 | Biosignal Inc. | Method of assaying compounds which affect the activity of g protein-coupled receptors based on measurement of receptor oligomerization |
US6555522B1 (en) | 1998-02-05 | 2003-04-29 | Mount Sinai School Of Medicine Of The City Of New York | Peptides and other small molecules derived from regions of interacting proteins and uses thereof |
US7105488B1 (en) | 1998-02-27 | 2006-09-12 | The United States Of America As Represented By The Department Of Health And Human Services | G protein-coupled receptor antagonists |
WO1999043711A1 (en) * | 1998-02-27 | 1999-09-02 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | G protein-coupled receptor antagonists |
WO2001016182A2 (en) * | 1999-08-27 | 2001-03-08 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | POLYPEPTIDES THAT BIND HIV gp120 AND RELATED NUCLEIC ACIDS, ANTIBODIES, COMPOSITIONS, AND METHODS OF USE |
US7304127B2 (en) | 1999-08-27 | 2007-12-04 | United States Of America As Represented By The Secretary, Department Of Health And Human Services | Polypeptides that bind HIV gp120 and related nucleic acids, antibodies, compositions, and methods of use |
WO2001016182A3 (en) * | 1999-08-27 | 2002-08-15 | Us Health | POLYPEPTIDES THAT BIND HIV gp120 AND RELATED NUCLEIC ACIDS, ANTIBODIES, COMPOSITIONS, AND METHODS OF USE |
WO2001066723A2 (en) * | 2000-03-10 | 2001-09-13 | Mcgill University | Hetero-oligomeric g protein-coupled receptors as drug target |
WO2001066723A3 (en) * | 2000-03-10 | 2004-02-26 | Univ Mcgill | Hetero-oligomeric g protein-coupled receptors as drug target |
AU2001257169B2 (en) * | 2000-04-21 | 2006-12-07 | Tufts Medical Center, Inc. | G-protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same |
EP2336169A1 (en) * | 2000-04-21 | 2011-06-22 | New England Medical Center Hospital | G protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same |
JP2003530875A (en) * | 2000-04-21 | 2003-10-21 | ニュー イングランド メディカル センター ホスピタル インコーポレイテッド | Agonists and antagonists of G protein-coupled receptors (GPCRs) and methods of using them to activate and inhibit GPCRs |
AU2001257169B9 (en) * | 2000-04-21 | 2007-05-24 | Tufts Medical Center, Inc. | G-protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same |
WO2001081408A3 (en) * | 2000-04-21 | 2002-07-18 | New England Medical Ct | G-protein coupled receptor (gpcr) agonists and antagonists and methods of activating and inhibiting gpcr using the same |
US7696168B2 (en) | 2000-04-21 | 2010-04-13 | Tufts Medical Center, Inc. | G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same |
AU2001257169C1 (en) * | 2000-04-21 | 2011-06-16 | Tufts Medical Center, Inc. | G-protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same |
US6864229B2 (en) | 2000-04-21 | 2005-03-08 | New England Medical Center Hospitals, Inc. | G protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same |
EP2348047A1 (en) * | 2000-04-21 | 2011-07-27 | New England Medical Center | G protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same |
US8324172B2 (en) | 2000-04-21 | 2012-12-04 | Tufts Medical Center, Inc. | G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same |
US8354378B2 (en) | 2000-04-21 | 2013-01-15 | Tufts Medical Center, Inc. | G protein coupled receptor antagonists and methods of activating and inhibiting G protein coupled receptors using the same |
US8389480B2 (en) | 2000-04-21 | 2013-03-05 | Tufts Medical Center, Inc. | G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same |
US8563519B2 (en) | 2000-04-21 | 2013-10-22 | Tufts Medical Center, Inc. | Methods of activating or inhibiting G protein coupled receptors (GPCRs) |
US8440627B2 (en) | 2004-11-04 | 2013-05-14 | Tufts Medical Center, Inc. | G protein coupled receptor agonists and antagonists and methods of use |
US10709763B2 (en) | 2017-12-19 | 2020-07-14 | Gpcr Therapeutics, Inc. | GPCR heteromer inhibitors and uses thereof |
US11857600B2 (en) | 2017-12-19 | 2024-01-02 | Gpcr Therapeutics, Inc. | GPCR heteromer inhibitors and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
AU3705097A (en) | 1998-01-21 |
EP0910640A2 (en) | 1999-04-28 |
WO1998000538A3 (en) | 1998-05-07 |
CA2259132A1 (en) | 1998-01-08 |
CA2205543A1 (en) | 1998-01-01 |
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