WO1994000492A1 - Angiotensin iv peptides and receptor - Google Patents

Angiotensin iv peptides and receptor Download PDF

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Publication number
WO1994000492A1
WO1994000492A1 PCT/US1993/006038 US9306038W WO9400492A1 WO 1994000492 A1 WO1994000492 A1 WO 1994000492A1 US 9306038 W US9306038 W US 9306038W WO 9400492 A1 WO9400492 A1 WO 9400492A1
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Prior art keywords
receptor
ain
binding
aiv
ligand
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PCT/US1993/006038
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French (fr)
Inventor
Joseph W. Harding
John W. Wright
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Washington State University Research Foundation
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Application filed by Washington State University Research Foundation filed Critical Washington State University Research Foundation
Priority to US08/360,784 priority Critical patent/US5854388A/en
Priority to AU46492/93A priority patent/AU4649293A/en
Priority to EP93916733A priority patent/EP0647239A4/en
Publication of WO1994000492A1 publication Critical patent/WO1994000492A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • 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
    • 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
    • C07K7/14—Angiotensins: Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the polypeptide ligand NYTHPF (angiotensin IN or AIN) and to related peptide ligands and polyaminoacid ligands that bind to, activate and/or antagonize a novel angiotensin AT4 receptor.
  • the ligands comprise at least three of the ⁇ -terminal amino acids of AIN, or AT4 receptor binding equivalents or analogs thereof. Engagement of the receptor by its ligand triggers acute physiological effects (e.g., vasodilation) and long-term effects in cells (e.g., hypertrophic growth).
  • the renin-angiotensin system has wide-ranging actions on numerous tissues in the body affecting blood pressure (pressor activity) and cardiovascular and electrolyte homeostasis. It is currently believed that angiotensins All and Affl are derived via enzymatic cleavage in the cascade depicted in Figure 1, steps 1, 2, and 3 (1). (Numbering herein of the amino acid residues in Al, All, AIII, and AIN is according to that appearing in Figure 1.) The renin-angiotensin cascade is thought to begin with the action of renin on angiotensinogen to release angiotensin I (Al), a biologically inactive decapeptide.
  • Angiotensin II (All), the bioactive octapeptide, is thought to be formed by the action of angiotensin converting enzyme (ACE) on circulating Al (2).
  • Des-AspAH Angiotensin III; Am
  • AHI in the adrenal gland (3) and brain (4). It has been reported that All and AUI are inactivated by enzymatic degradation through a series of smaller inactive fragments (5). Fragments smaller than AIII have been thought, for the most part, to be biologically inactive and of little physiological significance (6).
  • ATI ⁇ g ATI ⁇ g
  • AT2 receptors 9,10
  • An All receptor having components with molecular weights of 60-64kDa and 112-115kDa has reportedly been cloned from adrenal cortical cells as well as rat smooth muscle (11).
  • AII ⁇ -8 has been found to be much less active than All or AIII with regard to typical angiotensin-dependent pressor activity or stimulating water intake (9,10,12).
  • AII (3 . 8) while having little pressor activity or ability to stimulate aldosterone release, may under certain circumstances inhibit renin release from kidney (12,13).
  • Haberl et al. (14) reported a possible effect of AII ( 3_g ) on endothelium-dependent dilation in rabbit brain. Braszko et al.
  • the angiotensin field has often been fraught with complexity and conflicting information, particularly with regard to the levels of different All and AIII peptides required to elicit certain cellular responses, the concentrations predicted from receptor binding studies to be biologically active, and the levels of angiotensin peptides that may be measured in biological fluids. It has been reported that All and AIII are removed from, or destroyed in, circulation by enzymatic hydrolysis. Biological half- lives of the different metabolic fragments are reportedly quite short. Semple and co- workers (18) reportedly detected AHI, A ⁇ 3 .
  • AII ( 4_g ) in arterial and venous blood in man with half-lives for All, AIII, AII ( 3_g ) , and AII (4 .g ) of 4.4, 2.0, 1.9, and 2.4 minutes, respectively.
  • Blumberg et al. (19) reported that during transit through the kidney 72-76% of Al and All and 89% of A was metabolized.
  • Confusion has existed in the art as to how metabolic products of All and AIII can exhibit certain biological activities (e.g., inhibition of renin release and enhancement of cognitive function), while failing to bind to Al or All receptors.
  • Fragments of All smaller than A e.g., AII (3 .g ) and other smaller fragments, have not been reported to have specific saturable binding sites in tissues, and receptors for these fragments have not been identified previously.
  • the present invention provides partial explanation for certain previous confusing and contradictory findings, and provides novel AIN receptors (AT4), AIN ligands, peptides, analogs, agonists and antagonists that bind specifically to the AT4 receptor and not to Al (ATI) or All (AT2) receptors.
  • the AIV peptides and the AT4 receptor are labile and subject to proteolytic degradation.
  • the invention provides a specific angioteninase enzyme that converts All or AIII peptides to AIN peptides in a novel pathway. Summary of the Invention
  • AIN angiotensin AIN receptor
  • AT4 angiotensin AIN receptor
  • AIN ligand system for binding a small ⁇ -terminal hexapeptide fragment of Angiotensin II (referred to herein as AIN, with amino acid sequence Nal ⁇ -Tyr 2 -He 3 -His 4 -Pr ⁇ 5 -Phe 6 ) provides partial explanation for confusion in the prior art.
  • AIN binds saturably, reversibly, specifically, and with high affinity to membrane AT4 receptors in a variety of tissues and from many animal species.
  • the AT4 receptor is pharmacologically distinct from classic angiotensin receptors (ATI or AT2) in that the AT4 receptor displays no specificity for classic agonists (All and AIII) and antagonists (Sar ⁇ Ileg-AH).
  • ATI or AT2 classic angiotensin receptors
  • AT4 receptor displays no specificity for classic agonists (All and AIII) and antagonists (Sar ⁇ Ileg-AH).
  • the disclosure details the pharmacological and biochemical characterization of a newly discovered branch of the renin-angiotensin system that employs an AIN ligand as the signaling agent, and the AT4 plasma membrane receptor as the detection mechanism.
  • Angiotensin AIN appears to specifically mobilize calcium in vascular endothelial cells where AIN binding is evident. Binding to the endothelial AT4 receptor appears to trigger cellular proliferation. Binding of AIN to AT4 receptors in kidney and brain increases blood flow. In addition, binding of AIN to AT4 receptors in the brain facilitates learning and memory retention. AIN has also been shown to block the hypertrophic action of A on cardiocytes despite its inability to bind AT2 receptors. Since cardiocytes possess large numbers of AT4 receptors this action of AIN is most likely direct. Thus, in certain respects the action of AIN appears to neutralize, or act in apposition to the actions of All and AIII.
  • the invention provides an angiotensin AT4 receptor and receptor fragments (including the receptor binding site domain) that are capable of binding a VYIHPF angiotensin AIN ⁇ -terminal peptide, and related AIN ligands, but do not bind an angiotensin All or AIII ⁇ -terminal peptide, i.e., DRVYIHPF or RVYIHPF, respectively.
  • the AT4 receptor from adrenal cortical cells has a molecular size of about 140kD to about 150kD on SDS-PAGE following crosslinking, a K& of about 0.5nM for AIV peptides, and is widely expressed on the surface of adrenal cortical and medullary tissues in many mammalian species.
  • the receptor is expressed in all important organs and tissues including heart, lung, kidney, aorta, brain, liver, and uterus.
  • the invention further provides processes for identifying angiotensin AIV agonists and antagonists, and constructing diagnostic assays to specifically measure AIV and AT4 receptors.
  • FIGURE 1 is a schematic diagram depicting the amino acid sequence of angiotensinogen and its conversion by renin to Al, by angiotensin converting enzyme (ACE) to All, by angiopeptidase to AIII, and by a novel AIV angiotensinase, herein disclosed, to angiotensin AIV (AIV).
  • ACE angiotensin converting enzyme
  • AIV angiotensin AIV
  • FIGURE 2 A is a graphical representation of the results of equilibrium binding studies of 125 I-radiolabeled AIV to AT4 receptors isolated from bovine adrenal cortical membranes; as described in Example 1.
  • FIGURE 2B depicts graphically the structural requirements and specificity for binding of AIV ligand to the AT4 receptor from rabbit cardiac myocyte membranes; as described in Example 1.
  • FIGURE 3 compares AT2 and AT4 receptor localization in the Habenula region of the brain using receptor autoradiography with 125 I-Sar ⁇ ,Ileg-AII to localize AT2 receptors, and 125 I-ATV to localize AT4 receptors, as described in Example 2.
  • Panel A shows binding of 125 I-AIN to cells in the habenula, thalamus, cerebral cortex and hippocampus of guinea pig brain.
  • Panel B shows that the binding of 125 I-AIV is specifically competitively inhibited by lOOnM non-labeled AIV competitor.
  • Panel C shows that binding of 125 I-AIV is not competitively inhibited by lOOnM Sar ⁇ ,Ileg-AII.
  • Panel D shows a pattern of binding of 125 I-Sar ⁇ ,Ileg-AII to AT2 receptors that is different from the pattern observed with 125 I-AIV in Panel A.
  • Panel E shows that binding of 125 I-Sar ⁇ ,Ile 8 -AII is specifically inhibited by lOOnM of non-labeled All competitor.
  • Panel F shows that binding of 125 I-Sar ⁇ ,Ileg-AII is not inhibited by lOOnM non-labeled AIV competitor.
  • Panel G shows a "pseudo-color" photograph of 1 5 I-AIV binding.
  • Panel H shows a "pseudo-color photograph of 125 I-Sar ⁇ ,Ileg-AII binding.
  • Panel I shows a photomicrograph of a histology slide of a serial section of the same tissue as in Panels A-I.
  • FIGURES 5A and 5B are graphical representations of changes in blood flow that result from binding of agonist, Lys j AIN, to AT4 receptors in kidney, without changes in systemic blood pressure, as described in Example 4.
  • Figure 5A shows changes in arterial blood pressure following administration of Lys j AIV at 100pmole/25ml/min (open circles) or saline control (closed circles).
  • Figure 5B shows changes in renal blood flow following administration of Lysi AIN at 100pmole/25 ⁇ 1/min (open circles) or saline control (closed circles).
  • FIGURES 6A and 6B are graphical representations showing changes in blood flow that result from administering different doses of an agonist ⁇ orLeu j AIN (i.e., ⁇ orLeuYTHPF) that binds to AT4 receptors in kidney, without changes in systemic blood pressure, as described in Example 4.
  • ⁇ orLeuYTHPF an agonist ⁇ orLeu j AIN
  • FIGURES 6A and 6B are graphical representations showing changes in blood flow that result from administering different doses of an agonist ⁇ orLeu j AIN (i.e., ⁇ orLeuYTHPF) that binds to AT4 receptors in kidney, without changes in systemic blood pressure, as described in Example 4.
  • ⁇ orLeuYTHPF an agonist ⁇ orLeu j AIN
  • FIGURES 7A-7D, 8 and 9 are graphical representations of AIN binding, as described in Example 6.
  • Figure 7 A shows the results of kinetic analyses measuring binding of AIN to coronary venule endothelial cells (CVEC) showing maximal equilibrium binding in about 60 minutes with an apparent Ka of about 9.3 x 10 7 M- 1 .
  • Figure 7B shows the results of kinetic studies measuring the dissociation of AIV from CVEC endothelial cells with an apparent K- j of about 0.3nM.
  • Figure 7C shows the results of equilibrium binding of AIN to 2 separable types of AT4 receptor sites in coronary venule endothelial cells (CVEC).
  • Figure 7D shows the results of equilibrium binding of AIV to 2 separable types of AT4 receptor sites in aortic endothelial cells: one type of site with a K- j of about 4.4 +/- 0.8nM and a second type of site with a K- j of about 26.9 +/- 9pM.
  • Figure 8 shows competition of 125 I-AIV binding to coronary venule endothelial cells (CVEC) by non-radiolabeled AIV analogs.
  • Figure 9 shows association of AT2 receptors with G-protein in vascular smooth muscle cells (RVSMC), but non-association of AIV with G-proteins in endothelial cells (BAEC), as evidenced by the inhibility of GTP ⁇ S to inhibit AIV binding.
  • RVCSMC vascular smooth muscle cells
  • BAEC endothelial cells
  • FIGURES 10A and 10B show enhancement of cognitive function, i.e., learning, in AIV intracerebroventricularly (icv) injected animals but not in All-icv-injected animals. Testing of memory was conducted one day ( Figure 10 A), or one, two and three days ( Figure 10B), after the animals learned a passive avoidance response; as described in Example 7.
  • FIGURE 11 is a graphical representation of the comparative stability of
  • FIGURE 12 is a graphical representation of the effects of divalinal AIN (open squares), and divalinal AIV followed by LysiAIV (squares with dots), on blood pressure (Figure 12 A) and renal blood flow (Figure 12B), as compared to saline alone
  • Angiotensinogen is used herein to refer to a peptide having the sequence Asp 1 Arg2Val3Tyr4lle5His 6 Pro Phe His9Leu 10 Val 11 Ile 1 2His 1 3Ser 14 , abbreviated DRVYfflOPFHLVfflS (SEQ. ID. NO. 1)
  • Al and “angiotensin I” are terms used to refer to the decapeptide fragment of angiotensin having the N-terminal sequence
  • des-Asp Al “d-Asp Al” and “des-Asp angiotensin I” are terms used to refer to an angiotensin polypeptide having the N-terminal sequence
  • RVYIHPFHL (SEQ. ID. NO. 3).
  • angiotensin II is terms used to refer to an angiotensin, e.g., an octapeptide, having the N-terminal sequence Asp i Arg 2 Val3 Ty ⁇ IlesE ⁇ P ⁇ Pheg, abbreviated DRVYIHPF (SEQ. ID. NO. 4).
  • Am angiotensin m
  • Des-Asp AH Des-Asp AH
  • a ⁇ 2 . 8 are terms used to refer to the heptapeptide fragment of angiotensin having the N-terminal sequence abbreviated RVYIHPF (SEQ. ID. NO. 5).
  • AIV angiotensin IV
  • Des-Arg Am are terms used to refer to the hexapeptide fragment of angiotensin having the N-terminal sequence Val 1 Tyr2lle 3 His4Pr ⁇ 5Phe 6 , abbreviated VYIHPF (SEQ. ID. NO. 6).
  • AIV refers to physiological angiotensin II ( 3_ ) fragments formed in a variety of animal species.
  • An "AIV peptide ligand” is a ligand capable of binding to an AT4 receptor. AIV is a representative example of an AIV peptide ligand, as are AIV analogs.
  • Des-x also abbreviated “d-x,” is used to refer to an amino acid sequence that lacks the amino acid residue "x". Des-Asp All is used to refer to an angiotensin II lacking the N-terminal Asparagine residue; d-Nal ⁇ AIV is used to refer to AIV lacking the valine residue (position 1) at the ⁇ -terminus of AIV. " ⁇ -terminal” and “ ⁇ -terminus” are used interchangeably to refer to the
  • the N-terminal amino acid is the amino acid located at the NH terminus of the peptide.
  • Protein and polypeptide are used interchangeably to refer to a serial array of amino acids peptide bonded one to another of at least three amino acids in length to preferably six amino acids in length, but also up to many hundreds of amino acids in length.
  • AIV Ligand refers to a compound that is capable of filling the three-dimensional space in a receptor binding site so that electrostatic repulsive forces are minimized, electrostatic attractive forces are maximized, and hydrophobic and hydrogen bonding forces are maximized.
  • Representative ligands include "AIV peptides” and "ATV analogs”.
  • Ligands bind to their specific receptor in a specific saturable manner, e.g., specificity may determined by the ability of an AIV ligand to bind to an AT4 receptor in a manner that is not competitively inhibited in the presence of an excess (e.g., 1000-fold molar excess) of a competitor peptide (e.g., Al or All).
  • ATV peptide is used interchangeably with "angiotensin IV peptide” to refer to an AIN ligand that is a peptide having, or corresponding to, at least three of the ⁇ -terminal ten amino acid residues (preferably three of the ⁇ -terminal eight amino acid residues, and most preferably three of the ⁇ -terminal six amino acid residues), comprising three amino acids selected from among N, Y, I, H, P, F, L, K, A, H, ⁇ Nal, ⁇ Leu, or Orn; preferably from among N, Y, I, P, K, ⁇ Nal or ⁇ Leu; and most preferably from among N, Y, K, ⁇ Nal, or ⁇ Leu.
  • AIN peptides have an amino acid sequence related to the AIN ⁇ -terminal sequence NYIHPFX, i.e., by conservative and nonconservative substitutions of amino acids, or by derivatization or covalent modification, (as described below), and wherein X is any non-interfering amino acid.
  • Representative AIN peptides are polypeptides from 3 amino acids in length to many tens of amino acids in length.
  • AIN peptides include peptides that are capable of antagonizing binding of "AIN” to its receptor, i.e., “antagonists” (as defined below), and other "AIN ligands” are capable of binding to the AT4 receptor and exerting effects similar to “AIN", i.e., "agonists” (as defined below).
  • AIV analog is intended to mean a chemical compound that mimics or improves on the electronic, steric, hydrophobic, and 3 -dimensional space-filling requirements of the constituent amino acid residues involved in binding of the AIV peptide to the AT4 receptor (e.g., a mimetic chemical AIV composition).
  • AIV analogues may be polypeptides, i.e., having amino acids bonded by peptidic linkages, or may be non-peptides, i.e., having amino acids not bonded by peptidic linkages.
  • Representative examples of AIV analogs include chemical mimetic compounds that are capable of antagonizing binding of AIV to its receptor, i.e., antagonists (as defined below), and other AIV ligands are capable of binding to the AT4 receptor and exerting effects similar to ATV, i.e., agonists (as defined below).
  • Agonist as used herein means an AIV peptide or AJN analog that is capable of spacially conforming to the molecular space filled by an AIN ligand and that is further capable of combining with AT4 receptors to initiate an action that is initiated by a physiological AIN molecule when it binds to its specific AT4 receptors on cells in vivo or in vitro. Representative examples of actions initiated by AIN are illustrated in the Examples. Agonists possess binding affinity for AT4 receptor(s) and intrinsic activity for inducing the activities that are induced following the binding of AIN to AT4 receptor.
  • agonists include NYEHPFX, ⁇ vaYIHPFX, and OrnYTHPFX, wherein "X" is used to designate one or more non-interfering amino acids.
  • processes for recognizing agonists are described in Example 4.
  • Antagonist as used herein means an agent that spacially conforms to the molecular space filled by an AIN ligand and that is further capable of combining with the subject AT4 receptor(s) to inhibit, neutralize, impede or reverse, at least in part, an action of physiological AIN when it binds to its specific AT4 receptors on cells.
  • antagonists include KYIHPFX, and ⁇ Leu THPFX, wherein "X" is used to designate one or more non-interfering amino acids. Representative examples of processes for recognizing antagonists are described in Example 4.
  • AH ligand refers to a peptide having the ⁇ -terminal amino acid sequence DRNYTHPFX and capable of binding to an ATI or AT2 All receptor, where X is any non-interfering amino acid.
  • ⁇ on-interfering amino acid as used herein means any amino acid that when introduced into the C-terminus of an AIN peptide ligand does not interfere with binding of the AIN peptide ligand to its specific AT4 receptor.
  • ATI and “ATI receptor” and are terms used interchangeably to refer to a receptor subtype capable of binding AH.
  • AT2 and AT2 receptor are terms used interchangeably to refer to a second receptor subtype capable of binding AIL
  • AT4 receptor is the term used to refer to a receptor capable of binding an
  • AIN ligand but not an Al, All, or AIII ligand.
  • AT4 receptor fragments is a term used herein to refer to portions of the AT4 receptor that are smaller in size than an AT4 receptor isolated from a natural source, e.g., tissues, biological fluids and the like, but remain capable of binding AIN. Fragments may be prepared from an AT4 receptor isolated from a tissue and then subjected to proteolytic degradation or treatment with a chemical such as cyanogen bromide. In the latter case the fragments of the receptor are conveniently purified before use, e.g., by reverse-phase HPLC or immune affinity chromatography.
  • fragments of the AT4 receptor may be prepared by expression of a portion of a nucleotide sequence of a genomic or cD ⁇ A clone capable of expressing the AT4 receptor, e.g., a portion of the AT4 nucleotide sequence in an expression plasmid or vector introduced into a cell, wherein the cell manufactures the AT4 receptor fragment and the fragment can be purified (as above).
  • fragments of the AT4 receptor that contain the AIN ligand binding domain of the receptor may be soluble in biological fluids and aqueous solutions and may bind AIN ligand with a greater or less K-j than AT4 receptor under these conditions.
  • the binding affinity, expressed as the K-j, of the AT4 receptor fragment for an AIN ligand is about 30nM to about 0.003nM, preferably about lnM to about O.OlnM, and most preferably the binding affinity is about 0.5nM to about O.OlnM.
  • activation of the AT4 receptor are used interchangeably to refer to conformational and/or structural or activity changes resident in an AT4 receptor following binding of an AIN ligand; e.g., conformational changes may be evident by changes in the near UN spectra of the receptor or changes in the circular dichroism (CD) spectra; structural changes may be evident as covalent modification of the receptor, e.g., by phosphorylation; and, activity changes may be evident as an increase in enzyme activity, e.g., an innate tyrosine kinase activity.
  • conformational changes may be evident by changes in the near UN spectra of the receptor or changes in the circular dichroism (CD) spectra
  • structural changes may be evident as covalent modification of the receptor, e.g., by phosphorylation
  • activity changes may be evident as an increase in enzyme activity, e.g., an innate tyrosine kinase activity.
  • a receptor that has interacted with an AIN ligand and has undergone the process of "triggering” is also referred to herein as a "triggered AT4 receptor.”
  • “Substantially purified” as used herein refers to a preparation that contains a peptide, ligand, or receptor that is enriched greater than about 10-fold from the natural source material, e.g., membrane preparations of a tissue, and that also contains less than 5% impurities detectable by one-dimensional SDS-PAGE.
  • the substantially purified AT4 receptor approaches homogeneity at purification levels greater than about lOOOx.
  • ATV angiotensinase refers to a dipeptidylpeptidase capable of catalyzing hydrolysis of an arginine-valine peptide bond in an angiotensin, e.g., Al, All, or AIII, without catalyzing hydrolysis of any of the other peptide bonds in the angiotensin.
  • Pressure activity is used to refer to blood pressure changes induced by an agent, e.g., AH.
  • angiotensin II angiotensin II
  • bovine adrenal cortex During attempts to purify the angiotensin II (AT2) receptor from bovine adrenal cortex, the curious observation was made that as purification proceeded the apparent specificity of the receptor changed. While isolated membranes bound stable All analogs better than Am, the solubilized receptor exhibited the opposite order of ligand specificity with AHI binding better than All. Following purification it became apparent that the receptor had all but lost its ability to bind All, and was slowly losing its ability to bind Am, despite taking steps to inhibit proteases.
  • AT2 angiotensin II
  • a reevaluation of binding in purified membrane preparations demonstrated the presence of two different and distinct receptors, one for All and a second for A ⁇ -g ) . Further, neither All nor AII (3 _g ) ligand effectively displaced the other. The results, thus, strongly suggested the presence of two distinct receptors, one for All and a second for AII (3 _ 8) .
  • All ⁇ . ) is referred to as AIN and the novel AII (3 _ ) receptor is referred to as the AT4 receptor.
  • the notion of two separate and distinct receptors was confirmed by solubilizing, isolating, and substantially purifying the AT4 receptor under conditions that did not solubilize the AT2 receptor.
  • angiotensin IN angiotensin All
  • AII 3 _g
  • angiotensin IN angiotensin IN
  • AIN angiotensin IN
  • the angiotensin AT4 receptor is characterized in the Examples, with respect to structural requirements for ligand binding, species and tissue distribution of the receptor, physiological role of the AIN ligand- AT4 receptor system, intracellular messenger signaling pathways activated by the receptor, conditions for isolation and purification, and molecular size of the receptor.
  • compositions which comprise substantially purified angiotensin AT4 receptor or fragments thereof, that are capable of binding an angiotensin AIN ligand but not an angiotensin Al or All ligand.
  • the AT4 receptor binds AIN ligands, and does not bind to a peptide having the All ⁇ -terminal sequence, i.e., DRNYIHPF.
  • AT4 receptors of the invention are specific for AIN and AIN ligands, and are more fully characterized by the following properties: a) AT4 receptor has a K ⁇ for AIV of about 30nM to about 0.003nM, preferably about 3nM to about O.OlnM, and most preferably about InM to about 0.1 nM (representative examples of binding properties of AT4 receptors are summarized in Table 1); b) AT4 receptor binds to AIV ligands in a saturable and reversible manner; c) the binding of an AIV ligand to the AT4 receptor is competitively inhibited less than about 1% to about 10% by an angiotensin All preparation (e.g., Sar ⁇ Ileg-AII) that contains less than 0.1% of an AIV ligand when the competition of AIV binding is measured in the presence of about a 1000-fold molar excess concentration of the competing ligand using the assay conditions described in Example 1.
  • an angiotensin All preparation
  • AT4 receptors having these properties may be isolated from bovine adrenal cortical membranes (e.g., described in Example 1). Isolated AT4 receptors from this source have the kinetic, equilibrium binding, and physical properties set forth below in Example 1.
  • the AT4 receptor of the invention has a molecular size of about 120kD to about 200kD on SDS-PAGE, preferably about 140kD to about 160kD, and most preferably about 140kD to about 150kD.
  • an AT4 receptor of the invention is present in membrane preparations of adrenal glands of most mammalian species (e.g., cow, pig, horse, dog, cat, rabbit, and guinea pig) and, as purified from bovine adrenal membranes, the AT4 receptor has an apparent molecular size of about 146kDa on SDS-PAGE. AT4 receptors are also expressed in guinea pig aorta, heart, kidney, liver, lung, vascular smooth muscle, pituitary, and uterus, as well as vascular endothelial cells and brain.
  • mammalian species e.g., cow, pig, horse, dog, cat, rabbit, and guinea pig
  • AT4 receptors has an apparent molecular size of about 146kDa on SDS-PAGE. AT4 receptors are also expressed in guinea pig aorta, heart, kidney, liver, lung, vascular smooth muscle, pituitary, and uterus, as well as vascular end
  • the invention further provides AT4 receptor ligands that specifically bind to, activate and/or antagonize the AT4 receptor.
  • the ATV ligands generally comprise at least 3 of the N-terminal amino acid residues of AIN, or analogues or AT4 receptor binding equivalents thereof.
  • the amino acid residues of the ligands may be bonded by peptidic linkages, or may be bonded by non-peptidic linkages.
  • the ligands generally have a K d for the AT4 receptor below about 3 x 10 _6 M.
  • the AIN ligands of the invention are based on the structure of AIN.
  • the AIN ligands may be obtained by constructing AIN analogs that have one amino acid substituted for by another of like properties, i.e., a neutral polar amino acid for another neutral polar (e.g., G, A, N, I, L, F, P, or M), a neutral nonpolar amino acid for another neutral nonpolar (e.g., S, T, Y, W, ⁇ , Q, C), an acidic amino acid for another acidic (e.g., D or E), or a basic for a another basic (e.g., K, R, or H).
  • a neutral polar amino acid for another neutral polar e.g., G, A, N, I, L, F, P, or M
  • a neutral nonpolar amino acid for another neutral nonpolar e.g., S, T, Y, W, ⁇ , Q, C
  • an acidic amino acid for another acidic e.
  • the AIN ligands may alternatively be obtained by constructing an AIN analog that is covalently modified, e.g., wherein an amino acid residue is substituted by amidation, adenylation, methylation, acylation, phosphorylation, uridylation, fatty-acylation, glycosylation, and the like to form a "substituted amino acid residue".
  • the AIN ligands of the invention may contain one or more stereoisomers of the constituent amino acids residues; i.e., may contain one or more substituted or unsubstituted amino acid residues in the D-configuration.
  • the invention provides angiotensin AIN ligands and ligand compositions that include AIN analogs, AIN peptide derivatives, and covalently modified AIN peptides, all of which are capable of binding to an angiotensin AT4 receptor.
  • AIN ligands of the invention are generally defined by the formula
  • Rj is a substituted or unsubstituted amino acid residue having a neutral or positively charged aliphatic side chain Z j , said amino acid being selected from among N, I, L, A, G, F, P, M, K, norvaline, norleucine, and ornithine;
  • R 2 is a substituted or unsubstituted neutral nonpolar amino acid selected from the group consisting of Y, W, ⁇ , Q, F, or C;
  • R 3 is a substituted or unsubstituted neutral polar amino acid selected from the group consisting of G, A, N, I, L, F, P, or M;
  • X is nothing, R 4 , R 4 -R 5 , or R 4 -R 5 -R 6 , wherein R 4 is a substituted or unsubstituted basic amino acid residue selected from the group consisting of K, R and H, R 5 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, N, I, L, F, P, and M, and Rg is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, N, I, L, F, P, M, and polyamino acid residues containing one or amino acid residues which do not prevent binding of the AIN ligand with the AT4 receptor.
  • the AIN ligands of the invention are generally amino acid chains that contain 3, 4, 5, or 6 amino acid residues corresponding to the ⁇ -terminal 3, 4, 5 or 6 amino acid residues of AIN (the polypeptide, VYIHPF), or may optionally extended at the C-terminal end with one or more amino acid residues that do not prevent binding, due to spatial, conformational, electrostatic or other considerations, to the AT4 receptor.
  • the amino acid residues may be linked in the amino acid chain by peptidic linkages to form peptides, or the ATV ligands of the invention may contain one or more non-peptidic linkages, such as methylene or C- ⁇ linkages, to enhance metabolic stability or other properties of the AIN ligands, as is hereinafter further described.
  • Representative AIN ligands of the invention include, but are not limited to C-terminal truncated forms of AIN, such as AIN ( i_5 ) , AIN ⁇ ) , and AIN ( i.
  • AIN such as D-H 4 AIN, D-P 5 AIN, and D-F 6 AIN
  • full or truncated forms of AIN with modified amino acid residues such as G4 AIN, G 5 AIN, G 6 AIN, ⁇ lei AIN, K AIN, F AIN, Ii AIN, P, AIN, ⁇ va 2 AIN, O ⁇ AIN, Y 6 AIN, I 6 AIN, ⁇ leYI, KYI, and ⁇ leYI, derivatives of AIN with one or more non-peptide linkages between amino acid residues, such as ⁇ le al 1 AIN (wherein the designation al 1 refers to a methylene -CH 2 - linkage between the amino acid residue in position 1 ( ⁇ le) and the amino acid residue in position 2 (Y)), ⁇ le al 1 Nal 3 AIN, Kal 1 Nal 3 AIN, Kal 1 AIN, Nal 1 AIN, Nal 3 AIN, and Nal 1 Nal 3 AIN, and Nal 1 Nal 3
  • the physical properties of the AT4 receptors that determine binding of the AIN ligands were mapped using synthetic peptides and analogs, as described below in detail in the examples.
  • the structure of the ⁇ -terminus of AIN is most important for high affinity binding of an AIN peptide to an AT4 receptor.
  • the AT4 receptor binding site is a coordinated multidomain binding site wherein binding in one subdomain may be excluded by high affinity binding at a second subdomain through an induced conformation change in the AT4 receptor binding site hydrophobic pocket subdomain. At least three binding site subdomains in the AT4 receptor were mapped using synthetic peptides and analogs.
  • the binding site is stereospecific at a first subdomain for L-Naline in ⁇ -terminal amino acid position 1 (Nalj) of AIV; at a second subdomain for L-Tyrosine in position 2 (Ty ⁇ ) of AIV; and at a third site for L-isoleucine (Ile3) in position 3 in AIV.
  • Valj in AIV may interact laterally with the walls of the groove of the receptor while T3T2 in AIV may interact with the receptor binding site through van der Waals forces and hydrogen bonding.
  • AIV peptides having a weak hydrophobic amino acid at the N-terminus with an aliphatic side chain bind to the AT4 receptor with a higher binding affinity than AIV (binding of KYIHPF is 50-fold higher than AIV, and NleYHPF has a Kj of about 10" 12 M).
  • N-terminal extension of AIV is incompatible with binding, as is deletion of the N-terminal valine (Val residue. Deletion of Val reduced binding affinities 1000-fold; substitution of Val i with Sar decreased binding affinity; addition of D-arginine to the N-terminal Val x reduced affinity for the receptor by 100-fold.
  • the receptor binding site domain of the AT4 receptor contains a hydrophobic pocket conforming closely to the space filled by norleucine (i.e., engaging the Valj residue of ATV) and in close apposition with a negatively charged residue (i.e., engaging the primary amine of the N-terminus of Val j ). Removal of the N-terminal amino group decreases by 1000-fold.
  • the C-terminus of the AIN peptide is relatively less important in the receptor binding and C-terminal extension of AIN ligands of the invention with "X" is allowed. However, removal of both the Pro 5 and Ph ⁇ 6 residues from AIN reduced binding affinity by about 21-fold to a Kj of 500nM.
  • the C-terminus of the AIN peptide may determine receptor subtype specificity of binding.
  • AT4 receptors isolated from bovine adrenal cortical membranes do not effectively bind AIN peptides synthesized with an ⁇ -terminal extension with Sar or GABA.
  • the illustrative AT4 receptors effectively bind peptides having the N-terminal L-Nal replaced with D-Nal or Sar.
  • removal of the N-terminal L-Val from AIN all but eliminates binding to the AT4 receptor.
  • AT4 receptors of the invention have a receptor binding site that is stereospecific for L-Naline.
  • D-NaliYTHPF has 1000-fold lower binding affinity for the AT4 receptor than L-Va ⁇ YIHPF.
  • the illustrative AT4 receptor isolated from bovine adrenal cortical membranes contains a binding site that prefers weak hydrophobic amino acids in the number 1 position (i.e., Ri) of the AIN ligand, i.e., increasing hydrophobicity by replacing Nal j with Phe (i.e., FiYTHPF) decreases binding affinity 4-fold, but replacement of Nalj with another weak hydrophobic amino acid (i.e., IiYTHPF) results in only a slight change (an increase) in binding affinity.
  • Ri number 1 position
  • Phe i.e., FiYTHPF
  • IiYTHPF another weak hydrophobic amino acid
  • ⁇ -terminal extension is incompatible with binding, deletion of the terminal valine residue eliminates binding (Kj >10" 6 ), substitution with Sar decreased binding affinity, substitution with He results in equivalent binding, substitution with Phe resulted in a 5-10-fold decrease in the affinity of binding, Pro-substituted AIN peptides bind with 100-fold lower affinity, Lys-substituted AIV peptides bind with 10-fold higher affinity, and AIV ligands having a norleucine in the number 1 position (also abbreviated herein Nle, NLe, NLeu, NLeu j , or Nlei) bound with 1000-fold higher affinity.
  • Nle norleucine
  • the interaction between the AT4 receptor binding site and AIV ligand may be dictated by requirements for an AIV ligand containing a flexible aliphatic carbon side chain, (i.e., as opposed to a relatively rigid aromatic ring), rather than by the degree of hydrophobicity of the side chain.
  • substitution of Val ⁇ with Aspi results in an analog with no binding affinity for the AT4 receptor (i.e., has a K d > lO ⁇ M).
  • the AT4 receptor binding sites of the invention may prefer a flexible aliphatic carbon side chain having 4 carbon atoms that lack a positively charged residue.
  • Heptanoylj AIV with a 5 carbon side chain has reduced affinity as compared to Nlej AIV.
  • Nlei YTHPF has higher binding affinity for an illustrative AT4 receptor than Lysi YTHPF, which was higher than NVal j YIHPF, which is in turn higher than OrniYIHPF.
  • the AIV peptide ligands of the invention having norleucine substituted for Nal j i.e., ⁇ le j YIHPFX
  • the AT4 receptor binding site interacts specifically with the ⁇ -terminal amino acid residue (i.e., Ri ), and the latter interaction is specific with respect to both absolute space occupancy volume (i.e., of the receptor binding site) and charge (i.e., of the AIN ligand).
  • methylation of isoleucine in Ilei of IiYTHPF reduces affinity of the illustrative receptor for the peptide by 67-fold; substitution of the Nalj primary amine ( ⁇ H 3 ) with a secondary amine (-NH-; in this case by substituting Proj for Nal j , to form PYIHPF) reduces the affinity of binding to the illustrative receptor by 8-fold; substitution of Nal ⁇ with benzoic acid or 6-amino-hexanoic acid gives peptides with a K j >lmM; and r replacing Nal j ⁇ GABA (gamma-amino butyric acid; to form GAB A- YTHPF) decreases binding affinity by 250-fold for the illustrative receptor.
  • substitution of the Nalj primary amine ( ⁇ H 3 ) with a secondary amine (-NH-; in this case by substituting Proj for Nal j , to form PYIHPF) reduces the affinity
  • the AT4 receptor binding sites of the invention also appear to be stereospecific for Tyr 2 (i.e., Y) in the R 2 position of the subject AIN peptide ligands.
  • Y i.e., Y
  • substitution of D-Tyr 2 or Ph ⁇ 2 (with a benzyl ring) for Tyr 2 (with a phenolic ring) results in analogs (i.e., N[D-Y 2 ]IHPF, or NF 2 LHPF, respectively) with very low affinity for the illustrative adrenal cortical receptor.
  • Phenolic side chains in the Tyr 2 residue may also interact with residues in the subject AT4 receptors through hydrophobic and/or hydrogen-bonding.
  • the AT4 receptor binding sites of the invention tolerate replacement of the V 1 -Y 2 peptide bond with a non-carbonyl bond that has a similar bond length, but is non-planar and has a non-rigid carbon-nitrogen bond.
  • the latter replacement bond may preferably be resistant to proteolytic hydrolysis thereby conferring additional stability on the AIV ligand and enhancing utility in therapeutic compositions for oral delivery.
  • replacement of the N 1 -Y 2 peptide bond with a methylene bond reduces receptor binding affinity by only 5-fold; and, replacement of both the N 1 -Y 2 and I 3 -H 4 peptide bonds with methylene bonds results in N-N,-CH 2 -NH-Y 2 N 3 -CH 2 -NH-H 4 P 5 F 6 -C (also referred to herein as Vali Val 3 AIN or divalinal AIN) that has an affinity equal to or better than NYTHPF.
  • the binding site of the AT4 receptors of the invention is a coordinated, multidomain binding site wherein binding in one subdomain of the binding site may be enhanced or inhibited by binding at a distant second subdomain.
  • substitution of He for Phe at the R position of NYTHPF 6 results in an analog (i.e., NYIHPI 6 ) that binds to AT4 receptor (i.e., through the Nj subdomain sites) with a higher affinity than the parent VYIHPF molecule.
  • substitution of Ile 6 for Ph ⁇ 6 in KYTHPF 6 results in an analog (i.e., KYIHPI 6 ) that binds to the receptor (i.e., through the Nj subdomain site) with a lower affinity than the parent KYIHPF 6 molecule.
  • the C-terminus of the subject AIN peptide ligands appears to be relatively less important in receptor binding.
  • deletion of the C-terminal Pheg from VYIHPF i.e., to form Ni Y 2 I 3 H 4 P 5
  • C-terminal extension with histidine does not alter binding (i.e., to form N1Y 2 I 3 H 4 P 5 F 6 H 7 ); and, addition of both bis and leu reduces affinity only 2-fold (i.e., Vi Y 2 I 3 H 4 P 5 F 6 H 7 L 8 ).
  • Truncation of the C-terminus, i.e., at the R 5 position decreases binding.
  • removal of Pr ⁇ 5 from VYTHP to give NYTH decreases binding 21 -fold, and gives an analog with a Kj>500nM.
  • the binding site domains of the subject AT4 receptor of the invention recognize the ⁇ -terminus of the subject AIN peptide ligands with a high degree of specificity and while the receptor interacts less closely with the C-terminus this region of the subject AIN ligand may determine receptor subtype specificity.
  • antagonists of AIN are provided that bind to the AT4 receptor.
  • Presently particularly preferred antagonists of the invention include the non-peptide divalinal AIN and the C-terminal substituted tripeptide ⁇ leYi amide, as described in Example 4, although other antagonists will be readily apparent from the data and disclosure set forth herein.
  • aspects of the invention include processes for identifying AIN peptide ligands, i.e., by structural examination of the receptor binding requirements of test preparations (e.g., with respect to both blocking and/or promoting binding of the alternative peptide) to AT4 receptors such as those in heat-treated purified membrane preparation that are free of peptidase activity and devoid of other angiotensin receptors, i.e., ATI or AT2 receptors.
  • AIN peptide binding activity can be tested, e.g., using the receptor binding assays described herein, and that analogs, AIN peptide derivatives, and covalently modified AIN peptide or non-peptide ligands may exhibit activity as antagonists, agonists, promoters, or enhancers of AIN binding to its AT4 receptor.
  • AIN peptides may be prepared with substitution of other L-amino acids having different steric, electronic, and hydrophobic character for the L-Nal in the natural AIN ligand.
  • the first or second messenger intracellular pathways triggered in cells by interaction of an AIN ligand with an AT4 receptor may be used to test a series of peptides, analogs, derivatives, or covalently modified AIN peptides for their ability to bind to the AT4 receptor and trigger the intracellular signal. For instance, activities such as tyrosine kinase, guanylate cyclase, Protein kinase C, Ca “1”” * " flux changes, phospholipase C (PLC) activity, or prostaglandin or endocrine or exocrine hormone release from cells, may be monitored to determine whether the peptide triggered the AT4 receptor, and the receptor then signaled an increased or decreased activity in the cell.
  • activities such as tyrosine kinase, guanylate cyclase, Protein kinase C, Ca “1”” * " flux changes, phospholipase C (PLC) activity, or prostaglandin or endocrine or
  • the AIN peptides, AIN analogs, agonists and antagonists, and derivatives and covalently modified forms of the AIN peptides of the invention are recognized by their ability to bind the AT4 receptor with an equilibrium dissociation constant (K-i) below 3 x lO ⁇ M, more preferably below 3 x 10" 8 M and most preferably below 3 x 10 _9 M, and to a low binding affinity for ATI and AT2 receptors with a K (j greater than 1 x lO ⁇ M.
  • K-i equilibrium dissociation constant
  • processes are provided for identifying and characterizing a physiological effect of an angiotensin AIN peptide by assaying the effect(s) of the peptide on a selected in vitro cellular process.
  • an angiotensin AIN peptide by assaying the effect(s) of the peptide on a selected in vitro cellular process.
  • it may be convenient to assay renal blood flow, or in vitro cellular processes of endothelial cells and/or vascular smooth muscle cells.
  • assays may examine the effects of an AIV peptide on growth of a cardiomyocytes in vitro.
  • the processes disclosed herein are also useful in identifying how the in vitro activities of physiological AIV may be blocked or promoted by AIV peptides, AIV analogs, or derivatives or covalently modified forms of AIN peptides, as well as AT4 receptor fragments and the like.
  • Representative examples of useful assays for identifying the subject AIN peptide ligands and AIN ligands are provided in the examples.
  • cellular processes is intended to mean biological activities that may be measured in vitro or in vivo by quantitative and/or qualitative assay.
  • cell growth or metabolism may be measured (e.g., radiolabeled amino acid synthesis into protein, glycolytic activity, oncogene expression, and the like); or, proliferation (e.g., 3 H-thymidine synthesis into D ⁇ A); or, marker expression (e.g., mR ⁇ A by Northern, protein by Western blot, antigen by immunoassay, in vitro selectable drug-resistance marker by cell survival in toxic drug, and the like); or, electrical activity (e.g., in neural cells).
  • compositions and methods for promoting or inhibiting cellular activity of neural cells e.g., neural motor, cognitive or analgesic activity of neural cells in the brain.
  • the effect of the AIN compounds on motor activity may be observed by examining alterations in activity as measured with open-field techniques.
  • the cognitive activity may be observed by passive avoidance testing, Morris swimming maze performance, and various operant tasks.
  • To assay the effects of an AIN composition on a cellular process it may be useful, for example, to measure cellular processes before and after addition of AIN peptides to make comparative observations in parallel cell cultures. In this manner antagonists, agonists, inhibitors, promoters, enhancers, and the like may be identified and characterized with respect to their physiological effects in vitro and possible effects in vivo.
  • the route of delivery of the AIN ligands, AT4 receptor, AT4 receptor fragments, and AIV monoclonal antibodies of the invention is determined by the disease and site where treatment is required.
  • the compounds or compositions of the invention may be applied topically, or by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal injection, as well as by intrabronchial instillation (e.g., with a nebulizer), transdermal delivery (e.g., with a lipid-soluble carrier and skin patch), gastrointestinal delivery (e.g., with a capsule or tablet), intracerebroventricularly (icv) into brain, or intraspinally into cerebrospinal fluid (CSF).
  • the preferred therapeutic compositions will vary with the clinical indication.
  • AIV ligand per unit dose depends, among other things, on the particular ligand employed, on the body weight and the chosen inoculation regimen.
  • a unit dose of ligand refers to the weight of ligand without the weight of carrier, when a carrier is used.
  • An effective treatment will be achieved in the microenvironment of the cells at a tissue site as the concentration of AIV ligand approaches a concentration of 10" 5 M to 10 _11 M.
  • the most preferred method to achieve the therapeutic concentration is to gradually escalate the dosage and monitor both the biological effects and the concentration in the biological fluids (e.g., through the use of a diagnostic immunoassay, or radioisotopic or chemical label).
  • the initial dose, for such an escalating dosage regimen of therapy will depend upon the route of administration. For intravenous administration, for an agent with an approximate molecular weight of 10,000 daltons, an initial dosage of approximately 70mg/kg body weight is administered and the dosage is escalated at 10-fold increases in concentration for each interval of the escalating dosage regimen. Therapeutic efficacy in this example is achieved at 0.7-70mg/kg body weight of the theoretical 10,000 dalton peptide.
  • the compounds may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses.
  • suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various nontoxic organic solvents.
  • the pharmaceutical compositions formed by combining the ATV ligands or receptor fragments with the pharmaceutically acceptable carrier are then readily administered in a variety of dosage forms such as tablets, lozenges, syrups, injectable solutions, and the like.
  • These pharmaceutical carriers can, if desired, contain additional ingredients such as flavorings, binders, excipients, sweetening or flavoring agents, colored matter or dyes, emulsifying or suspending agents, and/or.
  • solutions of the AIN ligand or receptor fragment in sesame or peanut oil or in aqueous propylene glycol may be employed.
  • the present invention further provides processes for isolating inhibitors of an AT4 receptor- AIN ligand interaction by: a) selecting a cell type that expresses the AT4 receptor; b) adding an AIN ligand to a control culture of said cells; c) adding the AIN ligand and a putative inhibitor to a second test culture of the cells; and d) measuring the level of binding of the AIN peptide to the cells in said second test and control cultures. In the case that an inhibitor is present in the preparation, the level of binding in the test culture is lower than that in the control culture. (An example of such a process is provided in Example 1).
  • this process may be used to identify an inhibitor of an AT4 receptor- AIN ligand interaction in chromatographic fractions and the like during solubilization, isolation, and purification of said inhibitors, and that the subject inhibitors may act as agonists or antagonists of the action of AIN induced when AIN binds to its specific AT4 receptor.
  • the invention provides an AIN angiontensinase enzyme capable of hydrolyzing a peptide bond between an arginine and a valine residue in an angiotensin polypeptide, e.g., a polypeptide with a DR ⁇ VYIHPF ⁇ -terminal sequence, wherein " ⁇ " indicates the proteolytic cleavage site that gives rise to an AIV peptide, i.e., with an ⁇ -terminal sequence related (as described above) to the amino acid sequence VYTHPF.
  • Isolation and substantial purification of AIV angiotensinase may be conveniently accomplished, for example, by preparing an affinity resin having a non-cleavable or slowly-cleavable AIN ligand covalently bound to the resin, e.g., chemically modified derivatives of a peptide in an amino acid sequence selected from among DRNYIHPF, DRVYIHP, DRVYIH, DRVYI, DRNY, DRN, RNY, or ⁇ RNYIHPF, ⁇ RNYIHP, ⁇ RVYIH, ⁇ RVYI, ⁇ RVY, ⁇ RV.
  • an affinity resin having a non-cleavable or slowly-cleavable AIN ligand covalently bound to the resin, e.g., chemically modified derivatives of a peptide in an amino acid sequence selected from among DRNYIHPF, DRVYIHP, DRVYIH, DRVYI, DRNY
  • the peptide useful in this assay is selected based on its ability to bind the AIV angiotensinase and to be resistant to cleavage by the enzyme.
  • a test preparation of a cellular or tissue extract (or a biological fluid sample) is next chromatographed through the affinity resin; the bound polypeptide(s) is eluted, e.g., at low pH and high salt (e.g., pH2-3, and 2-3M ⁇ aCl, and the like), or the bound polypeptide is eluted by adding an excess of AIN ligand.
  • the presence of the AIN angiotensinase in the eluate can be determined by assaying for the ability of the column eluate to catalyze hydrolysis of an Arginine-Naline peptide bond in an angiotensin peptide (e.g., AIII), and subsequently confirming that the sequence of the product of the reaction has a Valine residue at ⁇ -terminal amino acid and an AIV peptide sequence.
  • the novel AIV ligand-AT4 receptor system of the invention is useful in a complementary or antagonistic role to All in mediating long-term effects of angiotensins, and in modulating the effects of Al, All, or AIII on cells.
  • AIV is derived from All (or AIII) directly (e.g., through the action of a specific AIV angiotensinase, and other peptidases); 2) AIN is very labile and will accumulate at physiologically significant concentrations only when high levels of All are present at the target site; 3) the AT4 receptor is specific for AIN ligand (with accompanying low affinity for the parent peptide, AIII). Under certain conditions, AIN begins to accumulate at angiotensin target tissues as the All levels rise. When ATV concentrations rise to near 0.5nM (i.e., the K-i of the receptor) auxiliary processes which modify the acute action of All will be engaged.
  • 0.5nM i.e., the K-i of the receptor
  • an intracellular signaling system different from that employed by AIL
  • the activation of such an intracellular system may potentiate or antagonize the target cell's short-term response to AIL
  • One physiological function of the AT4 receptor-ligand system may be to impart a longer- term response to high-level or chronic angiotensin stimulation in a tissue.
  • AIV angiotensinase
  • bovine adrenal tissues have shown that the AT4 receptor is specific, with almost no affinity for AIL
  • AIV is metabolized/hydrolyzed in bovine adrenal homogenates at 200 times the rate of All and 4 times the rate of AHI.
  • AIV may be derived directly from All by action of a dipeptidylaminopeptidase, termed herein AIV angiotensinase.
  • AIV angiotensinase The location of AT4 receptor sites in groups of cells in tissues allows a skilled artesian to predict likely functions for the AT4 receptor in different tissues.
  • All and/or Am
  • AT4 receptor-ligand interaction many activities previously attributed to the action of All (and/or Am) may be triggered or regulated instead by the AT4 receptor-ligand interaction.
  • ATV ligand acts as a negative-feedback agent thus enabling tighter control on the aldosterone release process.
  • the ATV ligand- receptor system may also be associated with a previously inexplicable up-regulation of the angiotensin receptor seen following chronic All exposure of cells in vitro.
  • Still other functions attributed to All that may be mediated instead by the AIN ligand- receptor system include altering the release of catecholamines from adrenal medullary cells or regulating adrenal blood flow.
  • the AIN ligand-receptor system modulates (i.e., increases or decreases) either the acute and/or the long-term synthesis and release of chromaffin catecholamines, e.g., by acting to stimulate intracellular expression of tyrosine hydroxylase (the rate limiting enzyme in the synthetic pathway).
  • AT4 receptor-ligand system may have a role as a mediator of long-term angiotensin effects on endothelial cells (e.g., cell growth; Example 5).
  • AIV ligand-receptor interactions also appear to activate processes in endothelial cells that are complementary or antagonistic to those activated by AIL
  • some of the ATV ligands that are embodiments of the invention are useful for increasing blood flow (e.g., renal blood flow as demonstrated in the examples).
  • Example 2 for physiological functions of AIV peptide ligands and AT4 receptors in controlling renal blood flow, for the cellular biology of AIV ligand-AT4 receptor interactions (e.g., second messenger pathways, G-proteins, phosphorylation, intracellular Ca* "1" , phosphoinositide turnover, and guanylate cyclase activity), for vascular effects on venular and aortic endothelial cells and vascular smooth muscle cells and G-protein linkage of certain ATN-receptors, for endocrine effects on adrenocortical cell catacholamine release for effects on cardiac myocytes (i.e., cardiocytes), and for characterization of brain AT4 receptors (e.g., in hippocampal cells and in cerebellum, hippocampus, piriform cortex, Par 1/2, Fr 1/2, caudate putamen, HDB, thalamus, and inferior coUuculus), as well as, neurological effects of intracerebroventricular injection of
  • AIN is active in endothelial cells in enhancing cellular proliferation (as evidenced by thymidine incorporation) and stimulating production of endothelial cell relaxing factor (EDRF).
  • EDRF endothelial cell relaxing factor
  • results set forth in the Examples further identify a role for the AIN ligand- AT4 receptor interactions in triggering normal and/or hyperplastic growth of endothelial cells in sites of tumors or traumatic or wound injury, and angiogenesis, and a therapeutic use for AIN analogs, agonists, antagonists, and derivatives and covalently modified AIN peptide ligands that are capable of inhibiting vascular smooth muscle cell growth in such hyperplastic states while at the same time promoting endothelial cell growth.
  • the agonist compositions are also useful for encouraging endothelial cell growth, e.g., in wound sites; antagonists for discouraging vascularization in tumor sites.
  • the AT4 receptor-ligand system may play a role in triggering vasodilation through a selective effect on subpopulations of endothelial cells that exist in particular vascular beds (e.g., in the heart, lung, liver, kidney, brain and the like).
  • endothelial cells that exist in particular vascular beds (e.g., in the heart, lung, liver, kidney, brain and the like).
  • vascular beds e.g., in the heart, lung, liver, kidney, brain and the like.
  • AIN ligand-receptor system mediates actions of angiotensin that fall within the bounds of cardiovascular regulation and body water homeostasis.
  • AIN analogs, ATV agonists and antagonists, and derivatives and covalently modified AIN peptide ligands include promoting renal blood flow (e.g., in chronic kidney diseases) or, alternatively, inhibiting renal blood flow (i.e., using inhibitors and antagonists of AIN), e.g., in conditions of hyperacute renal dysfunction and water loss, or during renal surgical procedures.
  • cardiac myocytes also termed herein “cardiocytes”
  • cardiac myocytes also termed herein “cardiocytes”
  • ACE angiotensin converting enzyme
  • AIN antagonizes the hypertrophic action of AIL
  • the control of cardiocyte growth may be regulated endogenously by a balance between the activating action of ALT and the inhibiting action of AIN.
  • AIN and AIN agonists will be effective in blocking the development of, and reversing the effects of, left ventricular hypertrophy in patients.
  • ACE inhibitor is due not to their inhibition of AH synthesis but to their ability to enhance the synthesis of AIN ligands such as results from the shunting of precursors from the All synthetic pathway into the AIN pathway.
  • beneficial effect of ACE inhibitors in treating cardiac hypertrophy may be due to ACE inhibitor enhancement of the formation of AIN.
  • AIN operate by separate receptors employing different intracellular signaling systems. It has been reported that ACE inhibitors may have a beneficial effect in reducing cardiac hypertrophy through effects at the level of All or AIII. Considering the results disclosed herein it is most likely that the long-term effects previously attributed to decreased ATI may in fact be mediated by the interaction of increased levels of endogenous AIN ligands with the AT4 receptor. Further, it is most likely that the antagonists and agonists of AIN ligands, disclosed herein, will provide improved pharmaceutical compositions for treating cardiac hypertrophy attributable to the renin-angiotensin system, e.g. ventricular hypertrophy. The inventors believe that the interaction between AIN and the AT4 receptor may trigger the receptor and inhibit growth in cardiomyocytes.
  • angiotensin In adrenal cells angiotensin All's role in the regulation of aldosterone release from the adrenal cortex is reportedly well established (27). As shown herein, certain activities (such as adrenocortical cell growth), previously attributed to ATT or ATI, are actually activated following AIN ligand binding to the AT4 receptor. All (and AIII) reportedly stimulates aldosterone release from adrenal glomerulosa cells.
  • the disclosure, herein, of high levels of AT4 receptors in adrenal cortical cells suggests a possible role of AIN ligand (i.e., rather than All or AIII) in triggering AT4 receptors on adrenal cells to inhibit All-mediated aldosterone release.
  • AT4 receptors may be to up-regulate the threshold level of All ligand required to trigger a cellular response by regulating the levels of cellular ATI and/or AT2 receptors and/or to regulate adrenal blood flow.
  • AT4 receptors are found at even higher levels in the adrenal medullary cells where All has previously been reported by others to potentiate catecholamine release.
  • AIN ligand may modulate release of catecholamines (i.e., increase or decrease the release) acutely (or possibly even long-term, e.g., by triggering the AT4 receptor and thereby stimulating increased or decreased expression of tyrosine hydroxylase, the rate- limiting enzyme in catecholamine synthesis.
  • AIN may act to inhibit growth of the cells thus opposing the action of AH.
  • Agonists of AIN binding to the AT4 receptor will be effective inhibitors of vascular smooth muscle growth and will be therapeutically useful in reducing neointimal growth which often occurs following angioplasty.
  • AIN ligands and the AT4 receptors may function as growth factors of the tyrosine kinase class indicates that certain inhibitors of tyrosine kinase growth factors may also serve as inhibitors of certain angiotensin AIN ligand-receptor system- mediated cellular hypertrophic processes (e.g., ventricular hypertrophy), and that nucleotide probes constructed for complementarity to portions of R ⁇ A encoding the AIN ligand and receptor sequence may be useful in identifying other members of the AIN family of growth factors.
  • the invention also provides diagnostic applications for the AIN peptide ligands and antibodies.
  • the role of the AT4 receptor-ligand system in cardiovascular regulation suggests a possible value to diagnostic tests for monitoring the levels of AIN ligand and AT4 receptor in biological fluids and tissues (i.e., rather than All or AIII).
  • Individuals with high renin-sodium profiles are reportedly at five times greater risk of myocardial infarction than individuals with low renin-sodium profiles despite adequate control of systemic blood pressure (28).
  • the AIN peptides, ligands, receptor fragments, and the like disclosed herein are useful in diagnostic assays, e.g., immunoassays, for the detection of the presence or amounts of AIN ligands or receptors in tissues, cells, and biological fluids of patients.
  • diagnostic assays e.g., immunoassays
  • the AIN peptides, ligands, analogs, derivatives, or covalently modified AIN peptides of the invention may be formulated in buffers with stabilizers, e.g., for use as positive or negative controls in diagnostic assay, or in reagent test kits for receptor- binding assays.
  • the AIN ligands of the invention may be readily employed using conventional techniques to produce polyclonal or monoclonal AIN ligand specific antibodies, and that the isolation and purification of the AT4 receptor provides materials useful for preparation of polypeptide fragments (e.g., using C ⁇ Br and proteolytic enzymes) that can be subjected to automated amino acid sequencing.
  • the amino acid sequence of the AT4 receptor provides the sequence data necessary for construction of conserved and degenerate nucleotide probes for cD ⁇ A or genomic molecular cloning of nucleic acids expressing the AT4 receptor, mutant AT4 receptor, or fragments of the AT4 receptor.
  • a convenient method for molecular cloning of the receptor is provided in Example 7. EXAMPLE 1
  • Table 3 The results of the equilibrium binding studies with membrane-bound AT4 receptor are summarized in Table 3.
  • 125 I-angiotensin IN binding to receptors in P2 membrane preparations from rabbit heart Comparisons were made of the binding of both AIN and of ATI, i.e., to the classical ATI receptor sites defined by binding of 125 I-Sar 1 ,Ile -AII. Binding studies were carried out in a buffer (below) containing an extensive cocktail of inhibitors that was designed to minimize metabolism of both the receptor and the test ligand, i.e., the buffer contained 5mM EDTA, 0.2% BSA, lO ⁇ M Bestatin, 50 ⁇ M Plummer's inhibitor, and lOO ⁇ M PMSF.
  • the buffer contained 5mM EDTA, 0.2% BSA, lO ⁇ M Bestatin, 50 ⁇ M Plummer's inhibitor, and lOO ⁇ M PMSF.
  • Angiotensin peptides (i.e., Al, All, AHI, or AIN) were stable in this buffer for 4 h at 37°C with less than 10% hydrolysis measured by reverse phase HPLC.
  • the studies were conducted as described in the Materials and Methods, below.
  • the association rate constant (k j ) for 125 I-AIN was determined to be 3.05 x 10 8 M _1 min -1 and the dissociation rate constant (k. ⁇ ) was 0.028 +/ 0.017 min" 1
  • the overall dissociation constant (K d ) measured under equilibrium binding conditions was determined to be 9.15 x 10 _11 M.
  • the results in Table 7, also include a summary of studies designed to analyze the structural features of the N-terminus of an AIN ligand that are required for binding to an AT4 receptor.
  • the results of these structural studies are also presented in Figure 2B.
  • the results show that modification of the ⁇ -terminal valine residue (i.e., by ⁇ -terminal shortening of AIN to AII (4 _ 8) ), or extending the ⁇ -terminus with a hydrophobic residue such as Sar or GABA, or changing the stereoisomer of the L-Nal to D-Nal, all drastically decrease binding of an AIN ligand to the AT4 receptor (Table 7).
  • the AT4 receptor also failed to bind DuP 743 (DuP, Figure 2B) or CGP 42112A (CGP, Figure 2B) and thus did not exhibit the pharmacological properties of a classic ATI binding site (26). As shown in Figure 2B, the ability of the various compounds to inhibit AIV binding to the solubilized AT4 receptor was tested.
  • DAAI1 desAsp angiotensin I (i.e., identical at the ⁇ -terminus to AIII; see open squares with a dot, Figure 2B); AIN, angiotensin IN (closed diamonds, Figure 2B); AH (open squares, Figure 2B); SIAII (All lacking the He residue at position 5, Figure 1; open diamonds, Figure 2B); DuP (Dup 743, an AH analog; open squares, Figure 2B); CGP (CGP 42112A, another All analog; closed triangles, Figure 2B); and, AIII (open triangles, Figure 2B.
  • ⁇ -terminal neutral polar amino acid e.g., valine
  • ⁇ -terminal extension is incompatible with binding
  • deletion of the terminal valine residue eliminates binding (K j >10" 6 )
  • substitution with Sar decreases binding affinity
  • substitution with Phe results in a 5- 10-fold decrease in the affinity of binding
  • Pro- substituted AIN peptides bind with 100-fold lower affinity, but substitution with He results in equivalent binding
  • Lys substitution results in 10-fold higher binding affinity of the KYIHPF AIN ligand (data not shown).
  • the D-substitution and glycine-substitution data confirms that positions 1-3 of the AIV molecule are critical for determining binding affinity to the receptor. Positions 4-6 are less critical. In fact removal of C-terminal groups appears to enhance binding affinity perhaps by reducing steric constraints. Ligands containing C-N nonpeptide bonds can be produced that possess high affinity. In general, highest affinity is obtainable by dual modifications at bonds between amino acids 1-2 and 3-4. Val(l)Val(3) AIV appears totally resistant to enzymatic degradation upon exposure to rat kidney homogenates. As further shown in Table 7, tripeptides containing straight chain aliphatic amino acids in position 1 exhibit high affinity.
  • Example 1 Materials and Methods: Peptide Synthesis The angiotensin analogs employed in this study were synthesized by the standard Merrifield method utilizing t-Boc protected amino acids and chloromethylated resins on a Vega 250 coupler automated synthesizer. Following synthesis, the crude peptides were purified by preparative reverse-phase HPLC using a 1 h gradient for elution at 9ml min. Initial conditions were 90% H 2 O, 10% acetonitrile, and 0.1% TFA and the final conditions at the top of the gradient were 65% H 2 O, 35% acetonitrile and 1% TFA. Purified peptides were amino acid analyzed to determine both peptide and total purity. Typically the peptides produced were greater than 99% pure and contain 20-25% acetate. Tissue Preparation: bovine adrenal cortical tissues
  • Adrenal cortex was removed from bovine adrenals obtained from a local slaughterhouse. The minced cortex was then homogenized in a Polytron as a 40:1 suspension in assay buffer at 10 sec/ml. The homogenate was then centrifuged at 500g for 10 min. to remove whole cells and nuclei. After a rehomogenization and recentrifugation the combined supernatants were spun at 40,000 x g for 20 min. The pellet was rehomogenized and respun at 40,000 rpm for 30 min. This final pellet was resuspended in assay buffer and layered on a discontinuous sucrose gradient (0.8M/1.2M). After a 100,000 x g spin for 90 min. the purified membranes were located at the density interface and were removed.
  • sucrose containing membrane suspension was diluted 1 :10 in assay buffer and spun a last time at 40,000 x g for 30 min.
  • the pellet was resuspended in assay buffer at a concentration of lOmg protein/ml and heat treated at 60°C for 30 min. in the presence of 20mM MgC ⁇ .
  • Binding studies bovine adrenal cortical membranes
  • Solubilization and characterization of the receptor from bovine adrenal membranes was accomplished by homogenizing the membranes (above) in hypertonic buffer followed by fractionation of the membranes by sucrose density gradient centrifugation.
  • the membrane preparation was then heat treated at 60°C in the presence of MgC-2 (to inactivate ATI receptors). The heat treatment also reduced endogenous peptidase activity in the preparations by 90-95%.
  • the AT4 receptor in the preparations was then solubilized using 1% zwitterionic detergent 3-[(3-cholamidopropyl) dimethyl ammonio]-l-propanesulfonic acid (CHAPS).
  • Binding studies rabbit heart P2 membranes from rabbit heart were prepared by homogenization and differential centrifugation at 4°C.
  • Binding was carried out in the presence of 5mM (EDTA), 0.2% heat-treated bovine serum albumin (HTBSA), lO ⁇ M Bestatin, 50 ⁇ M Plummer's inhibitor, lOO ⁇ M phenylmethylsulfonylfluoride (PMSF), and 50mM Tris, pH7.4, at 22°C. Binding was initiated by the addition of lOOmg protein and appropriate amounts of labeled ligand. (For kinetic binding studies the samples were incubated for 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, and 220 minutes at 37°C.
  • Dissociation (i.e., of ligand from receptors) experiments were conducted by adding lmM unlabeled AIV ligand competitor to the assay at 120 minutes after initiating binding (at 37°C) with 0.5nM 125 I-AIV.
  • N.Det. not detectable, i.e., less than 1.8 finol mg protein.
  • Bacitracin is a polypeptide antibiotic with the sequence ICLEIKOIFHDD (i.e., O is ornithine), and it is often included in angiotensin binding assays to inhibit the action of nonspecific proteases (i.e., as an alternative substrate for the proteases).
  • bacitracin interference is of potential significance for at least two reasons: 1) previous investigators who have included bacitracin in their assay buffers may have inhibited AIV ligand binding to the AT4 receptor; and, 2) inhibition of AIN binding by this polypeptide (notably at a very high molar concentration) may provide an indication of amino acid sequences that may contribute to electrostatic interactions in the AT4 receptor binding site (e.g., R 1 IR 2 HR 2 , where R j is an amino acid with an aromatic side chain such as OH, SH, or ⁇ H, and R 2 is a polar amino acid).
  • R 1 IR 2 HR 2 where R j is an amino acid with an aromatic side chain such as OH, SH, or ⁇ H, and R 2 is a polar amino acid
  • Receptor autoradiography is a useful extension of radioligand binding studies since it provides detailed anatomical information about the location of receptors in tissues and groups of cells in tissues, and thus it facilitates understanding the function of the AIN ligands and AT4 receptors in those sites.
  • Autoradiographic analyses of serial sections of guinea pig brain (20mM thickness) were performed. The autoradiographs showed a pattern of distribution for AH receptors and AT4 receptors in the Habenula (Figure 3), Hippocampus (data not shown) Cerebellum (data not shown), Prefrontal Cortex (data not shown), and Thalamus (data not shown).
  • the receptor distribution in the tissue was determined by binding of 1 5 I-Sar ⁇ ,Ile -AII, or 125 I-AIN, respectively. Specificity of ligand binding in these autoradiographic studies was demonstrated by competing the binding of the specific ligand (i.e., All or AIN) with unlabeled lOOnM Sarj-Heg-AII, or lOOnM AIN, respectively.
  • the data demonstrate that while specific All and AT4 receptors are located at similar sites in the Habenula, Hippocampus, and Cerebellum of guinea pig brain, the two receptors are distinct with regard to exact groups of cells that express the two different receptors.
  • the findings of AT4 receptors in the Hippocampus suggest that the AIN ligand-receptor interactions in the Hippocampus may mediate unique angiotensin-dependent functions including memory enhancement.
  • the AIN ligand-receptor system may provide a link between the Hippocampus and memory.
  • the mutually exclusive cellular distribution of AIN and All receptors is demonstrated in the autoradiograph shown in Figure 3.
  • Panel A reveals intense 125 I-AIN binding in the habenula
  • Panel D indicates that 125 I-Sar ⁇ ,Ile 8 -AII binding is localized primarily to fiber tracts including the visual tegmental relay zone and the medial lemniscus.
  • Example 7 Quantitative aspects of binding in brain is presented in Example 7, and other tissues data is presented in Example 1, above.
  • the results show that all important cardiovascular tissues in guinea pigs contain the AT4 receptor. This result is not surprising in light of the observation (above) that vascular endothelial cells contain high concentrations of receptors, but this is not responsible for tissue binding of AIN ligand because every vascularized tissue will possess AT4 receptors, i.e., skin and skeletal muscle has low levels of receptor.
  • tissue sections were incubated in the radioligand in the presence and absence of lOOnM unlabeled ATI or AIN peptide. After appropriate washing, autoradiograms were prepared by apposing the slide-mounted tissue sections to X-ray film (Hyperfilm, Amersham) for an appropriate exposure time. The amount of radioligand binding in a tissue was quantified using densitometric techniques and 125 I standards (Microscales, Amersham, Arlington Hts, IL).
  • EXAMPLE 3 Receptor Isolation. Purification, and Properties and Production of Monoclonal Antibodies Receptor Isolation and Purification:
  • the AT4 receptor was solubilized in high yield from purified bovine adrenal membranes using the zwitterionic detergent CHAPS (1%) at 4°C over 4 h under conditions where peptidase activity and differential solubilization of the AT4 receptor (but not the ATI receptors) is permitted (see also Example 4, Materials and Methods, below).
  • membranes from a variety of different tissues and cells were incubated for 4 h in Hepes buffer (20mM, pH7.4) containing 1% CHAPS and a cocktail of protease inhibitors and alternative protease substrates, i.e., lO ⁇ M bestatin; 50 ⁇ M Plummers' inhibitor; 0.2% BSA (bovine serum albumin); and lOO ⁇ M PMSF (phenylmethylsulfonyl fluoride).
  • a most useful component of any AH receptor purification scheme was including a step wherein the solubilized membrane proteins were subjected to a heat treatment at 60°C, e.g., for 20 minutes and in the presence of 20mM Mg "1" *. This step was useful in destroying any residual All receptor leaving the AT4 receptor intact.
  • the AT4 receptor was stable to chromatofocusing and SDS-PAGE, allowing isoelectric focusing, or one- or two-dimensional PAGE or SDS-PAGE to be used for purification. Due to the slow-off rate of 125 I-AIN binding, the receptor was radiolabeled with 125 I-AIN ligand to allow ease of identification during purification. As an additional aid to purification, the receptor was successfully cross-linked to a 125 I-radiolabeled AIN analog ligand having a C-terminal extension, i.e., from residue 8, with lysine residues (i.e., 125 I-Lysu-AIN).
  • the Lys n -AIN analog binds to the AT4 receptor with a K, j that is similar to AIN ligand.
  • BS3 Bis (sulfosuccinimidly) suberimidate
  • the 125 I-Lys ⁇ j-AIN analog of AIV was bound to the AT4 receptor and then cross-linked to the AT4 receptor through the e-amino group of Lys.
  • Purification of the AT4 receptor may also be achieved, for example, by ion exchange, lectin chromatography, hydrophobic chromatography with conventional techniques, HPLC, or FPLC.
  • Identification of the family to which a receptor belongs commonly permits predictions to be made about possible improvements in purification, useful methods for stabilizing the receptor during purification, cellular sources and assays useful for molecular cloning of the receptor, and identification of novel physiological roles for a receptor.
  • neurotransmitters and hormones are known to interact with four types of plasma membrane receptors: 1) multisubunit receptors that regulate an intrinsic ion channel; 2) G-protein linked receptors that, via the G-protein, can activate membrane channels and enzymes; 3) guanylate cyclase receptors that possess intrinsic guanylate cyclase activity in a single membrane spanning polypeptide chain; and, 4) protein tyrosine kinase receptors that have intrinsic tyrosine kinase activity capable of phosphorylating multiple protein substrates.
  • the AH receptor may be a member of the G-protein-linked family of cellular receptors.
  • the majority of known peptide receptors belonging to this family are characterized by seven membrane-spanning alpha-helical regions and when stimulated are capable of activating membrane-bound enzymes like adenylate cyclase, phosphodiesterase, and phospholipase C. (30). Additionally, membrane channel or ion transporter properties can be indirectly modified by the intervening G-protein (31).
  • many strategies have been devised to test a particular receptor's linkage with a G-protein, three strategies seem to predominate.
  • these include the following approaches: 1) GTP and its analogs are known to alter the binding affinity of agonists to their receptors. Therefore, the ability of GTP or analogs to change agonist-binding affinity is diagnostic of a G-protein-linked receptor. In the presence of GTP, dissociation of the G-protein subunits from the receptor results in a lowered affinity for agonists. This was examined (see Example 5) by the direct assessment of GTP (of GTP ⁇ S) effects on agonist binding via changes in dissociation rates or total binding over a range of GTP concentrations, or indirectly by monitoring shifts in IC50 values for agonists during competition for antagonist binding.
  • G-protein linkage Another indication of G-protein linkage is the ability of agonists to stimulate the intrinsic GTPase activity of the alpha subunit of G-proteins. This GTPase activity is triggered following receptor occupation and subsequent dissociation of alpha and beta-gamma subunits. 3) A final approach is to determine whether an agonist can facilitate nucleotide cycling. A crucial step in G-protein signal transduction is the agonist-stimulated dissociation of GDP from the alpha-subunit and its replacement with GTP. Changes in cycling are often assessed by comparing the binding of radiolabeled irreversibly bound GTP analogs before and after agonist stimulation.
  • AT4 receptor may be G-protein linked in certain cells (see Example 5) the AT4 receptor does not belong to the classical family of G-protein-linked receptors for at least three reasons: namely, 1.) Solubilization and stability characteristics of the All receptor (i.e., binding 125 I-Sar 1 ,He 8. AII) and the AT4 receptors (i.e., binding 125 I-AIN) are significantly different which is consistent with: a) large structural differences between the two receptors, and, b) differences in the structural basis of receptor integration into membranes. Thus, it is reasonable to assume that if the ATI/ ATI receptor is a member of the G-protein linked family of receptors, then the AT4 receptor probably is not.
  • BS3 cross-linked bovine adrenal AT4 receptor These molecular sizes are significantly different from the molecular weights of 55KDa to 65KDa that are commonly associated with members of the G-protein-linked family of receptors.
  • the AIN site is not a classical G-protein-linked receptor, then to what family of receptors does it belong?
  • Evidence in recent years indicates the presence of peptide receptors with intrinsic guanylate cyclase activity.
  • These receptors best exemplified by the mammalian A ⁇ P receptor, consist of a single polypeptide chain with one membrane-spanning region that possesses guanylate cyclase activity that resides near the intracellular C-terminus (32). Since only two such mammalian receptors have been identified (to date), the A ⁇ P and rat intestinal enterotoxin receptor, it is difficult to speculate on the probability that the AT4 receptor is a member of such a family of receptors. Nevertheless, the similarity in the molecular weights and in ion requirements of the ANP and AT4 receptors necessitates the consideration that the AT4 receptor may be a member of such a family.
  • the final receptor family to which the AT4 receptor may belong is the tyrosine-kinase growth factor family of receptors. These receptors are characterized by a protein kinase activity which preferentially phosphorylates tyrosine residues. Among the substrates of phosphorylation are the receptor itself and phospholipase C, which when phosphorylated initiates the inositol phosphate cascade (33).
  • the tissue response to prototypical peptides which act as tyrosine kinase receptors includes long- term alterations that invariably involve changes in the transcription rate of selective mRNAs.
  • tyrosine kinase receptors often "cross talk" with other cellular receptor types (34) in response to physiological and chemical stimuli. This type of role is precisely the function envisaged for the ATV ligand-receptor system.
  • a comparison of the solubilization, physical properties, and functional activities of the AT4 receptor with the cellular biology of members of the tyrosine kinase family of growth factor receptors suggests a closer relationship of the AT4 receptor to this family of receptors than to the guanylate cyclase family of receptors.
  • both the AT4 receptor and the FGF receptor have related biochemical characteristics, e.g., the FGF receptor has a molecular weight of about 140-150kDa (35), is relatively heat stable (i.e., at 56°C), and has divalent ion requirements (28).
  • AT4 receptors appear to have growth factor activity on at least endothelial cells and myocytes. (In the latter case, the tissue distribution and the activities of AT4 receptors are also consistent with a role for AT4 receptors in growth regulation. For instance, as disclosed above, high concentration of AT4 receptors is present in cardiovascular tissues where angiotensins are reported to enhance tissue growth.)
  • the molecular weight of the AT4 receptor is in the range of members of the tyrosine kinase families of receptors.
  • the AT4 receptor like members of both the tyrosine kinase families of receptors, is characterized by divalent cation binding sites (i.e., Mg "1""1" ).
  • the AT4 receptor like members of the tyrosine kinase and guanylate cyclase families of receptors, is characterized by relatively high heat stability (i.e., 60°C/20minutes).
  • the epidermal growth factor receptor (EGF) is heat stable at 50°C for 30 min., and has specific binding sites for Mn "1"1” and Mg "1-1” [28]).
  • the AT4 receptor appears to be a member of the tyrosine kinase family of receptors, and not the G-protein-linked family of receptors.
  • Experimental approaches to validate this vision are presented below, the experiments examine the ability of AIN ligand to stimulate phosphorylation of tyrosine residues in membrane proteins.
  • Cross-linking 125 I-AIN to the AT4 receptor can be accomplished with Bis (sulfosuccinimidyl) suberimidate (BS3) as discussed above.
  • the cross-linked receptor (approx. mw of 146,000) can then be electroeluted from PAGE gel slices in a substantially pure form for use as an electrophoretic standard.
  • For cross-linking one milligram of total solubilized membrane protein containing AT4 receptor was incubated with 30 x 10-6cpm of 125 I-AIN in 50mM Tris, pH7.4 and 150mM ⁇ aCl containing a cocktail of protease/peptidase inhibitors for 2 hr at 37°C (final volume 0.5ml).
  • Monoclonal antibodies are useful for purification of receptor, and for identifying the receptor (and fragments thereof) in tissues, cells, and biological fluids.
  • Purified or semi-purified AT4 receptor (preferably nondenatured) can be used as an in vivo or in vitro immunogen.
  • Those skilled artisans will recognize a variety of options available to them for evoking monoclonal and polyclonal antibodies, e.g., see Harlow, E. and D. Lane, Eds. "Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory, 1988).
  • antigen can be incubated in picogram quantities with murine, rat, or human lymphocytes.
  • Production of antibodies can be screened by testing for the ability of 125 I-AIV-prelabeled receptor to bind to antibodies adsorbed on a polystyrene plastic surface, e.g., in 96 well plates; or, alternatively, by testing the ability of the antibody to inhibit binding of a purified labeled receptor to AIN ligand adsorbed to a solid phase. In either case, antibody producing cells are identified, cultured, and cloned. The monoclonal antibody product of the cloned cell lines can bind the AT4 receptor in ligand-binding and non- binding domains of the AT4 receptor.
  • ⁇ on-binding domains can include structural regions of the molecule as well as enzyme active sites, phosphorylation sites, glycosylation sites, and the like.
  • the presence of antibodies specific for the ligand- binding domain can be assessed directly via the ability of the mono to competitively inhibit in binding assays.
  • antibodies are useful for receptor purification and immunohistochemical studies designed to elucidate the cellular location of receptors and also in structure/activity studies designed to map functional domains in the receptor.
  • AIN Receptor Antagonists and Agonists To test the ability of synthesized AIN ligands to competitively inhibit for 125 I-AIN ligand binding to the AT4 receptor, displacement curves were constructed using heat-treated (60°C for 20 min. in 20mM MgCl 2 ) purified bovine adrenal cortical membranes using Methods described in Example 1, above. Effects of AIN analogues on renal blood flow were determined as described in Example 6. The design of AIN analogs followed a question based approach. The unifying question: What are the essential ligand domains for receptor binding and activation? Individual chemical modifications were made to ask specific questions about spacial orientation of molecular surfaces, charge, hydrophobicity and occupancy of space (volume occupied at a specific location).
  • Agonist versus antagonist activity was assessed using a laser doppler to monitor renal cortical blood flow following infusion of a test analog into the renal artery (see Example 6). Maximal response was compared to the response (increased in flow) to AIN. (Note that under these conditions All produces a decrease in blood flow in this assay.) Interprestation of physiologic and binding data was based on the precept of a lock and key model of receptor binding and that dynamic change of the receptor upon interaction of the ligand was required for activation (full agonist activity; with second messenger activation).
  • Ligands with the highest affinity, when modeled in an energy minimized conformation offer a visual representation of the receptor binding site field surface (i.e., hydrophobic charge interactions) and charge locations in the "pre-binding state” and "non-activated state” (i.e., just as a clay imprint on a well fitting key represents the interaction surface of the lock).
  • Specific ligand domains induce changes in the receptor upon binding that produce cellular responses.
  • Ligands that fit the "pre-binding" receptor with high affinity may not activate the receptor, i.e., they may act as antagonists, while structures that induce changes in the conformation of the receptor may be compatible with, and part of, the changes that produce high affinity binding, i.e., they may act as agonists.
  • Fragments containing less than positions #1 through #4 have K i *s>500nM.
  • Figure 5 A shows no significant changes in arterial blood pressure following adminstration of KYTHPF at 100pmole/25ml min (open circles) or saline control (closed circles).
  • Figure 5B shows changes in renal blood flow following adminstration of KYIHPF at 100pmole/25ml min (open circles) or saline control (closed circles), with the increased blood flow being equal to 38% of the maximum attainable with a strong vasodilatory agent (i.e., bradykinin, as described above).
  • a strong vasodilatory agent i.e., bradykinin, as described above.
  • the receptor contains a binding site sub-domain that closely interacts with the primary amine function in the Ri residue with respect to absolute space occupancy (volume) and probably a electrostatic charge, i.e., the receptor non-planar ⁇ H3-binding component of the Ri -binding sub-domain (the same non-planar sub-domain component described in response to Question #1 above), most likely is a negatively charged residue that resides adjacent to the l°-amine when the R j group is engaging the receptor sub-domain.
  • Nle-substituted AIV had a 4-fold greater affinity for the AT4 receptor than Orn-substituted AIV.
  • Nle-substituted ATV had a remarkable binding affinity 60-fold higher than Lys-substituted AIV: i.e., NlejYIHPF had a K j of ⁇ 1 x 10" 12 M, a virtually irreversible binding ligand and indicative of partial-agonist activity.
  • NlejYIHPF had a K j of ⁇ 1 x 10" 12 M, a virtually irreversible binding ligand and indicative of partial-agonist activity.
  • Figure 6A shows changes in arterial blood pressure following adminstration of NorLeu YTHPF at 100pmole/25ml/min (open circles), 50pmole/25ml min (open squares) or saline control (closed squares).
  • Figure 6B shows changes in renal blood flow following adminstration of NorLeu YTHPF at 100pmole/25ml min (open circles), 50pmole/25ml/min (open squares) or saline control (closed squares).
  • the infusion of 0.05pmole NorLeui THPF had no effect on mean arterial pressure (Figure 6A) but increased renal blood flow in a dose-dependent manner: a maximum of 19% increase in renal blood flow was observed with infusions of 0.05pmole (Figure 6B); 19% also at O.l pmole (Figure 6B); 21% at lOpmole ( Figure 6B); and, lOOpmole NorLeui YTHPF increased renal blood flow by 30% ( Figure 6B).
  • Infusion of 0.15M NaCl in control animals were without any significant effect.
  • R-group protection was: Tosyl for His and 2,6-dichlorobenzyl for Tyr. Synthesis occurred on a t-Boc-Phe substituted resin (0.76mmol gram of 1% cross- linked divinyl benzene resin from Peninsula).
  • methylene chloride wash 1X1 min; 45% w/v trifluoroacetic acid and 0.08% indole in methylene chloride deprotection: 1X3 min and 1X30 min; methylene chloride wash: 5X1 min; isopropanol wash: 3X1 min; methylene chloride wash: 3X1 min; 10% v/v triethylamine in methylene chloride neutralization: 1X1 min and 1X5 min; methylene chloride wash: 2X1 min; isopropanol wash: 2X1 min; methylene chloride wash: 2X1 min; isopropanol wash: 2X1 min; methylene chloride wash: 3X1 min; amino acid coupling with a 2.5 or 5-fold excess of amino acid and EDC in methylene chloride: reaction times of 1.5 to 3.5 hours; methylene chloride wash: 3X1 min; isopropanol wash: 3X1 min; methylene chloride wash: 3X1 min; amino
  • Nalinal ( ⁇ -t-Boc-L valine aldehyde from Peninsula) was linked to the free amino-terminal of the growing peptide by formation of a Schiffs base intermediate with subsequent bond reduction.
  • the above protocol was utilized with the following alterations: prior to coupling, the resin was washed with dimethyl formamide 3X1 min; a 5-fold excess of valinal was added in 1% acetic acid/dimethyl formamide; a 10-fold mole ratio excess of sodium cynoborohydride (Sigma) was dissolved in 3 ml 1% acetic acid dimethyl formamide and added in equal aliquots at 0,3,5,10,15,20,25,30,40 and 50 min with concurrent nitrogen purge; the coupling was allowed to continue for 70 additional min; the resin was washed with dimethyl formamide 3X1 min. Linkage was assessed with the Kaiser test and revealed a slightly reddish color of the beads when greater than 94%.
  • the finished N-terminal deprotected resin-linked peptide was cleaved from the resin and side chain deprotected with anhydrous HF containing 10% anisole at 0°C for 40 min.
  • the HF and anisole were removed under vacuum and the peptide washed with anhydrous ether.
  • the peptide was extracted with 20% glacial acetic acid and lyophilized.
  • the crude peptide was then purified by preparative reversed phase HPLC in two steps, the first an isocratic method using acetonitrile:triethylamine-phosphate, pH3 followed by a second gradient method using acetonitrile:water (0.1% TFA).
  • the purified product was analyzed by analytical reversed phase HPLC (acetonitrile:triethylamine-phosphate, pH3) gradient method (12-18% over 60 min at 2ml/min).
  • the metabolish of 125 I-ATV and 125 I-Dival AIV by rat kidney membranes was determined as follows: Rat membranes (25 ⁇ g protein) were incubated with .6nM 125 I-peptide at room temperature in a buffer containing Tris, 50mM, pH7.4; ⁇ aCl, 150mM; BSA, 0.1%; EDTA. 5mM; bestatin, 20 ⁇ M; and Plummer's inhibitor, 50 ⁇ M. Metabolism was stopped by the addition of acetonitrile (final concentration 50%), and the samples were analyzed by reverse phase (C 18 ) HPLC. As can be seen in Figure 11, ATN is rapidly degraded while Dival AIN remains 100% intact after 4 hr of incubation.
  • AT4 receptor binding site domain binds analogues in which the peptide bond has been replaced with a non-carbonyl (non- peptidase sensitive) bond that has a similar bond length, and that is non-planar and has a non-rigid carbon-nitrogen bond.
  • ⁇ on-peptide bonds offer pharmacological advantages for a therapeutic composition, i.e., prolonged half-life.
  • the model capable of explaining this behavior has the following component parts: a) The receptor binding site sub-domain interactions with the side groups (i.e., of Ri) determines receptor activation; b) The interaction at the R j -sub-domain binding site involves a hydrophobic pocket; c) The space in the latter hydrophobic pocket conforms very closely with the 4 carbon side chain of norleucine; d) Nlej (i.e., in NleiYTHPF) interacts with the hydrophobic pocket without changing the conformation of the pocket; e) Vali (i.e., in VYIHPF) must occupy an "expanded" hydrophobic pocket, i.e., where the receptor hydrophobic pocket is displaced laterally to accomodate the branched carbon side chain in these residues.
  • Lys ⁇ i.e., in KYIHPF
  • Lys ⁇ must similarly occupy an "expanded" hydrophobic pocket because of the charge repulsion from the hydrophobic "walls" of the pocket; and, f)
  • the process of "expanding" the hydrophobic pocket constitutes a molecular trigger for the process transitioning the receptor from the "pre-binding state" to the "binding state".
  • Nle 1 -ATV (1 . 5) i.e., NleYIHP
  • Nle r AIN i.e., ⁇ leYIH
  • ⁇ le ⁇ -ATV i_3
  • NleYI may be useful for testing space-filling modifications that can be made to alter binding in the receptor binding site sub-domains. It is considered highly likely that independent modifications that can be made to alter the binding of the latter small Nlei peptides into the AT4 receptor binding site sub-domains will be paralleled when the modification are incorporated into larger AIV ligands.
  • Nal ⁇ lle 6 -AIN had a higher binding affinity for the AT4 receptor than AIN (i.e., VYfflPI >VYHTPF); and Lys 1 Ile 6 -AIV had a lower affinity than Lys r AIV (i.e., KYIHPI ⁇ KYIHPF).
  • the AT4 receptor binding site is a multi-domain binding site with interactions such that binding in one sub-domain (e.g., within the hydrophobic pocket of the Ri sub-domain) can be excluded by high affinity binding at a distant sub-domain site (e.g., within the subdomain with specificity for the C-terminal Ile6 or Pr ⁇ 5 residues; i.e., at the R ⁇ subdomain binding site in the receptor).
  • the induced-fit model supplied above in response to Question #8 is compatible with the observed exclusionary binding properties: i.e., binding of R hydrophobic pocket that constitutes the Ri -binding subdomain requires flexibility of expansion in the pocket, and binding of Rg in the R sub-domain binding site confers a rigidity to the receptor that inhibits flexibility in the R j -binding subdomain.
  • Binding was carried out as described in Example 1, above, in siliconized glass culture tubes containing 0.2nM 125 I-AIV, 25 ⁇ g of membrane protein, and the desired analogue over a concentration range of 10" 12 to lO ⁇ M using half-log dilutions. All binding incubations were carried out in duplicate at 37°C for 2 h in a buffer containing: 50mM Tris, 150mM NaCl, 5mM EDTA, lO ⁇ M bestatin, 50 ⁇ M Plummer's Reagent, lOO ⁇ M PMSF and 2% BSA (Assay buffer) in a total volume of 0.25ml.
  • AIN analogues that are peptides were synthesized by the standard Merrifield method utilizing t-Boc protected amino acids and chloromethylated resins on a Nega 250 coupler automated synthesizer (as described in Example 1, above). Following synthesis, the crude peptides were purified by preparative reverse-phase HPLC. The amino acid composition of the purified peptides was determined with respect to both composition and total purity. Typically the peptides used in these studies were greater than 99% pure and contained about 20-25% acetate. EXAMPLE 5
  • endothelial cells such as bovine coronary venular endothelial cells
  • endothelial cells such as bovine coronary venular endothelial cells
  • angiotensins were reported to be capable of stimulating angiogenesis (22).
  • studies in the inventors' laboratory over the past ten years have failed no less than six times to demonstrate detectable levels of
  • the competitive binding affinities of analogues to the receptor showed affinites that (in decreasing order) were ATV >AII (3 _ 7) >AHI >AII >Sar ⁇ Ile 8 -AII, or AII (4 _ 8) »DUP 753, or CGP42112A.
  • the AT4 receptor in endothelial cells may not be G-protein linked because the non-hydrolyzable GTP analog GTP ⁇ S had no effect on 1 5 I-AIN binding to receptors in BAEC cells.
  • association rat constant (1 ⁇ ) 0.084 +/- 0.013.
  • RVSMC rat vascular smooth muscle cells
  • BAEC bovine aortic endothelial cells
  • This study is the first to describe a novel angiotensin binding site in vascular endothelium that exhibits high affinity and specificity for the hexapeptide AIV fragment of angiotensin AIL
  • the AT4 receptor is distinct from the ATI or AT2 receptors in vascular tissue. Analysis of the binding characteristics indiactes that the AT4 receptor binds AIV in a saturable and reversible manner, and that 125 I-AIV reaches equilibrium in binding to the AT4 receptor in membrane preparations in approximately 60 min. at 37°C. Binding of AIN to its receptor remains stable for at least 4 h ( Figure 7 A) with less than 10% degradation of the ligand under these binding conditions.
  • the first component is a high affinity component that exhibits K d 's of 14 and 27pM with B max 'S of 6 and lOfmol/mg protein for receptors in CVEC and BAEC membrane preparations, respectively.
  • the second binding component is a lower affinity component with K ⁇ s of 1.4 and 4.4nM (i.e., in CVEC and BAEC, respectively).
  • the second component displays a high concentration of Ugand binding commensurate with large numbers of such receptor sites in the membrane preparations: i.e., these sites bind 594 and 434fmol/mg protein in CVEC and BAEC membrane preparations, respectively.
  • the overall binding affinity (i.e., K d , single or composite site fit produced by LIGAND) was calculated to be 0.7nM for CVEC and l.OnM for BAEC. These results are in good agreement with the K d calculated from the results of kinetic binding studies (0.3nM).
  • AIV bound to these AT4 receptors in vascular tissues is presented in Figure 8 and Table 11, above.
  • This profile reveals a strict structural requirement for the N-terminus of the AIV ligand, i.e., removal of the N-terminus (Vali) of the ATV ligand results in a 200-fold decrease in affinity of the AIN ligand for the AT4 receptor in vascular tissues (i.e., an increase in the Kj).
  • ⁇ -terminal extension i.e., beyond Nali, is detrimental to the binding of AIN ligands to the vascular AT4 receptor as indicated by the inability of All and Sarj -es-AII to competitively inhibit binding of AIN to the AT4 receptor, (i.e., note the 200-fold increase in Kj seen with All and Sar ⁇ fleg-AH, when compared with AIN in Table 11).
  • the apparent affinity of AIII for the vascular AT4 receptor i.e., 20-fold higher Kj than AIN, Table 11
  • 125 I-AHI binding to bovine adrenal AT4 receptors was directly proportional to the amount of Am hydrolyzed to AIN.
  • the vascular AT4 receptor appears to exhibit less specificity for the C-terminus than exhibited for the ⁇ -terminus: i.e., the AIN ( i_ 7) fragment (with the C-terminal Phe 8 deleted still bound with reasonable affinity to the receptor (i.e., only a 7-fold increase in Kj over AIN).
  • the vascular AT4 receptors do not apparently bind either DUP 753 or CGP 42112A (i.e., Kj >10" 4 ), but ATI or AT2 receptors are well-known to do so (Timmermans, P. et al. ⁇ PS 12:55-62, 1991; Whitebread, S. et al. Biochem. Biophys. Res. Comm. 163:284-291, 1989). (This property of failure to bind either DUP 753 or CGP 42112A distinguishes AT4 receptors of the invention from ATI and AT2 receptors.)
  • Binding of 125 I-AIN to vasular endothelial AT4 receptors was not sensitive to inhibition by guanine nucleotides.
  • binding of All to ATI and AT2 receptors in membrane preparations of rat vascular smooth muscle cells was sensitive to inhibition by guanine nucleotides in a dose-dependent manner, i.e., the affinity of the ATI receptor for AH was shifted to a lower value when the receptor was uncoupled from G-proteins by the presence of the GTP analogue GTP ⁇ S ( Figure 9).
  • This shift in binding affinity in response to gunaine nucleotides is a characteristic of the high affinity form of the ATI receptor (Glossmann, H. et al. J.
  • AIV ATV-like fragments of All may have unique biological attributes.
  • AIN-like fragments of AH In cultured chick myocytes, AIN-like fragments of AH have been reported to antagonize the effects of All-induced increases in cytosolic free calcium, protein synthesis, and hypertrophic cell growth while being unable to competitively inhibit for 125 I-AII binding (Baker, K.M. et al. Am. J. Physiol. 259:H610-H618, 1990).
  • Topical application of both All and AIN-like fragments of ATI have been reported to mediate endothelium-dependent vasodilation in rabbit brain arterioles.
  • the following protocols are useful for: 1) confirming the vasodilating potential of an AIN ligand, demonstrating that ligand action is dependent on an AT4 receptor, and showing that the action is independent of Al or All receptors; 2) establishing that any observed vasodilation is endothelium dependent; 3) determining whether the mechanism of vasodilation involves prostaglandins, EDRF, or other factors like EDHF as second messengers; and 4) determining the functionality of the many AIN analogues (i.e., such as those synthesized in Example 4) as either AIN ligands or as agonists, antagonists, inhibitors, or promoters of the AIN ligand-receptor interaction.
  • AIN analogues i.e., such as those synthesized in Example 4
  • AIN and ATI ligands and various analogues (Example 4) in the presence or absence of angiotensin inhibitors (e.g., Sar ls Ile 8 -AII, DUP 753, and CGP42112A) were screened for the vasodilating activity using rabbit aorta and inferior vena cava rings or spiral strips suspended in 20ml organ baths containing Krebs solution at 37°C and continuously gassed with 5% CO 2 in oxygen. After a 1 h equilibration period, cumulative dose-response curves were constructed for the analogues over a concentration range of 10" 10 M to 10" 5 M.
  • angiotensin inhibitors e.g., Sar ls Ile 8 -AII, DUP 753, and CGP42112A
  • AIN ligand on endothelial cells was examined by measuring growth of bovine endothelial cells.
  • Cells were grown at 37°C in 35mm culture plates CO 2 /air under 5% CO 2 /95% air in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5 ⁇ g/ml insulin and 10% (v/v) newborn bovine serum ( ⁇ BBS).
  • DMEM Dulbecco's Modified Eagle's Medium
  • ⁇ BBS newborn bovine serum
  • the test medium was supplemented with 3 H-thymidine and either All ligand (50nM) or AIN ligand (50nM) or lOng/ml acidic or basic FGF (as a positive control). Negative controls were also included using ethidium bromide (lmM).
  • the cells were harvested at various times, and cellular lysates were prepared for scintillation counting by lysing and washing the cells on glass fiber filters.
  • VYIHPF VYIHPF
  • AII (3 _ 7) VYHTP
  • AII (4 . 8) YIHPF
  • All reagents and other peptides were obtained from Sigma Chemical Co., with the exception of: Plummer's inihibitor (Calbiochem); bestatin (Peninsula Biochem); DUP 753 was a gift from Dr. Ron Smith of Dupont Merck and CGP 42112A was a gift from Dr. Marc de Gasparo of Ciba- Geigy. Angiotensin fragments numbering was based on the sequence of All ( Figure 1).
  • Bovine coronary venular endothelial cells were isolated by a bead- perfusion technique and characterized as described previously (Schelling, M.E. et al. Am. T. Physiol. 254:H1211-H1217, 1988).
  • Bovine aortic endothelial cells were a gift from Dr. Stephen Schwartz (University of Washington).
  • Cells were grown to confluence in 100mm culture dishes. Dishes were washed once in Ca ++ /Mg ++ -free PBS, pH7.4 at 37°C follwed by the addition of 2ml of cold isotonic assay buffer (150mM NaCl, 50mM Tris, ImM PMSF, lO ⁇ M bestatin, 50 ⁇ M Plummer's inhibitor, pH7.4 at 4°C). Cells were then removed from the plates with a rubber policeman and homogenized in 5ml assay buffer for approximately 10 sec (Polytron, Brinkman Inst. Co.).
  • AIV AIV Iodination of AIV AIV (and other peptides) were iodinated using an immobilized lactoperoxidase-glucose oxidase system (Enzymobeads, Biorad Laboratories) to a specific activity of 2176Ci mmole.
  • 125 I-AIV was separated from unlabeled peptide by HPLC (Beckman) using a reverse phase C ⁇ 8 column (5mm x 250mm; Adsorbosphere, Alltech, Associates). Receptor binding assays
  • the apparent pseudo-first order association rate constant k ⁇ was deterimined by the non-linear curve fitting program LIGAND.
  • the apparent dissociation rate constant, k.j was determined by LIGAND.
  • Bovine aortic endothelial cells were grown at 37°C in 35mm culture plates under 5% CO 2 in air in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5 ⁇ g/ml insulin and 10% (v/v) newborn bovine serum (NBBS). The medium was aspirated 10-12 hours after seeding and replaced with serum-free medium. The medium was again aspirated 10-12 hours later and replaced with either test or control medium. Control medium was DMEM with 5g/ml insulin and 2%, 5%, or 10% (v/v) NBBS as indicated. The test medium was supplemented with either All or AIN ligand ⁇ the antagonist Sar ⁇ ,He 8 -AH at various concentrations. The medium was changed every 48 h (i.e., with supportive DMEM medium for the remainder of the experiment).
  • DMEM Dulbecco's Modified Eagle's Medium
  • NBBS newborn bovine serum
  • cells can be harvested on various days during the culture period by washing the plates with calcium free medium (CMF) two times for 5 min. followed by incubation in 0.1% trypsin in CMF for 5 min. The cells can then be washed free from the plate and aspirated by Pasteur pipet into 15ml centrifuge tubes containing 3 ml DMEM with 20% (v/v) ⁇ BBS. The plates can be washed with an additional 1ml DMEM 20% ⁇ BBS which was transferred to the appropriate centrifuge tube and spun at 300 x g for 10 min. Excess medium was aspirated and the pellet resuspended in a final volume of 1ml of the control medium. Aliquots can then be counted using a hemocytometer and cell number expressed as cells/plate.
  • CMF calcium free medium
  • thymidine incorporation was measured.
  • D ⁇ A synthesis [methyl- 3 H]thymidine (60Ci/mmol, lOmCi per plate) was added to cultures 12 h after addition of the All or AIN. Twelve h later, medium was removed and 1ml of a 1% aqueous solution of Triton X-100 was added. The cells were incubated with this solution for 5 min. and the entire contents of the plate transferred to 10ml of absolute ethanol. This material was then filtered under vacuum through 2.4cm glass fiber filters (GF/A, Whatman), and the filters were washed twice with 10ml of absolute ethanol and assayed for radioactivity by scintillation counting.
  • GF/A 2.4cm glass fiber filters
  • EXAMPLE 6 Physiological Function of Angiotensin IN Receptor and Ligand Angiotensins Al, AH, and Am are reported to have a wide variety of effects on target issues, some of which are acute while others appear more long-term. All reportedly has a cellular effect of increasing c-fos levels in cultured vascular smooth muscle cells (17), and c-fos is reported to be one common pathway for triggering cell growth. Considering the widespread distribution of AT4 receptors in many organs and tissues (EXAMPLES 1 and 2, above), it is Ukely that AIN has multiple functions, including long-term effects on cells by triggering increased expression of c-fos, i.e., activities previously mistakenly attributed to All and AHI.
  • AIN ligand-receptor system may play in three organs enriched in AT4 receptors: blood vessels, kidney, and adrenal glands. (Other organs such as brain or heart which also possess high levels of specifically localized AT4 receptors can be studied in a similar manner.) Renal Blood Flow: The AIN Receptor and All Receptor Have Physiologically Distinct and Opposing Activities:
  • AIN ligand in the regulation of renal blood flow.
  • the rationale for initially choosing to examine the kidney was at least two-fold.
  • the AT4 receptor is found in high concentrations in kidney and endothelial cells (Example 1 and 2, above).
  • Second, vascular endothelial cells are reported to regulate vascular tone and to play a role in the control of renal blood flow.
  • Superficial blood flow in the rat kidney was assessed using laser doppler methods in anesthetized rats following direct infusion of a test substance into the renal artery.
  • the infusion of experimental compounds and saline had no effect on systemic arterial blood pressure (see results in Example 4).
  • AIV ligand-receptor effects on renal functions was provided by analyzing distribution of radio-labeled insulin and p-aminohippicuric acid; in combination with measurements of urine flow, urine osmolality, urine ⁇ a + and K + , and hematocrit.
  • the effects of AIV ligand, All, and other AIV analogues were determined, i.e., a) on renal blood flow, b) glomerular filtration rate, c) osmolal clearance, d) filtration fraction, and e) tubular function.
  • Dose-response curves for AIV ligand and AH ligand were constructed in the presence and absence of the AH antagonist Sar ⁇ Iles-AII.
  • AIV analogues with special in vitro properties were tested in a similar manner (above) to determine their effects on renal function. Studies were carried out as acute preparations in anesthetized rabbits and using jugular and urethral catheters.
  • AIV Ugand is produced locally in neural tissues, i.e., by synthesis of Al and conversion to ATV.
  • ATV ligand is produced locally from precursors synthesized in the tissue.
  • circulating ATV precursors e.g., AT, All or AHI
  • the first or second scenario is an operative mechanism in a particular tissue can be determined by introducing radiolabeled precursors (i.e., 125 I-AI) into the bodily fluid bathing the tissue (e.g., plasma or CNS fluid), and by then collecting samples of the fluid at different times and assaying by reverse-phase HPLC to determine if the AIV precursor has been converted to ATV ligand in the fluid. If it has been converted, the second scenario is operative; if it has not been converted a second series of experiments is conducted. In the second series of experiments biosynthesis of ATV precursors is evaluated (i.e., with radiolabeled amino acids) and conversion of the precursor into AIV ligand is examined in pulse-chase type experiments. If biosynthetically radiolabeled AIN precursor chases into AIN ligand, then the first scenario is operative in the tissue. Changes in the AIN-Ligand-Receptor System in Response to Neurological Effects:
  • AT4 receptors in brain A representative experimental protocols for showing changes in the AIN- ligand-receptor system in response to neurological and physiological effects is described in the Materials and Methods, below.
  • AT4 receptors in brain A representative experimental protocols for showing changes in the AIN- ligand-receptor system in response to neurological and physiological effects is described in the Materials and Methods, below.
  • AT4 receptors in brain A representative experimental protocols for showing changes in the AIN- ligand-receptor system in response to neurological and physiological effects is described in the Materials and Methods, below. AT4 receptors in brain:
  • Hippocampal AT4 receptors identified in tissues by receptor autoradiography in Example 2, above, were evaluated further by isolating hippocampal membranes (i.e., including hypothalamus, thalamus, septum, anteroventral third ventricular area, HSTA, above) and then solubilizing the receptor. (A similar approach may be employed with AT4 receptors in other tissues.) The results presented below show that the guinea pig hippocampal AT4 receptor binds AIN ligand with a high affinity (K d 1.29 ⁇ 0.18nM, mean ⁇ SD, Hill Coeff.
  • Memory Retrieval The effects of AIN ligand on learning and memory were tested in rats by measuring the passive avoidance response, i.e., the mean latency period (time in seconds) for which the animal avoided a dark compartment. Training was conditioned to avoid the dark compartment by administering a 0.25mA foot shock over a period of 2 seconds with the door to a lighted compartment closed. On day 2 retrieval of the cognitive memory was tested 5 minutes after intracerebroventribular (icv) injection of AH or AIN.
  • icv intracerebroventribular
  • the tissue was homogenized in 40 volumes of hypotonic buffer containing 50mM Tris, pH7.4 and 5mM EDTA, and spun at lOOOg for 10 min. The supernatant was removed and recentrifuged at 40,000g for 30 min. The pellet was rehomogenized in hypotonic buffer and recentrifuged. The 40,000g pellet was homogenized in isotonic buffer (50mM Tris, pH7.4, 5mM EDTA, 150mM ⁇ aCl, 20mM bestatin, 50mM Plummer's inhibitor, lOOmM PMSF, and 0.1% heat treated BSA) and recentrifuged a final time at 40,000 x g.
  • hypotonic buffer containing 50mM Tris, pH7.4 and 5mM EDTA, and spun at lOOOg for 10 min. The supernatant was removed and recentrifuged at 40,000g for 30 min. The pellet was rehomogenized in hypotonic buffer and recentr
  • the pellet was resuspended at a concentration of 2.5mg protein/ml as determined by the Lowry protein assay.
  • Binding assays which totaled 250ml, contained 10ml 125 I-AIN ligand (sp. act-2176 Ci mmol), 10ml tissue homogenate, 10ml unlabeled peptide (if employed), and the remainder isotonic buffer. Incubations were carried out for 2 h at 37°C. Preliminary experiments demonstrated that incubation for 1 h at 37°C was necessary for equilibrium to be reached and that binding was stable for at least 4 h. At that time less than 10% of the 125 I-AIN was shown to be by HPLC analysis.
  • tissue sections mounted on slides. Slices were initially preincubated in isotonic buffer for 30 min at room temperature, then incubated in labeled ligand (O. ⁇ nM) for 2 h, rinsed, dried, and exposed to X-ray film as previously described.
  • labeled ligand O. ⁇ nM
  • AIII to AIV may result from the action of nonspecific proteases but these enzymes may also cleave all angiotensins at sites other than the All R 2 -N 3 . and are not termed herein AIV angiotensinase.). In either case, cleavage of the AH Arg 2 -Val 3 peptide bond in Al, AH, or AIII generates AIV.
  • AIN angiotensinase enzyme i.e., that cleaves Al, All, and AHI in an efficient manner to permit regulatable formation of AIN.
  • the AIN angiotensinase enzyme may be identified, isolated, and purified using the experimental approaches described below, in the Materials and Methods, in combination with the assays described in the Examples above (see Example 1). Data presented herein indicate that AH and Am are exceUent and specific inhibitors of 125 I-AIN formation from 125 I-AI. Materials and Methods: Experiment #1. Formation of AIN Ligand From AIN Precursors in Circulation.
  • 125 I-labeled angiotensins (10 7 dpm) - Al, All, or AHI, and tetradecapeptide can be injected into the carotid artery of a guinea pig and blood samples (50 ⁇ l) can be collected at 30-sec or 1-min. intervals from a second cannula in the femoral artery into lOO ⁇ l of 20% TCA for 10 min. Samples may be analyzed by reverse-phase HPLC utilizing methods that have been reported previously (47). The data are analyzed to determine the rate of formation of AIN Ugand from potential AIN precursors. Experiment #2: Formation of AIN from Precursors via Action of Adrenal Enzymes.
  • Guinea pig adrenals were excised and homogenized in a Krebs-Ringer buffer containing the full complement of ions (as above in Example 1). After a low speed spin at 500g for 10 min. to remove whole cells and nuclei, the supernatant is centrifuged at 40,000g for 30 min. The supernatant is recentrifuged at 100,000g for 90 min. yielding both a soluble (100,000g supernatant) and a microsomal (100,000g pellet) fraction. The 40,000 x g pellet is rehomogenized and fractionated on a discontinuous sucrose fractionated gradient (0.4M-1.2M sucrose, in 0.2M steps).
  • the membranes at the 0.8M to l.M and IM to 1.2M interfaces can be collected and combined, resuspended in a 10X excess of Krebs buffer. The membranes were then centrifuged at 40,000 x g for 30 min. After a final resuspension in Krebs buffer and centrifugation at 40,000 x g for 30 min., the final plasma membrane fraction is ready for the assay. Soluble, membrane, and microsomal fractions may be incubated at various protein concentrations and times at 37°C with 10 6 cpm of 125 I-AI, AH, AIII, and tetradecapeptide.
  • guinea pig adrenal tissue (as expected) possesses an AIN angiotensinase
  • the specificity of the enzyme(s), its activity on various substrates, and metal ion requirements can be established by incubating preparations of the isolated enzyme with angiotensins (e.g., in the presence of inhibitors of nonspecific proteases), and followed by examination of the hydrolytic products on reverse-phase HPLC.
  • the sequence of the hydrolytic products may be determined by automated amino acid sequencing. Incubation conditions with varying concentrations of the angiotensin substrate were used to develop data for double reciprocal plots thus allowing the affinity of enzyme(s) for the different angiotensins to be determined.
  • Pandiella A., Bequinot, L., Nincentini, L.M., and Meldotesi, I, TIPS 10,

Abstract

A unique and novel angiotensin AT4 receptor and AIV ligand system for binding a small N-terminal hexapeptide fragment of Angiotensin II (referred to as AIV, with amino acid sequence Val1-Tyr2-Ile3-His4-Pro5-Phe6) is disclosed. AIV ligand binds saturably, reversibly, specifically, and with high affinity to membrane AT4 receptors in a variety of tissues, including heart, lung, kidney, aorta, brain, liver, and uterus, from many animal species. The AT4 receptor is pharmacologically distinct from classic angiotensin receptors (AT1 or AT2). The system employs AIV or C-terminally truncated or extended AIV-like peptides (e.g. VYIHPFX) as the signaling agent, and the AT4 plasma membrane receptor as the detection mechanism. The angiotensin AT4 receptor and receptor fragments (including the receptor binding site domain) are capable of binding a VYIHPF angiotensin AIV N-terminal peptide but not an angiotensin AII or AIII N-terminal peptide, i.e., DRVYIHPF or RVYIHPF, respectively. Also disclosed are processes for isolating angiotensin AT4 receptor and AIV angiotensinase, identifying angiotensin AIV agonists and antagonists, and constructing diagnostic assays to specifically measure AIV and AI-specific angiotensinase in biological fluids.

Description

ANGIOTENSIN IV PEPTIDES AND RECEPTOR
Field of the Invention This invention relates to the polypeptide ligand NYTHPF (angiotensin IN or AIN) and to related peptide ligands and polyaminoacid ligands that bind to, activate and/or antagonize a novel angiotensin AT4 receptor. The ligands comprise at least three of the Ν-terminal amino acids of AIN, or AT4 receptor binding equivalents or analogs thereof. Engagement of the receptor by its ligand triggers acute physiological effects (e.g., vasodilation) and long-term effects in cells (e.g., hypertrophic growth).
Background of the Invention The renin-angiotensin system has wide-ranging actions on numerous tissues in the body affecting blood pressure (pressor activity) and cardiovascular and electrolyte homeostasis. It is currently believed that angiotensins All and Affl are derived via enzymatic cleavage in the cascade depicted in Figure 1, steps 1, 2, and 3 (1). (Numbering herein of the amino acid residues in Al, All, AIII, and AIN is according to that appearing in Figure 1.) The renin-angiotensin cascade is thought to begin with the action of renin on angiotensinogen to release angiotensin I (Al), a biologically inactive decapeptide. Angiotensin II (All), the bioactive octapeptide, is thought to be formed by the action of angiotensin converting enzyme (ACE) on circulating Al (2). Des-AspAH (Angiotensin III; Am) is derived from All, and certain reports have suggested possible activities for AHI in the adrenal gland (3) and brain (4). It has been reported that All and AUI are inactivated by enzymatic degradation through a series of smaller inactive fragments (5). Fragments smaller than AIII have been thought, for the most part, to be biologically inactive and of little physiological significance (6). This assumption has been based on the lack of pressor and certain endocrine activities (i.e., aldosterone release) of small angiotensin fragments (7) and the finding that Ν-terminal deleted fragments, i.e., smaller than AIII, reportedly exhibit low binding affinity for angiotensin Al or All receptors (known as ATI and AT2, respectively) as determined in radiolabeled ligand studies (8).
Certain studies have used ATI^g) as one of several controls in structure- activity studies of ATI and AT2 receptors (9,10). An All receptor having components with molecular weights of 60-64kDa and 112-115kDa has reportedly been cloned from adrenal cortical cells as well as rat smooth muscle (11).
In general, AIIπ-8) has been found to be much less active than All or AIII with regard to typical angiotensin-dependent pressor activity or stimulating water intake (9,10,12). However, certain reports have suggested that AII(3.8), while having little pressor activity or ability to stimulate aldosterone release, may under certain circumstances inhibit renin release from kidney (12,13). Haberl et al. (14) reported a possible effect of AII(3_g) on endothelium-dependent dilation in rabbit brain. Braszko et al. (15,16) reported possible effects of All^.g) or AII(3_7) on motor activity, memory, and learning when administered intracerebroventricularly (icv) into rat brain and suggested that these effects should be considered "unspecific," i.e., not mediated by receptors (Braszko et al. (17), p. 195).
The angiotensin field has often been fraught with complexity and conflicting information, particularly with regard to the levels of different All and AIII peptides required to elicit certain cellular responses, the concentrations predicted from receptor binding studies to be biologically active, and the levels of angiotensin peptides that may be measured in biological fluids. It has been reported that All and AIII are removed from, or destroyed in, circulation by enzymatic hydrolysis. Biological half- lives of the different metabolic fragments are reportedly quite short. Semple and co- workers (18) reportedly detected AHI, Aπ 3. ), and AII(4_g) in arterial and venous blood in man with half-lives for All, AIII, AII(3_g), and AII(4.g) of 4.4, 2.0, 1.9, and 2.4 minutes, respectively. Blumberg et al. (19) reported that during transit through the kidney 72-76% of Al and All and 89% of A was metabolized.
Confusion has existed in the art as to how metabolic products of All and AIII can exhibit certain biological activities (e.g., inhibition of renin release and enhancement of cognitive function), while failing to bind to Al or All receptors. Fragments of All smaller than A , e.g., AII(3.g) and other smaller fragments, have not been reported to have specific saturable binding sites in tissues, and receptors for these fragments have not been identified previously. The present invention provides partial explanation for certain previous confusing and contradictory findings, and provides novel AIN receptors (AT4), AIN ligands, peptides, analogs, agonists and antagonists that bind specifically to the AT4 receptor and not to Al (ATI) or All (AT2) receptors. The AIV peptides and the AT4 receptor are labile and subject to proteolytic degradation. In other aspects, the invention provides a specific angioteninase enzyme that converts All or AIII peptides to AIN peptides in a novel pathway. Summary of the Invention
The discovery, herein, of a unique and novel angiotensin AIN receptor (AT4) and AIN ligand system for binding a small Ν-terminal hexapeptide fragment of Angiotensin II (referred to herein as AIN, with amino acid sequence Nalι-Tyr2-He3-His4-Prθ5-Phe6) provides partial explanation for confusion in the prior art. AIN binds saturably, reversibly, specifically, and with high affinity to membrane AT4 receptors in a variety of tissues and from many animal species. The AT4 receptor is pharmacologically distinct from classic angiotensin receptors (ATI or AT2) in that the AT4 receptor displays no specificity for classic agonists (All and AIII) and antagonists (Sar^Ileg-AH). Thus, the disclosure details the pharmacological and biochemical characterization of a newly discovered branch of the renin-angiotensin system that employs an AIN ligand as the signaling agent, and the AT4 plasma membrane receptor as the detection mechanism.
Angiotensin AIN appears to specifically mobilize calcium in vascular endothelial cells where AIN binding is evident. Binding to the endothelial AT4 receptor appears to trigger cellular proliferation. Binding of AIN to AT4 receptors in kidney and brain increases blood flow. In addition, binding of AIN to AT4 receptors in the brain facilitates learning and memory retention. AIN has also been shown to block the hypertrophic action of A on cardiocytes despite its inability to bind AT2 receptors. Since cardiocytes possess large numbers of AT4 receptors this action of AIN is most likely direct. Thus, in certain respects the action of AIN appears to neutralize, or act in apposition to the actions of All and AIII.
The invention provides an angiotensin AT4 receptor and receptor fragments (including the receptor binding site domain) that are capable of binding a VYIHPF angiotensin AIN Ν-terminal peptide, and related AIN ligands, but do not bind an angiotensin All or AIII Ν-terminal peptide, i.e., DRVYIHPF or RVYIHPF, respectively. The AT4 receptor from adrenal cortical cells has a molecular size of about 140kD to about 150kD on SDS-PAGE following crosslinking, a K& of about 0.5nM for AIV peptides, and is widely expressed on the surface of adrenal cortical and medullary tissues in many mammalian species. The receptor is expressed in all important organs and tissues including heart, lung, kidney, aorta, brain, liver, and uterus. The invention further provides processes for identifying angiotensin AIV agonists and antagonists, and constructing diagnostic assays to specifically measure AIV and AT4 receptors.
Brief Description of the Drawings FIGURE 1 is a schematic diagram depicting the amino acid sequence of angiotensinogen and its conversion by renin to Al, by angiotensin converting enzyme (ACE) to All, by angiopeptidase to AIII, and by a novel AIV angiotensinase, herein disclosed, to angiotensin AIV (AIV).
FIGURE 2 A is a graphical representation of the results of equilibrium binding studies of 125I-radiolabeled AIV to AT4 receptors isolated from bovine adrenal cortical membranes; as described in Example 1.
FIGURE 2B depicts graphically the structural requirements and specificity for binding of AIV ligand to the AT4 receptor from rabbit cardiac myocyte membranes; as described in Example 1. FIGURE 3 compares AT2 and AT4 receptor localization in the Habenula region of the brain using receptor autoradiography with 125I-Sarι,Ileg-AII to localize AT2 receptors, and 125I-ATV to localize AT4 receptors, as described in Example 2. Panel A shows binding of 125I-AIN to cells in the habenula, thalamus, cerebral cortex and hippocampus of guinea pig brain. Panel B shows that the binding of 125I-AIV is specifically competitively inhibited by lOOnM non-labeled AIV competitor. Panel C shows that binding of 125I-AIV is not competitively inhibited by lOOnM Sarι,Ileg-AII. Panel D shows a pattern of binding of 125I-Sarι,Ileg-AII to AT2 receptors that is different from the pattern observed with 125I-AIV in Panel A. Panel E shows that binding of 125I-Sarι,Ile8-AII is specifically inhibited by lOOnM of non-labeled All competitor. Panel F shows that binding of 125I-Sarι,Ileg-AII is not inhibited by lOOnM non-labeled AIV competitor. Panel G shows a "pseudo-color" photograph of 1 5I-AIV binding. Panel H shows a "pseudo-color photograph of 125I-Sarι,Ileg-AII binding. Panel I shows a photomicrograph of a histology slide of a serial section of the same tissue as in Panels A-I. FIGURE 4 graphically depicts the percentage change in renal blood flow after infusion of lOOpmol of AIV (n=13 experiments); 0.15M saline (n=9); lOOpmol of D-Val -AIV (i.e., AIN with a D-valine residue in the 1 position); or lOOpmol of All (n=8) into the renal artery at a rate of 25ml/min, as described in Example 6.
FIGURES 5A and 5B are graphical representations of changes in blood flow that result from binding of agonist, Lys j AIN, to AT4 receptors in kidney, without changes in systemic blood pressure, as described in Example 4. Figure 5A shows changes in arterial blood pressure following administration of LysjAIV at 100pmole/25ml/min (open circles) or saline control (closed circles). Figure 5B shows changes in renal blood flow following administration of Lysi AIN at 100pmole/25μ 1/min (open circles) or saline control (closed circles). FIGURES 6A and 6B are graphical representations showing changes in blood flow that result from administering different doses of an agonist ΝorLeujAIN (i.e., ΝorLeuYTHPF) that binds to AT4 receptors in kidney, without changes in systemic blood pressure, as described in Example 4. A therapeutically effective dose for increasing renal blood flow was achieved when doses greater than 50finole/25μl/min were infused. Figure 6 A shows changes in arterial blood pressure following administration of ΝorLeuYTHPF at 100pmole/25μl/min (open circles), 50fmole/25μ 1/min (open squares) or saline control (closed squares). Figure 6B shows changes in renal blood flow following administration of ΝorLeuY-HPF at 100pmole/25μl/min (open circles), 50fmole/25μl/min (open squares) or saline control (closed squares). FIGURES 7A-7D, 8 and 9 are graphical representations of AIN binding, as described in Example 6. Figure 7 A shows the results of kinetic analyses measuring binding of AIN to coronary venule endothelial cells (CVEC) showing maximal equilibrium binding in about 60 minutes with an apparent Ka of about 9.3 x 107 M-1. Figure 7B shows the results of kinetic studies measuring the dissociation of AIV from CVEC endothelial cells with an apparent K-j of about 0.3nM. Figure 7C shows the results of equilibrium binding of AIN to 2 separable types of AT4 receptor sites in coronary venule endothelial cells (CVEC). One type of site with a K-j of about 1.4 +/- 0.2nM and a second type of site with a Kd of about 14.6 +/- 26.5pM. Figure 7D shows the results of equilibrium binding of AIV to 2 separable types of AT4 receptor sites in aortic endothelial cells: one type of site with a K-j of about 4.4 +/- 0.8nM and a second type of site with a K-j of about 26.9 +/- 9pM. Figure 8 shows competition of 125I-AIV binding to coronary venule endothelial cells (CVEC) by non-radiolabeled AIV analogs. Figure 9 shows association of AT2 receptors with G-protein in vascular smooth muscle cells (RVSMC), but non-association of AIV with G-proteins in endothelial cells (BAEC), as evidenced by the inhibility of GTPγS to inhibit AIV binding.
FIGURES 10A and 10B show enhancement of cognitive function, i.e., learning, in AIV intracerebroventricularly (icv) injected animals but not in All-icv-injected animals. Testing of memory was conducted one day (Figure 10 A), or one, two and three days (Figure 10B), after the animals learned a passive avoidance response; as described in Example 7. FIGURE 11 is a graphical representation of the comparative stability of
125I-AIV (closed dots) and 125I-divalinal (or
Figure imgf000008_0001
open squares) following exposure to rat kidney, as described in Example 4.
FIGURE 12 is a graphical representation of the effects of divalinal AIN (open squares), and divalinal AIV followed by LysiAIV (squares with dots), on blood pressure (Figure 12 A) and renal blood flow (Figure 12B), as compared to saline alone
(triangles), saline followed by AIN (closed circles) and saline followed by LysiAIN
(open circles), as described in Example 4.
Detailed Description of the Preferred Embodiment As used herein the following terms are intended to mean the following, namely:
"Angiotensinogen" is used herein to refer to a peptide having the sequence Asp1Arg2Val3Tyr4lle5His6Pro Phe His9Leu10Val11Ile12His13Ser14, abbreviated DRVYfflOPFHLVfflS (SEQ. ID. NO. 1) "Al" and "angiotensin I" are terms used to refer to the decapeptide fragment of angiotensin having the N-terminal sequence
Asp i Arg2 Na^Tyfyl^HisgProTPhe HisoLeu ιo> abbreviated DRVYIHPFHL (SEQ. ID. NO. 2).
"des-Asp Al", "d-Asp Al" and "des-Asp angiotensin I" are terms used to refer to an angiotensin polypeptide having the N-terminal sequence
ArgiVa^Ty^I^HissProgPhe HisgLeucj, abbreviated RVYIHPFHL (SEQ. ID. NO. 3).
"All" and "angiotensin II" are terms used to refer to an angiotensin, e.g., an octapeptide, having the N-terminal sequence Asp i Arg2 Val3 Ty^IlesE^Pπ^Pheg, abbreviated DRVYIHPF (SEQ. ID. NO. 4).
"Am," "angiotensin m," "Des-Asp AH," and "Aπ 2.8 " are terms used to refer to the heptapeptide fragment of angiotensin having the N-terminal sequence
Figure imgf000008_0002
abbreviated RVYIHPF (SEQ. ID. NO. 5).
"AIV," "angiotensin IV,"
Figure imgf000008_0003
or "Des-Arg Am" are terms used to refer to the hexapeptide fragment of angiotensin having the N-terminal sequence Val1Tyr2lle3His4Prθ5Phe6, abbreviated VYIHPF (SEQ. ID. NO. 6). In the context of usage herein "AIV" refers to physiological angiotensin II(3_ ) fragments formed in a variety of animal species. An "AIV peptide ligand" is a ligand capable of binding to an AT4 receptor. AIV is a representative example of an AIV peptide ligand, as are AIV analogs. "Des-x," also abbreviated "d-x," is used to refer to an amino acid sequence that lacks the amino acid residue "x". Des-Asp All is used to refer to an angiotensin II lacking the N-terminal Asparagine residue; d-Nal^AIV is used to refer to AIV lacking the valine residue (position 1) at the Ν-terminus of AIV. "Ν-terminal" and "Ν-terminus" are used interchangeably to refer to the
ΝH2-amino terminus of a peptide. The N-terminal amino acid is the amino acid located at the NH terminus of the peptide.
"Peptide" and "polypeptide" are used interchangeably to refer to a serial array of amino acids peptide bonded one to another of at least three amino acids in length to preferably six amino acids in length, but also up to many hundreds of amino acids in length.
"AIV Ligand" as used herein refers to a compound that is capable of filling the three-dimensional space in a receptor binding site so that electrostatic repulsive forces are minimized, electrostatic attractive forces are maximized, and hydrophobic and hydrogen bonding forces are maximized. Representative ligands include "AIV peptides" and "ATV analogs". Ligands bind to their specific receptor in a specific saturable manner, e.g., specificity may determined by the ability of an AIV ligand to bind to an AT4 receptor in a manner that is not competitively inhibited in the presence of an excess (e.g., 1000-fold molar excess) of a competitor peptide (e.g., Al or All). "ATV peptide" is used interchangeably with "angiotensin IV peptide" to refer to an AIN ligand that is a peptide having, or corresponding to, at least three of the Ν-terminal ten amino acid residues (preferably three of the Ν-terminal eight amino acid residues, and most preferably three of the Ν-terminal six amino acid residues), comprising three amino acids selected from among N, Y, I, H, P, F, L, K, A, H, ΝNal, ΝLeu, or Orn; preferably from among N, Y, I, P, K, ΝNal or ΝLeu; and most preferably from among N, Y, K, ΝNal, or ΝLeu. Representative examples of AIN peptides have an amino acid sequence related to the AIN Ν-terminal sequence NYIHPFX, i.e., by conservative and nonconservative substitutions of amino acids, or by derivatization or covalent modification, (as described below), and wherein X is any non-interfering amino acid. Representative AIN peptides are polypeptides from 3 amino acids in length to many tens of amino acids in length. Other representative examples of "AIN peptides" include peptides that are capable of antagonizing binding of "AIN" to its receptor, i.e., "antagonists" (as defined below), and other "AIN ligands" are capable of binding to the AT4 receptor and exerting effects similar to "AIN", i.e., "agonists" (as defined below). As used herein the term "AIV analog" is intended to mean a chemical compound that mimics or improves on the electronic, steric, hydrophobic, and 3 -dimensional space-filling requirements of the constituent amino acid residues involved in binding of the AIV peptide to the AT4 receptor (e.g., a mimetic chemical AIV composition). AIV analogues may be polypeptides, i.e., having amino acids bonded by peptidic linkages, or may be non-peptides, i.e., having amino acids not bonded by peptidic linkages. Representative examples of AIV analogs include chemical mimetic compounds that are capable of antagonizing binding of AIV to its receptor, i.e., antagonists (as defined below), and other AIV ligands are capable of binding to the AT4 receptor and exerting effects similar to ATV, i.e., agonists (as defined below).
"Agonist" as used herein means an AIV peptide or AJN analog that is capable of spacially conforming to the molecular space filled by an AIN ligand and that is further capable of combining with AT4 receptors to initiate an action that is initiated by a physiological AIN molecule when it binds to its specific AT4 receptors on cells in vivo or in vitro. Representative examples of actions initiated by AIN are illustrated in the Examples. Agonists possess binding affinity for AT4 receptor(s) and intrinsic activity for inducing the activities that are induced following the binding of AIN to AT4 receptor. Representative examples of agonists include NYEHPFX, ΝvaYIHPFX, and OrnYTHPFX, wherein "X" is used to designate one or more non-interfering amino acids. Representative examples of processes for recognizing agonists are described in Example 4.
"Antagonist" as used herein means an agent that spacially conforms to the molecular space filled by an AIN ligand and that is further capable of combining with the subject AT4 receptor(s) to inhibit, neutralize, impede or reverse, at least in part, an action of physiological AIN when it binds to its specific AT4 receptors on cells. Representative examples of antagonists include KYIHPFX, and ΝLeu THPFX, wherein "X" is used to designate one or more non-interfering amino acids. Representative examples of processes for recognizing antagonists are described in Example 4.
"AH ligand" as used herein refers to a peptide having the Ν-terminal amino acid sequence DRNYTHPFX and capable of binding to an ATI or AT2 All receptor, where X is any non-interfering amino acid.
"Νon-interfering amino acid" as used herein means any amino acid that when introduced into the C-terminus of an AIN peptide ligand does not interfere with binding of the AIN peptide ligand to its specific AT4 receptor. "ATI" and "ATI receptor" and are terms used interchangeably to refer to a receptor subtype capable of binding AH.
"AT2" and "AT2 receptor" are terms used interchangeably to refer to a second receptor subtype capable of binding AIL "AT4 receptor" is the term used to refer to a receptor capable of binding an
AIN ligand but not an Al, All, or AIII ligand.
"AT4 receptor fragments" is a term used herein to refer to portions of the AT4 receptor that are smaller in size than an AT4 receptor isolated from a natural source, e.g., tissues, biological fluids and the like, but remain capable of binding AIN. Fragments may be prepared from an AT4 receptor isolated from a tissue and then subjected to proteolytic degradation or treatment with a chemical such as cyanogen bromide. In the latter case the fragments of the receptor are conveniently purified before use, e.g., by reverse-phase HPLC or immune affinity chromatography. Alternatively, fragments of the AT4 receptor may be prepared by expression of a portion of a nucleotide sequence of a genomic or cDΝA clone capable of expressing the AT4 receptor, e.g., a portion of the AT4 nucleotide sequence in an expression plasmid or vector introduced into a cell, wherein the cell manufactures the AT4 receptor fragment and the fragment can be purified (as above). For example, fragments of the AT4 receptor that contain the AIN ligand binding domain of the receptor may be soluble in biological fluids and aqueous solutions and may bind AIN ligand with a greater or less K-j than AT4 receptor under these conditions. The binding affinity, expressed as the K-j, of the AT4 receptor fragment for an AIN ligand is about 30nM to about 0.003nM, preferably about lnM to about O.OlnM, and most preferably the binding affinity is about 0.5nM to about O.OlnM. "Triggering the AT4 receptor," "activating the AT4 receptor," or
"activation of the AT4 receptor" are used interchangeably to refer to conformational and/or structural or activity changes resident in an AT4 receptor following binding of an AIN ligand; e.g., conformational changes may be evident by changes in the near UN spectra of the receptor or changes in the circular dichroism (CD) spectra; structural changes may be evident as covalent modification of the receptor, e.g., by phosphorylation; and, activity changes may be evident as an increase in enzyme activity, e.g., an innate tyrosine kinase activity. A receptor that has interacted with an AIN ligand and has undergone the process of "triggering" is also referred to herein as a "triggered AT4 receptor." "Substantially purified" as used herein refers to a preparation that contains a peptide, ligand, or receptor that is enriched greater than about 10-fold from the natural source material, e.g., membrane preparations of a tissue, and that also contains less than 5% impurities detectable by one-dimensional SDS-PAGE. The substantially purified AT4 receptor approaches homogeneity at purification levels greater than about lOOOx. The term "ATV angiotensinase" as used herein refers to a dipeptidylpeptidase capable of catalyzing hydrolysis of an arginine-valine peptide bond in an angiotensin, e.g., Al, All, or AIII, without catalyzing hydrolysis of any of the other peptide bonds in the angiotensin.
"Pressor activity" is used to refer to blood pressure changes induced by an agent, e.g., AH.
As discussed above, the angiotensin field has often been fraught with complexity and conflicting information. During attempts to purify the angiotensin II (AT2) receptor from bovine adrenal cortex, the curious observation was made that as purification proceeded the apparent specificity of the receptor changed. While isolated membranes bound stable All analogs better than Am, the solubilized receptor exhibited the opposite order of ligand specificity with AHI binding better than All. Following purification it became apparent that the receptor had all but lost its ability to bind All, and was slowly losing its ability to bind Am, despite taking steps to inhibit proteases. Previously it had been reasoned that 10% hydrolysis of ligand was inconsequential; however, considering the strange behavior of the receptor during purification, the possibility was considered that a portion of the confounding data could be explained by metabolic conversion of the active All and AIII ligands to previously undiscerned metabolite(s) and interaction of the metabolite(s) with a novel receptor(s). The N-terminus of both All and Am are reportedly labile to proteolytic hydrolysis, but previous studies did not satisfactorily control for such hydrolysis. Thus, the possibility was considered that previous studies may have been confounded by the conversion of one ligand (i.e., Al or All) into another (i.e., AHI or AIV), e.g., mediated by renin, angiotensin converting enzyme (ACE), aminopeptidases, endopeptidases, and/or carboxypeptidases (e.g., through reactions such as those depicted in Figure 1). Considering the previous studies in this light, physiological activities reportedly triggered by the All or Am peptides, on re-examination, seemed to require higher levels of peptides than would be predicted from the physical binding properties of the respective ATI or AT2 receptors. However, if a previously unrecognized receptor existed it might be reasoned that the relatively high levels of All or AHI are required to provide precursors for conversion to metabolites that would bind to the novel receptor. To approach this problem an improved assay system was developed. After an exhaustive search, a rapid spin chromatographic technique was developed to separate bound from free ligand, and an assay buffer was discovered that minimized N-terminal degradation of angiotensins. In addition, a stable N-substituted analog of All was used as a control (i.e., Sar^Ileg-AII), so that classic All binding sites (20) could be identified. Under these conditions, the possible conversion of All to AIII or other metabolites is eliminated, and All binding sites are accurately identified. When assays were conducted under these conditions to determine what angiotensin fragments bound to bovine adrenal cells, it was surprisingly observed that the 125I-AII or 125I-AII radiolabel "specifically bound" to the cells was a hexapeptide fragment of AIII consisting of residues 3 through 8, i.e., AII(3.g). Further, it was discovered that the amount of binding in a given All (or AIII) preparation was directly proportional to the amount of the hexapeptide in the preparation. The use of 125I-radiolabeled hexapeptide as the ligand in the receptor assay dramatically increased binding. A reevaluation of binding in purified membrane preparations demonstrated the presence of two different and distinct receptors, one for All and a second for Aϋπ-g). Further, neither All nor AII(3_g) ligand effectively displaced the other. The results, thus, strongly suggested the presence of two distinct receptors, one for All and a second for AII(3_8). Hereinafter All^. ) is referred to as AIN and the novel AII(3_ ) receptor is referred to as the AT4 receptor. The notion of two separate and distinct receptors was confirmed by solubilizing, isolating, and substantially purifying the AT4 receptor under conditions that did not solubilize the AT2 receptor.
The experimental results described in the Examples, demonstrate for the first time the existence of a distinct high-affinity cellular receptor that specifically binds the hexapeptide fragment of angiotensin All, i.e., AII(3_g), termed herein angiotensin IN, or simply AIN. The angiotensin AT4 receptor is characterized in the Examples, with respect to structural requirements for ligand binding, species and tissue distribution of the receptor, physiological role of the AIN ligand- AT4 receptor system, intracellular messenger signaling pathways activated by the receptor, conditions for isolation and purification, and molecular size of the receptor.
In one embodiment of the invention, compositions are provided which comprise substantially purified angiotensin AT4 receptor or fragments thereof, that are capable of binding an angiotensin AIN ligand but not an angiotensin Al or All ligand. The AT4 receptor binds AIN ligands, and does not bind to a peptide having the All Ν-terminal sequence, i.e., DRNYIHPF. AT4 receptors of the invention are specific for AIN and AIN ligands, and are more fully characterized by the following properties: a) AT4 receptor has a K^ for AIV of about 30nM to about 0.003nM, preferably about 3nM to about O.OlnM, and most preferably about InM to about 0.1 nM (representative examples of binding properties of AT4 receptors are summarized in Table 1); b) AT4 receptor binds to AIV ligands in a saturable and reversible manner; c) the binding of an AIV ligand to the AT4 receptor is competitively inhibited less than about 1% to about 10% by an angiotensin All preparation (e.g., Sar^Ileg-AII) that contains less than 0.1% of an AIV ligand when the competition of AIV binding is measured in the presence of about a 1000-fold molar excess concentration of the competing ligand using the assay conditions described in Example 1.
In a representative embodiment, AT4 receptors having these properties may be isolated from bovine adrenal cortical membranes (e.g., described in Example 1). Isolated AT4 receptors from this source have the kinetic, equilibrium binding, and physical properties set forth below in Example 1. The AT4 receptor of the invention has a molecular size of about 120kD to about 200kD on SDS-PAGE, preferably about 140kD to about 160kD, and most preferably about 140kD to about 150kD. For example, an AT4 receptor of the invention is present in membrane preparations of adrenal glands of most mammalian species (e.g., cow, pig, horse, dog, cat, rabbit, and guinea pig) and, as purified from bovine adrenal membranes, the AT4 receptor has an apparent molecular size of about 146kDa on SDS-PAGE. AT4 receptors are also expressed in guinea pig aorta, heart, kidney, liver, lung, vascular smooth muscle, pituitary, and uterus, as well as vascular endothelial cells and brain.
TABLE 1 Bindin Pro erties of AIV Rece tors
Figure imgf000015_0001
c.) 61035.= maximal binding under equilibrium binding conditions, (fmol/mg protein); d.) sol. receptor= solubilized receptor;
«.) vase. endo.= vascular endothelial cells CVEC; f) aortic endo.= aortic endothelial cells BAEC; g.) hippocampus= hippocampal solubilized receptor; and, h.) HSTA= hypothalamus, thalamus, septum, antereoventral third ventricular area of brain.
The invention further provides AT4 receptor ligands that specifically bind to, activate and/or antagonize the AT4 receptor. The ATV ligands generally comprise at least 3 of the N-terminal amino acid residues of AIN, or analogues or AT4 receptor binding equivalents thereof. The amino acid residues of the ligands may be bonded by peptidic linkages, or may be bonded by non-peptidic linkages. The ligands generally have a Kd for the AT4 receptor below about 3 x 10_6M.
Generally, the AIN ligands of the invention are based on the structure of AIN. The AIN ligands may be obtained by constructing AIN analogs that have one amino acid substituted for by another of like properties, i.e., a neutral polar amino acid for another neutral polar (e.g., G, A, N, I, L, F, P, or M), a neutral nonpolar amino acid for another neutral nonpolar (e.g., S, T, Y, W, Ν, Q, C), an acidic amino acid for another acidic (e.g., D or E), or a basic for a another basic (e.g., K, R, or H). The AIN ligands may alternatively be obtained by constructing an AIN analog that is covalently modified, e.g., wherein an amino acid residue is substituted by amidation, adenylation, methylation, acylation, phosphorylation, uridylation, fatty-acylation, glycosylation, and the like to form a "substituted amino acid residue". In addition, the AIN ligands of the invention may contain one or more stereoisomers of the constituent amino acids residues; i.e., may contain one or more substituted or unsubstituted amino acid residues in the D-configuration.
In other embodiments, the invention provides angiotensin AIN ligands and ligand compositions that include AIN analogs, AIN peptide derivatives, and covalently modified AIN peptides, all of which are capable of binding to an angiotensin AT4 receptor. AIN ligands of the invention are generally defined by the formula
1" 2~ 3~ wherein Rj is a substituted or unsubstituted amino acid residue having a neutral or positively charged aliphatic side chain Zj, said amino acid being selected from among N, I, L, A, G, F, P, M, K, norvaline, norleucine, and ornithine; R2 is a substituted or unsubstituted neutral nonpolar amino acid selected from the group consisting of Y, W, Ν, Q, F, or C;
R3 is a substituted or unsubstituted neutral polar amino acid selected from the group consisting of G, A, N, I, L, F, P, or M; and
X is nothing, R4, R4-R5, or R4-R5-R6, wherein R4 is a substituted or unsubstituted basic amino acid residue selected from the group consisting of K, R and H, R5 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, N, I, L, F, P, and M, and Rg is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, N, I, L, F, P, M, and polyamino acid residues containing one or amino acid residues which do not prevent binding of the AIN ligand with the AT4 receptor. Thus, the AIN ligands of the invention are generally amino acid chains that contain 3, 4, 5, or 6 amino acid residues corresponding to the Ν-terminal 3, 4, 5 or 6 amino acid residues of AIN (the polypeptide, VYIHPF), or may optionally extended at the C-terminal end with one or more amino acid residues that do not prevent binding, due to spatial, conformational, electrostatic or other considerations, to the AT4 receptor. The amino acid residues may be linked in the amino acid chain by peptidic linkages to form peptides, or the ATV ligands of the invention may contain one or more non-peptidic linkages, such as methylene or C-Ν linkages, to enhance metabolic stability or other properties of the AIN ligands, as is hereinafter further described. Representative AIN ligands of the invention include, but are not limited to C-terminal truncated forms of AIN, such as AIN(i_5), AIN^), and AIN(i.3); stereoisomerically modified forms of AIN, such as D-H4 AIN, D-P5 AIN, and D-F6 AIN; full or truncated forms of AIN with modified amino acid residues, such as G4 AIN, G5 AIN, G6 AIN, Νlei AIN, K AIN, F AIN, Ii AIN, P, AIN, Νva2 AIN, Oπ^ AIN, Y6 AIN, I6 AIN, ΝleYI, KYI, and ΝleYI, derivatives of AIN with one or more non-peptide linkages between amino acid residues, such as Νle al1 AIN (wherein the designation al1 refers to a methylene -CH2- linkage between the amino acid residue in position 1 (Νle) and the amino acid residue in position 2 (Y)), Νle al1 Nal3 AIN, Kal1 Nal3 AIN, Kal1 AIN, Nal1 AIN, Nal3 AIN, and Nal1 Nal3 AIN, and substitued AIN ligands, such as propanoyl-Ν ornj AIN, O-me Y2 AIN, isobutyl-Ν ornj AIN, Ν-me lj AIN, ΝleYI amide, KYI amide, ΝleYΝle amide, ΝleYΝva amide, Νle al Ν-me YI amide, benzyl C AIN and the like.
The physical properties of the AT4 receptors that determine binding of the AIN ligands were mapped using synthetic peptides and analogs, as described below in detail in the examples. The structure of the Ν-terminus of AIN is most important for high affinity binding of an AIN peptide to an AT4 receptor. The AT4 receptor binding site is a coordinated multidomain binding site wherein binding in one subdomain may be excluded by high affinity binding at a second subdomain through an induced conformation change in the AT4 receptor binding site hydrophobic pocket subdomain. At least three binding site subdomains in the AT4 receptor were mapped using synthetic peptides and analogs. The binding site is stereospecific at a first subdomain for L-Naline in Ν-terminal amino acid position 1 (Nalj) of AIV; at a second subdomain for L-Tyrosine in position 2 (Ty^) of AIV; and at a third site for L-isoleucine (Ile3) in position 3 in AIV. The results suggest that Valj in AIV may interact laterally with the walls of the groove of the receptor while T3T2 in AIV may interact with the receptor binding site through van der Waals forces and hydrogen bonding. AIV peptides having a weak hydrophobic amino acid at the N-terminus with an aliphatic side chain (e.g., KYIHPF, NleYIHPF, OrnYIHPF) bind to the AT4 receptor with a higher binding affinity than AIV (binding of KYIHPF is 50-fold higher than AIV, and NleYHPF has a Kj of about 10"12M). N-terminal extension of AIV is incompatible with binding, as is deletion of the N-terminal valine (Val residue. Deletion of Val reduced binding affinities 1000-fold; substitution of Val i with Sar decreased binding affinity; addition of D-arginine to the N-terminal Valx reduced affinity for the receptor by 100-fold. The receptor binding site domain of the AT4 receptor contains a hydrophobic pocket conforming closely to the space filled by norleucine (i.e., engaging the Valj residue of ATV) and in close apposition with a negatively charged residue (i.e., engaging the primary amine of the N-terminus of Valj). Removal of the N-terminal amino group decreases by 1000-fold.
The C-terminus of the AIN peptide is relatively less important in the receptor binding and C-terminal extension of AIN ligands of the invention with "X" is allowed. However, removal of both the Pro5 and Phβ6 residues from AIN reduced binding affinity by about 21-fold to a Kj of 500nM. The C-terminus of the AIN peptide may determine receptor subtype specificity of binding.
In addition, it has been found that AT4 receptors isolated from bovine adrenal cortical membranes do not effectively bind AIN peptides synthesized with an Ν-terminal extension with Sar or GABA. Nor do the illustrative AT4 receptors effectively bind peptides having the N-terminal L-Nal replaced with D-Nal or Sar. Also, removal of the N-terminal L-Val from AIN all but eliminates binding to the AT4 receptor. AT4 receptors of the invention have a receptor binding site that is stereospecific for L-Naline. In one illustrative example, D-NaliYTHPF has 1000-fold lower binding affinity for the AT4 receptor than L-Va^YIHPF. The illustrative AT4 receptor isolated from bovine adrenal cortical membranes contains a binding site that prefers weak hydrophobic amino acids in the number 1 position (i.e., Ri) of the AIN ligand, i.e., increasing hydrophobicity by replacing Nalj with Phe (i.e., FiYTHPF) decreases binding affinity 4-fold, but replacement of Nalj with another weak hydrophobic amino acid (i.e., IiYTHPF) results in only a slight change (an increase) in binding affinity. For high affinity binding of an ATV peptide to an AT4 receptor the structure of the Ν-terminal neutral polar amino acid is most important. Ν-terminal extension is incompatible with binding, deletion of the terminal valine residue eliminates binding (Kj >10"6), substitution with Sar decreased binding affinity, substitution with He results in equivalent binding, substitution with Phe resulted in a 5-10-fold decrease in the affinity of binding, Pro-substituted AIN peptides bind with 100-fold lower affinity, Lys-substituted AIV peptides bind with 10-fold higher affinity, and AIV ligands having a norleucine in the number 1 position (also abbreviated herein Nle, NLe, NLeu, NLeuj, or Nlei) bound with 1000-fold higher affinity. The interaction between the AT4 receptor binding site and AIV ligand may be dictated by requirements for an AIV ligand containing a flexible aliphatic carbon side chain, (i.e., as opposed to a relatively rigid aromatic ring), rather than by the degree of hydrophobicity of the side chain. In a representative example, substitution of Val^ with Aspi (i.e., to form Ai YTHPF) results in an analog with no binding affinity for the AT4 receptor (i.e., has a Kd > lO^M). Further, the AT4 receptor binding sites of the invention may prefer a flexible aliphatic carbon side chain having 4 carbon atoms that lack a positively charged residue. Heptanoylj AIV with a 5 carbon side chain has reduced affinity as compared to Nlej AIV. In a representative example, Nlei YTHPF has higher binding affinity for an illustrative AT4 receptor than Lysi YTHPF, which was higher than NValjYIHPF, which is in turn higher than OrniYIHPF. The AIV peptide ligands of the invention having norleucine substituted for Nalj (i.e., ΝlejYIHPFX) are partial agonists of NYfflPFX binding to the subject AT4 receptor and have an apparent Kj of about 1 x 10"12M.
The AT4 receptor binding site interacts specifically with the Ν-terminal amino acid residue (i.e., Ri ), and the latter interaction is specific with respect to both absolute space occupancy volume (i.e., of the receptor binding site) and charge (i.e., of the AIN ligand). In representative examples, methylation of isoleucine in Ilei of IiYTHPF (i.e., to form CH3-I1 YTHPF) reduces affinity of the illustrative receptor for the peptide by 67-fold; substitution of the Nalj primary amine (ΝH3) with a secondary amine (-NH-; in this case by substituting Proj for Nalj, to form PYIHPF) reduces the affinity of binding to the illustrative receptor by 8-fold; substitution of Nalχ with benzoic acid or 6-amino-hexanoic acid gives peptides with a Kj>lmM; andr replacing Nalj ^ GABA (gamma-amino butyric acid; to form GAB A- YTHPF) decreases binding affinity by 250-fold for the illustrative receptor. The AT4 receptor binding sites of the invention also appear to be stereospecific for Tyr2 (i.e., Y) in the R2 position of the subject AIN peptide ligands. In representative examples, substitution of D-Tyr2 or Phβ2 (with a benzyl ring) for Tyr2 (with a phenolic ring) results in analogs (i.e., N[D-Y2]IHPF, or NF2LHPF, respectively) with very low affinity for the illustrative adrenal cortical receptor. Phenolic side chains in the Tyr2 residue may also interact with residues in the subject AT4 receptors through hydrophobic and/or hydrogen-bonding. The AT4 receptor binding sites of the invention tolerate replacement of the V1-Y2 peptide bond with a non-carbonyl bond that has a similar bond length, but is non-planar and has a non-rigid carbon-nitrogen bond. The latter replacement bond may preferably be resistant to proteolytic hydrolysis thereby conferring additional stability on the AIV ligand and enhancing utility in therapeutic compositions for oral delivery. In a representative example, replacement of the N1-Y2 peptide bond with a methylene bond reduces receptor binding affinity by only 5-fold; and, replacement of both the N1-Y2 and I3-H4 peptide bonds with methylene bonds results in N-N,-CH2-NH-Y2N3-CH2-NH-H4P5F6-C (also referred to herein as Vali Val3 AIN or divalinal AIN) that has an affinity equal to or better than NYTHPF.
The binding site of the AT4 receptors of the invention is a coordinated, multidomain binding site wherein binding in one subdomain of the binding site may be enhanced or inhibited by binding at a distant second subdomain. In one representative example, substitution of He for Phe at the R position of NYTHPF6 results in an analog (i.e., NYIHPI6) that binds to AT4 receptor (i.e., through the Nj subdomain sites) with a higher affinity than the parent VYIHPF molecule. In a second representative example, substitution of Ile6 for Phβ6 in KYTHPF6 results in an analog (i.e., KYIHPI6) that binds to the receptor (i.e., through the Nj subdomain site) with a lower affinity than the parent KYIHPF6 molecule. The C-terminus of the subject AIN peptide ligands appears to be relatively less important in receptor binding. In representative examples disclosed below, deletion of the C-terminal Pheg from VYIHPF (i.e., to form Ni Y2I3H4P5) does not alter binding significantly; C-terminal extension with histidine does not alter binding (i.e., to form N1Y2I3H4P5F6H7); and, addition of both bis and leu reduces affinity only 2-fold (i.e., Vi Y2I3H4P5F6H7L8). Truncation of the C-terminus, i.e., at the R5 position decreases binding. In a representative example removal of Prθ5 from VYTHP to give NYTH, decreases binding 21 -fold, and gives an analog with a Kj>500nM. The binding site domains of the subject AT4 receptor of the invention recognize the Ν-terminus of the subject AIN peptide ligands with a high degree of specificity and while the receptor interacts less closely with the C-terminus this region of the subject AIN ligand may determine receptor subtype specificity.
In another embodiment of the invention, antagonists of AIN are provided that bind to the AT4 receptor. Presently particularly preferred antagonists of the invention include the non-peptide divalinal AIN and the C-terminal substituted tripeptide ΝleYi amide, as described in Example 4, although other antagonists will be readily apparent from the data and disclosure set forth herein. Other aspects of the invention include processes for identifying AIN peptide ligands, i.e., by structural examination of the receptor binding requirements of test preparations (e.g., with respect to both blocking and/or promoting binding of the alternative peptide) to AT4 receptors such as those in heat-treated purified membrane preparation that are free of peptidase activity and devoid of other angiotensin receptors, i.e., ATI or AT2 receptors. (Examples of such heat-treated membrane preparations and assay methods are provided in the examples, below.) Those skilled in the art will recognize that the binding activity of any AIN peptide can be tested, e.g., using the receptor binding assays described herein, and that analogs, AIN peptide derivatives, and covalently modified AIN peptide or non-peptide ligands may exhibit activity as antagonists, agonists, promoters, or enhancers of AIN binding to its AT4 receptor. Candidate AIN peptides may be prepared with substitution of other L-amino acids having different steric, electronic, and hydrophobic character for the L-Nal in the natural AIN ligand. Skilled artisans will also recognize that a similar approach may be used to characterize further the role of C-terminal amino acid residues in binding of a peptide to the AT4 receptor, (i.e., other than the C-terminal P and F). Substitutions and modifications of internal amino acids (i.e., Y, I, or H) can also be examined by constructing the appropriate series of D-substituted, covalently modified, derivatized, or deleted peptides. The first or second messenger intracellular pathways triggered in cells by interaction of an AIN ligand with an AT4 receptor may be used to test a series of peptides, analogs, derivatives, or covalently modified AIN peptides for their ability to bind to the AT4 receptor and trigger the intracellular signal. For instance, activities such as tyrosine kinase, guanylate cyclase, Protein kinase C, Ca"1""*" flux changes, phospholipase C (PLC) activity, or prostaglandin or endocrine or exocrine hormone release from cells, may be monitored to determine whether the peptide triggered the AT4 receptor, and the receptor then signaled an increased or decreased activity in the cell.
In all cases, the AIN peptides, AIN analogs, agonists and antagonists, and derivatives and covalently modified forms of the AIN peptides of the invention are recognized by their ability to bind the AT4 receptor with an equilibrium dissociation constant (K-i) below 3 x lO^M, more preferably below 3 x 10"8M and most preferably below 3 x 10_9M, and to a low binding affinity for ATI and AT2 receptors with a K(j greater than 1 x lO^M.
In still other embodiments of the invention, processes are provided for identifying and characterizing a physiological effect of an angiotensin AIN peptide by assaying the effect(s) of the peptide on a selected in vitro cellular process. For instance, to identify and characterize the physiological effects of an AIN peptide on blood flow, it may be convenient to assay renal blood flow, or in vitro cellular processes of endothelial cells and/or vascular smooth muscle cells. To identify and characterize a physiological effect of an AIN peptide on cardiac ventricular hypertrophy, assays may examine the effects of an AIV peptide on growth of a cardiomyocytes in vitro. The processes disclosed herein are also useful in identifying how the in vitro activities of physiological AIV may be blocked or promoted by AIV peptides, AIV analogs, or derivatives or covalently modified forms of AIN peptides, as well as AT4 receptor fragments and the like. Representative examples of useful assays for identifying the subject AIN peptide ligands and AIN ligands are provided in the examples.
As used herein the term "cellular processes" is intended to mean biological activities that may be measured in vitro or in vivo by quantitative and/or qualitative assay. For example, cell growth or metabolism may be measured (e.g., radiolabeled amino acid synthesis into protein, glycolytic activity, oncogene expression, and the like); or, proliferation (e.g., 3H-thymidine synthesis into DΝA); or, marker expression (e.g., mRΝA by Northern, protein by Western blot, antigen by immunoassay, in vitro selectable drug-resistance marker by cell survival in toxic drug, and the like); or, electrical activity (e.g., in neural cells). Other aspects of the invention provide compositions and methods for promoting or inhibiting cellular activity of neural cells, e.g., neural motor, cognitive or analgesic activity of neural cells in the brain. The effect of the AIN compounds on motor activity may be observed by examining alterations in activity as measured with open-field techniques. The cognitive activity may be observed by passive avoidance testing, Morris swimming maze performance, and various operant tasks. To assay the effects of an AIN composition on a cellular process it may be useful, for example, to measure cellular processes before and after addition of AIN peptides to make comparative observations in parallel cell cultures. In this manner antagonists, agonists, inhibitors, promoters, enhancers, and the like may be identified and characterized with respect to their physiological effects in vitro and possible effects in vivo.
When used for therapeutic purposes, the route of delivery of the AIN ligands, AT4 receptor, AT4 receptor fragments, and AIV monoclonal antibodies of the invention is determined by the disease and site where treatment is required. For example, the compounds or compositions of the invention may be applied topically, or by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal injection, as well as by intrabronchial instillation (e.g., with a nebulizer), transdermal delivery (e.g., with a lipid-soluble carrier and skin patch), gastrointestinal delivery (e.g., with a capsule or tablet), intracerebroventricularly (icv) into brain, or intraspinally into cerebrospinal fluid (CSF). The preferred therapeutic compositions will vary with the clinical indication.
Some variation in dosage will necessarily occur depending on the condition of the patient being treated, and the physician will, in any event, determine the appropriate dose for the individual patient. The effective amount of AIV ligand per unit dose depends, among other things, on the particular ligand employed, on the body weight and the chosen inoculation regimen. A unit dose of ligand refers to the weight of ligand without the weight of carrier, when a carrier is used. An effective treatment will be achieved in the microenvironment of the cells at a tissue site as the concentration of AIV ligand approaches a concentration of 10"5M to 10_11M. Since the pharmacokinetics and pharmacodynamics of these agents will vary in different species and different patients, the most preferred method to achieve the therapeutic concentration is to gradually escalate the dosage and monitor both the biological effects and the concentration in the biological fluids (e.g., through the use of a diagnostic immunoassay, or radioisotopic or chemical label). The initial dose, for such an escalating dosage regimen of therapy, will depend upon the route of administration. For intravenous administration, for an agent with an approximate molecular weight of 10,000 daltons, an initial dosage of approximately 70mg/kg body weight is administered and the dosage is escalated at 10-fold increases in concentration for each interval of the escalating dosage regimen. Therapeutic efficacy in this example is achieved at 0.7-70mg/kg body weight of the theoretical 10,000 dalton peptide.
The compounds may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various nontoxic organic solvents. The pharmaceutical compositions formed by combining the ATV ligands or receptor fragments with the pharmaceutically acceptable carrier are then readily administered in a variety of dosage forms such as tablets, lozenges, syrups, injectable solutions, and the like. These pharmaceutical carriers can, if desired, contain additional ingredients such as flavorings, binders, excipients, sweetening or flavoring agents, colored matter or dyes, emulsifying or suspending agents, and/or. For parenteral administration, solutions of the AIN ligand or receptor fragment in sesame or peanut oil or in aqueous propylene glycol may be employed.
The present invention further provides processes for isolating inhibitors of an AT4 receptor- AIN ligand interaction by: a) selecting a cell type that expresses the AT4 receptor; b) adding an AIN ligand to a control culture of said cells; c) adding the AIN ligand and a putative inhibitor to a second test culture of the cells; and d) measuring the level of binding of the AIN peptide to the cells in said second test and control cultures. In the case that an inhibitor is present in the preparation, the level of binding in the test culture is lower than that in the control culture. (An example of such a process is provided in Example 1). Those skilled in the art will recognize that this process may be used to identify an inhibitor of an AT4 receptor- AIN ligand interaction in chromatographic fractions and the like during solubilization, isolation, and purification of said inhibitors, and that the subject inhibitors may act as agonists or antagonists of the action of AIN induced when AIN binds to its specific AT4 receptor.
In other embodiments the invention provides an AIN angiontensinase enzyme capable of hydrolyzing a peptide bond between an arginine and a valine residue in an angiotensin polypeptide, e.g., a polypeptide with a DR\ VYIHPF Ν-terminal sequence, wherein "\" indicates the proteolytic cleavage site that gives rise to an AIV peptide, i.e., with an Ν-terminal sequence related (as described above) to the amino acid sequence VYTHPF. Isolation and substantial purification of AIV angiotensinase may be conveniently accomplished, for example, by preparing an affinity resin having a non-cleavable or slowly-cleavable AIN ligand covalently bound to the resin, e.g., chemically modified derivatives of a peptide in an amino acid sequence selected from among DRNYIHPF, DRVYIHP, DRVYIH, DRVYI, DRNY, DRN, RNY, or ΝRNYIHPF, ΝRNYIHP, ΝRVYIH, ΝRVYI, ΝRVY, ΝRV. Operationally, the peptide useful in this assay is selected based on its ability to bind the AIV angiotensinase and to be resistant to cleavage by the enzyme. A test preparation of a cellular or tissue extract (or a biological fluid sample) is next chromatographed through the affinity resin; the bound polypeptide(s) is eluted, e.g., at low pH and high salt (e.g., pH2-3, and 2-3M ΝaCl, and the like), or the bound polypeptide is eluted by adding an excess of AIN ligand. The presence of the AIN angiotensinase in the eluate can be determined by assaying for the ability of the column eluate to catalyze hydrolysis of an Arginine-Naline peptide bond in an angiotensin peptide (e.g., AIII), and subsequently confirming that the sequence of the product of the reaction has a Valine residue at Ν-terminal amino acid and an AIV peptide sequence. The novel AIV ligand-AT4 receptor system of the invention is useful in a complementary or antagonistic role to All in mediating long-term effects of angiotensins, and in modulating the effects of Al, All, or AIII on cells. Although not being limited by any particular theory of action, it is believed that: 1) AIV is derived from All (or AIII) directly (e.g., through the action of a specific AIV angiotensinase, and other peptidases); 2) AIN is very labile and will accumulate at physiologically significant concentrations only when high levels of All are present at the target site; 3) the AT4 receptor is specific for AIN ligand (with accompanying low affinity for the parent peptide, AIII). Under certain conditions, AIN begins to accumulate at angiotensin target tissues as the All levels rise. When ATV concentrations rise to near 0.5nM (i.e., the K-i of the receptor) auxiliary processes which modify the acute action of All will be engaged. These actions will be mediated by an intracellular signaling system(s) different from that employed by AIL The activation of such an intracellular system may potentiate or antagonize the target cell's short-term response to AIL One physiological function of the AT4 receptor-ligand system may be to impart a longer- term response to high-level or chronic angiotensin stimulation in a tissue. Studies support the hypothesis that the ATV ligand-receptor system possesses the characteristics set forth above and is, therefore, in a position to serve a short- or long- term modulatory role on the activities of AIL Studies using bovine adrenal tissues have shown that the AT4 receptor is specific, with almost no affinity for AIL In addition, AIV is metabolized/hydrolyzed in bovine adrenal homogenates at 200 times the rate of All and 4 times the rate of AHI. Data suggest that AIV may be derived directly from All by action of a dipeptidylaminopeptidase, termed herein AIV angiotensinase. The location of AT4 receptor sites in groups of cells in tissues allows a skilled artesian to predict likely functions for the AT4 receptor in different tissues. In addition, it will be recognized that many activities previously attributed to the action of All (and/or Am) may be triggered or regulated instead by the AT4 receptor-ligand interaction. For example, it is likely that ATV ligand acts as a negative-feedback agent thus enabling tighter control on the aldosterone release process. The ATV ligand- receptor system may also be associated with a previously inexplicable up-regulation of the angiotensin receptor seen following chronic All exposure of cells in vitro. Still other functions attributed to All that may be mediated instead by the AIN ligand- receptor system include altering the release of catecholamines from adrenal medullary cells or regulating adrenal blood flow. It is, therefore, likely the AIN ligand-receptor system modulates (i.e., increases or decreases) either the acute and/or the long-term synthesis and release of chromaffin catecholamines, e.g., by acting to stimulate intracellular expression of tyrosine hydroxylase (the rate limiting enzyme in the synthetic pathway).
Experiments described below demonstrate that the AT4 receptor-ligand system may have a role as a mediator of long-term angiotensin effects on endothelial cells (e.g., cell growth; Example 5). AIV ligand-receptor interactions also appear to activate processes in endothelial cells that are complementary or antagonistic to those activated by AIL For instance, some of the ATV ligands that are embodiments of the invention are useful for increasing blood flow (e.g., renal blood flow as demonstrated in the examples).
Because of the widespread distribution of AT4 receptors in many tissues (see examples, below) it is impossible within the scope of this specification to detail every one of AIV's actions in angiotensin-sensitive tissues. However, representative data is provided in the following examples (below), for the physical characteristics of AT4 receptors (see esp. Example 1), for AT4 receptor tissue distribution and species distribution (see esp. Example 2), for physiological functions of AIV peptide ligands and AT4 receptors in controlling renal blood flow, for the cellular biology of AIV ligand-AT4 receptor interactions (e.g., second messenger pathways, G-proteins, phosphorylation, intracellular Ca*"1", phosphoinositide turnover, and guanylate cyclase activity), for vascular effects on venular and aortic endothelial cells and vascular smooth muscle cells and G-protein linkage of certain ATN-receptors, for endocrine effects on adrenocortical cell catacholamine release for effects on cardiac myocytes (i.e., cardiocytes), and for characterization of brain AT4 receptors (e.g., in hippocampal cells and in cerebellum, hippocampus, piriform cortex, Par 1/2, Fr 1/2, caudate putamen, HDB, thalamus, and inferior coUuculus), as well as, neurological effects of intracerebroventricular injection of AIN (e.g., on learning and memory). The disclosures made herein for assays and processes relating to endothelial cells, adrenal cortical cells, cardiac myocytes (cardiocytes), and vascular smooth muscle cells are discussed briefly below. As shown in the examples AIN is active in endothelial cells in enhancing cellular proliferation (as evidenced by thymidine incorporation) and stimulating production of endothelial cell relaxing factor (EDRF). These results also show the non-interaction of G-proteins with vascular AT4 receptors in bovine aortic or coronary venous endothelial cells. The results set forth in the Examples further identify a role for the AIN ligand- AT4 receptor interactions in triggering normal and/or hyperplastic growth of endothelial cells in sites of tumors or traumatic or wound injury, and angiogenesis, and a therapeutic use for AIN analogs, agonists, antagonists, and derivatives and covalently modified AIN peptide ligands that are capable of inhibiting vascular smooth muscle cell growth in such hyperplastic states while at the same time promoting endothelial cell growth. The agonist compositions are also useful for encouraging endothelial cell growth, e.g., in wound sites; antagonists for discouraging vascularization in tumor sites. In addition, the AT4 receptor-ligand system may play a role in triggering vasodilation through a selective effect on subpopulations of endothelial cells that exist in particular vascular beds (e.g., in the heart, lung, liver, kidney, brain and the like). As shown in the examples, increased renal blood flow occurs in rats following infusion of AIN ligands and taken together with the demonstrated ability of AIN to stimulate EDRF production in vascular endothelial cells, the AIN ligand-receptor system mediates actions of angiotensin that fall within the bounds of cardiovascular regulation and body water homeostasis. Thus, therapeutic uses for AIN analogs, ATV agonists and antagonists, and derivatives and covalently modified AIN peptide ligands include promoting renal blood flow (e.g., in chronic kidney diseases) or, alternatively, inhibiting renal blood flow (i.e., using inhibitors and antagonists of AIN), e.g., in conditions of hyperacute renal dysfunction and water loss, or during renal surgical procedures.
In cardiac myocytes (also termed herein "cardiocytes") it has been speculated previously that angiotensin II may somehow be involved in the development of left ventricular hypertrophy since patients treated with angiotensin converting enzyme (ACE) blockers to block blood pressure changes, show less tendency to develop left ventricular hypertrophy (25,26). As shown herein, AIN antagonizes the hypertrophic action of AIL Accordingly, the control of cardiocyte growth may be regulated endogenously by a balance between the activating action of ALT and the inhibiting action of AIN. It is further believed that AIN and AIN agonists will be effective in blocking the development of, and reversing the effects of, left ventricular hypertrophy in patients. Additionally, it is believed that the action of ACE inhibitor is due not to their inhibition of AH synthesis but to their ability to enhance the synthesis of AIN ligands such as results from the shunting of precursors from the All synthetic pathway into the AIN pathway. Contrary to current popular belief, the beneficial effect of ACE inhibitors in treating cardiac hypertrophy may be due to ACE inhibitor enhancement of the formation of AIN.
The data presented herein also indicates that All and AIN operate by separate receptors employing different intracellular signaling systems. It has been reported that ACE inhibitors may have a beneficial effect in reducing cardiac hypertrophy through effects at the level of All or AIII. Considering the results disclosed herein it is most likely that the long-term effects previously attributed to decreased ATI may in fact be mediated by the interaction of increased levels of endogenous AIN ligands with the AT4 receptor. Further, it is most likely that the antagonists and agonists of AIN ligands, disclosed herein, will provide improved pharmaceutical compositions for treating cardiac hypertrophy attributable to the renin-angiotensin system, e.g. ventricular hypertrophy. The inventors believe that the interaction between AIN and the AT4 receptor may trigger the receptor and inhibit growth in cardiomyocytes.
In adrenal cells angiotensin All's role in the regulation of aldosterone release from the adrenal cortex is reportedly well established (27). As shown herein, certain activities (such as adrenocortical cell growth), previously attributed to ATT or ATI, are actually activated following AIN ligand binding to the AT4 receptor. All (and AIII) reportedly stimulates aldosterone release from adrenal glomerulosa cells. The disclosure, herein, of high levels of AT4 receptors in adrenal cortical cells (Examples 1-2) suggests a possible role of AIN ligand (i.e., rather than All or AIII) in triggering AT4 receptors on adrenal cells to inhibit All-mediated aldosterone release. Another role of AT4 receptors in adrenocortical cells may be to up-regulate the threshold level of All ligand required to trigger a cellular response by regulating the levels of cellular ATI and/or AT2 receptors and/or to regulate adrenal blood flow. In addition to being found in high concentrations in the adrenal cortex, AT4 receptors are found at even higher levels in the adrenal medullary cells where All has previously been reported by others to potentiate catecholamine release. AIN ligand may modulate release of catecholamines (i.e., increase or decrease the release) acutely (or possibly even long-term, e.g., by triggering the AT4 receptor and thereby stimulating increased or decreased expression of tyrosine hydroxylase, the rate- limiting enzyme in catecholamine synthesis.
In vascular smooth muscle cells the role of AIN and its specific receptor appears to be similar to that articulated above for AIN in cardiocytes: AIN may act to inhibit growth of the cells thus opposing the action of AH. Agonists of AIN binding to the AT4 receptor will be effective inhibitors of vascular smooth muscle growth and will be therapeutically useful in reducing neointimal growth which often occurs following angioplasty.
As disclosed herein, high levels of AT4 receptors are present in cardiac and vascular tissue, including cultured bovine endothelial cells. The disclosure, herein, that AIN ligands and the AT4 receptors may function as growth factors of the tyrosine kinase class indicates that certain inhibitors of tyrosine kinase growth factors may also serve as inhibitors of certain angiotensin AIN ligand-receptor system- mediated cellular hypertrophic processes (e.g., ventricular hypertrophy), and that nucleotide probes constructed for complementarity to portions of RΝA encoding the AIN ligand and receptor sequence may be useful in identifying other members of the AIN family of growth factors.
The invention also provides diagnostic applications for the AIN peptide ligands and antibodies. The role of the AT4 receptor-ligand system in cardiovascular regulation suggests a possible value to diagnostic tests for monitoring the levels of AIN ligand and AT4 receptor in biological fluids and tissues (i.e., rather than All or AIII). Individuals with high renin-sodium profiles are reportedly at five times greater risk of myocardial infarction than individuals with low renin-sodium profiles despite adequate control of systemic blood pressure (28).
The AIN peptides, ligands, receptor fragments, and the like disclosed herein are useful in diagnostic assays, e.g., immunoassays, for the detection of the presence or amounts of AIN ligands or receptors in tissues, cells, and biological fluids of patients. The AIN peptides, ligands, analogs, derivatives, or covalently modified AIN peptides of the invention may be formulated in buffers with stabilizers, e.g., for use as positive or negative controls in diagnostic assay, or in reagent test kits for receptor- binding assays. Those skilled in the art will recognize that the AIN ligands of the invention may be readily employed using conventional techniques to produce polyclonal or monoclonal AIN ligand specific antibodies, and that the isolation and purification of the AT4 receptor provides materials useful for preparation of polypeptide fragments (e.g., using CΝBr and proteolytic enzymes) that can be subjected to automated amino acid sequencing. The amino acid sequence of the AT4 receptor, in turn, provides the sequence data necessary for construction of conserved and degenerate nucleotide probes for cDΝA or genomic molecular cloning of nucleic acids expressing the AT4 receptor, mutant AT4 receptor, or fragments of the AT4 receptor. A convenient method for molecular cloning of the receptor is provided in Example 7. EXAMPLE 1
Physical Characterization of the AIN Receptor Kinetic binding studies: bovine adrenal cortical membranes
In kinetic binding studies, conducted as set forth in Example 1 Materials and Methods described below, both 125I Sarι,Ileg-AII and 125I-AIN binding were characterized by slow association rates (kι=1.01±12xl0"2 and 5.58±0.64xl0"2 nM-1 min"1, respectively), very slow dissociation rates (k.1=2.36±0.49xl0-2 and 2.57±0.05xl0"2 nM-1 min"1, respectively), and high affinity binding (calculated
Kd=2.25±0.26xlO"10M and 4.42±0.46xlO"10M, respectively; number of experiments
(n) = 4) (Table 2).
TABLE 2
Kinetic constants for 125I Sarι,Ileg-Ang II and 125I-AIV bindin to bovine adrenal cortical membranes. *
Figure imgf000030_0001
* n = 3, mean ± SD
Equilibrium binding studies: bovine adrenal cortical membranes
Equilibrium binding studies were conducted to evaluate the binding of 125I-ATV to receptors in bovine cortical membrane preparations. Comparisons were made of the binding of both AIN and of All, i.e., to the classical ATI receptor sites defined by binding of 125I-Sarι,Ileg-AII. Binding studies were carried out in buffer containing 5mM EDTA, lOμM Bestatin, 50μM Plummer's inhibitor, and lOOμM PMSF, developed specifically to inhibit metabolism of angiotensin fragments and receptors during the assay.
Saturation isotherms for 125I-ATI and 125I-AIN indicated the presence of two distinct separable high-affinity binding sites in bovine adrenal cortical membrane preparations, i.e., one for All ligand and a second for AIN ligand. The equilibrium constants calculated from this data were as follows: a) for AH receptor-ligand binding (i.e., 125I-Sar, ιIle8-AII) the Kd=0.54±0.14nM, Bmax=1.03±0.26pmol/mg membrane protein (n=4); and b) for the AT4 receptor ligand binding (i.e., 125I-AIN) the Kd=0.74±0.14nM, 6-^=3.8.^1.12pmol/mg membrane protein (n=4). The results of the equilibrium binding studies with membrane-bound AT4 receptor are summarized in Table 3.
TABLE 3
Equilibrium Binding Constants for 125I Sari, Ile -Ang and 125I-AIV Binding to Bovine Adrenal Cortical Membranes.
Figure imgf000031_0001
n=4, mean ± SD The results of these kinetic and equilibrium binding studies show: (a) two separable high affinity binding sites, one for All and a second for AIV; (b) large differences in the maximal binding (Bm^) per mg membrane protein, i.e., with more than three-fold more AT4 receptor in this preparation than All receptor; and, (c) no cross-displacement of All binding by AIV or vice versa. The results provide convincing evidence for the existence of two separable receptors; one for AH and a second for ATV. However, the theoretical possibility existed that a single receptor might have differing affinities for All and AIV. Since it was known that ATI and AT2 are commonly destroyed during extraction from membranes, and are also heat labile (i.e., at 60°C) a scheme was devised to rule out the latter possibility by testing for AT4 receptors in solubilized and heat-treated membrane preparations. The results of these studies are presented below. Equilibrium binding studies: solubilized bovine adrenal cortical AT4 receptor
Initial studies, conducted as described in Example 1 Materials and Methods, confirmed that 125I-AIV bound to solubilized receptors in membrane preparations which would not be expected to contain ATI or AT2 receptors. The kinetics of binding of 125I-AIN to the solubilized bovine adrenocortical receptor, at an AIV ligand concentration equal to 25% of the apparent Kd (with 25 μg of membrane protein), indicated that equilibrium was reached in approximately 100 min. at 37°C.) The plateau region of binding to the solubilized receptor for 125I-AII or 125I-AIV, (after reaching equilibrium), was stable for at least one additional hour. The off-rate of the AT4 receptor, as determined following the addition of 1000-fold excess of unlabeled AIN, was exceedingly slow, with an average tιy2 =292.4 min (n=5).
Equilibrium binding studies were next conducted at 37°C with a 120-minute incubation (as in the Materials and Methods) with the solubilized membrane receptor preparations. Saturation isotherms for 125I-AIN (Figure 2 A) and 125I-AII (not shown) were developed to compare the equilibrium binding constants of the solubilized AT4 receptor. A concentration range of about 5 x lO^M to about 5 x 10"12M AIN was employed in a typical experiment using 25μg of total protein. The best fit for the transformed data using the LIGAND program revealed a single AIN binding site with no apparent cooperactivity. A summary of the binding data for AIN ligand to solubilized receptor is found in Table 4.
TABLE 4 Equilibrium Binding Data for 125I-AIN to Bovine Adrenal Cortical Solubilized Receptor.
*KH(M) Brn-tv(fmol/mg protein) r(Scatchard Plot) Hill Coefficient
5.06 ± .57xl0"10 87.9 ± 9.7 0.991 ± .009 0.995 ± .039
*Ν=4, mean ± SD The data presented in Table 4 shows that the solubilized receptor, like the membrane receptor (Table 3), has an extraordinarily high binding affinity for AIV. Competition binding studies: bovine adrenal cortical membranes
To establish the specificity of the AT4 receptor, competition curves were developed with several different angiotensin analogs using a concentration range of lO^M to lO'^M. Comparisons were also made of the binding specificity of classical ATI receptor binding sites (i.e., 125I-Sar1,Ile8-AII binding sites). Competition analysis (the summarized results of which are presented below in Table 5) also clearly distinguished the existence of two distinct receptors based on their specificity for different ligand structures in the angiotensin analogs. (The r values for log-logits transformations of the competition data were typically >0.98.) Binding of 125I-AII ligand to the AH receptor (as characterized by binding of 125I-Sar1,Ileg-AII) was effectively competitively inhibited by Sar^Ileg-AII, AIII, and DuP 743. In contrast, AIN ligand, AII(4_8), and CGP42112A demonstrated very little affinity for the ATI binding site (Table 5.) The pattern of binding at the AH site is consistent with a Type I classic AH binding site (20,25). (Binding Sarls Ile8-Ang II, Sar Ile8-Ang II, All, AHI, and DuP 753 with high affinity is a pattern of binding specificity consistent with an ATI site.) In contrast to the AH receptor, the binding site for 125I-AIN ligand was effectively competitively inhibited only by AIN ligand and to a lesser extent by the peptides in the AIII preparation (Table 5). TABLE 5
Competition of 125I-Sarι ,Ile8-AII and 125I-AIV Binding to
Bovine Adrenal Cortical Membranes.
Figure imgf000033_0001
Binding studies: two receptor binding states in rabbit heart membranes Studies were next conducted to examine the kinetic parameters of
125I-angiotensin IN binding to receptors in P2 membrane preparations from rabbit heart. Comparisons were made of the binding of both AIN and of ATI, i.e., to the classical ATI receptor sites defined by binding of 125I-Sar1,Ile -AII. Binding studies were carried out in a buffer (below) containing an extensive cocktail of inhibitors that was designed to minimize metabolism of both the receptor and the test ligand, i.e., the buffer contained 5mM EDTA, 0.2% BSA, lOμM Bestatin, 50μM Plummer's inhibitor, and lOOμM PMSF.
Angiotensin peptides (i.e., Al, All, AHI, or AIN) were stable in this buffer for 4 h at 37°C with less than 10% hydrolysis measured by reverse phase HPLC. The studies were conducted as described in the Materials and Methods, below. The association rate constant (kj) for 125I-AIN was determined to be 3.05 x 108M_1 min-1 and the dissociation rate constant (k.ι) was 0.028 +/ 0.017 min"1 The overall dissociation constant (Kd) measured under equilibrium binding conditions was determined to be 9.15 x 10_11M. (The results represent the mean values from the results of 4 experiments conducted using duplicate samples.) Saturation isotherms and Scatchard analysis produced data best resolved in a two-site model using non¬ linear curve fitting methods (LIGAND program curve fitting options). The Kd for site #1 was determined to be 10.3 +/- 3mM with Bmax =1747 +/- 393 fmol/mg; the Kd for site #2 was 10.1 +/- 5pM with Bmax =15 +/- 4 fmol/mg. Binding to the rabbit heart membranes was competitively inhibited in a specific manner by AIV but not All, AIII, 125I Sarι,Ile8-AII, DuP753, CGP, AII(4.8), or DAAI (see Table 6). TABLE 6 Competition of Binding of 125I-AIN to Rabbit Heart Membrane Receptors
Figure imgf000034_0001
The data in Table 6 was calculated from competition displacement curves for binding of 0.5nM 125I-ATV to membrane fractions; membranes were incubated for 120 min. at 37°C in the presence of lOpM to lmM competitor; possible conversion of AIV to other (smaller) fragments was evaluated after 120 min at 37°C by adding 20% TCA to stop the incubation, and then evaluating the percentage of AIV by reverse-phase HPLC on a C18 column with a 20% ACΝ/TEAP3 mobile phase; greater than 92% of the 125I label present at the conclusion of the incubation was present as ATV. Structural requirements for ligand binding to bovine adrenal AT4 receptors
The results in Table 7, also include a summary of studies designed to analyze the structural features of the N-terminus of an AIN ligand that are required for binding to an AT4 receptor. The results of these structural studies are also presented in Figure 2B. The results show that modification of the Ν-terminal valine residue (i.e., by Ν-terminal shortening of AIN to AII(4_8)), or extending the Ν-terminus with a hydrophobic residue such as Sar or GABA, or changing the stereoisomer of the L-Nal to D-Nal, all drastically decrease binding of an AIN ligand to the AT4 receptor (Table 7). The AT4 receptor also failed to bind DuP 743 (DuP, Figure 2B) or CGP 42112A (CGP, Figure 2B) and thus did not exhibit the pharmacological properties of a classic ATI binding site (26). As shown in Figure 2B, the ability of the various compounds to inhibit AIV binding to the solubilized AT4 receptor was tested. The following compounds are shown in Figure 2B as follows: DAAI1, desAsp angiotensin I (i.e., identical at the Ν-terminus to AIII; see open squares with a dot, Figure 2B); AIN, angiotensin IN (closed diamonds, Figure 2B); AH (open squares, Figure 2B); SIAII (All lacking the He residue at position 5, Figure 1; open diamonds, Figure 2B); DuP (Dup 743, an AH analog; open squares, Figure 2B); CGP (CGP 42112A, another All analog; closed triangles, Figure 2B); and, AIII (open triangles, Figure 2B. The results presented in Figure 2B and the Kj values summarized below in Table 7 show that: (a) only AIN, and peptides in the DAAI1 (i.e., AIII Ν-terminal sequence), and AIII preparations effectively competitively inhibit binding of the 125I-AIN ligand to the AT4 receptor; and (b) the peptides in the AHI, Sar^AIII, and DAATl preparations are approximately 100 times less effective than AIN in competing binding to the AT4 receptor.
TABLE 7 Competition of 125I-AIV Bindin to Solubilized Bovine Adrenal Cortical Receptor.
Figure imgf000036_0001
*Ν=4, mean ± SD The combined results show the importance of the valine at the 3 position for binding of an ATV ligand to the solubilized bovine adrenal gland AT4 receptors (i.e., D-Valj; des-Valj-AIV are inactive). The results also show the apparent insignificance of Phe8 for AIV binding to this AT4 receptor (i.e., desPhe6-AIV still retained AIN binding activity). Thus, a minimal AIN penta peptide ligand having a sequence NYTHP retained AT4 receptor binding activity.
For high affinity binding of an AIN ligand to an AT4 receptor, the structure of the Ν-terminal neutral polar amino acid (e.g., valine) is most important. Ν-terminal extension is incompatible with binding, deletion of the terminal valine residue eliminates binding (Kj >10"6), substitution with Sar decreases binding affinity, substitution with Phe results in a 5- 10-fold decrease in the affinity of binding, Pro- substituted AIN peptides bind with 100-fold lower affinity, but substitution with He results in equivalent binding, and Lys substitution results in 10-fold higher binding affinity of the KYIHPF AIN ligand (data not shown). In the studies presented in Tables 6-7, and Figure 2B (above), a minimal level of binding of peptides in AIII preparations to the AT4 receptor was observed. This most probably represents an artifact in the assay and/or ligand preparations that is easily accounted for by a low level of Ν-terminal hydrolysis of AIII to AIV, e.g., the apparent affinity of peptides in AHI preparations for the AT4 receptor (i.e., at 22.8nM) can easily be explained by approximately 2.5% hydrolysis of AIII to AIN. This notion was supported by the results of studies conducted with density-gradient- purified heat-treated membranes (i.e., heat-treated and purified to minimize enzymatic activity in the membrane preparations). In these studies the amount of AIII conversion to AIN was substantially reduced and the apparent affinity of Am for the AT4 receptor was also reduced (Ki=29.3±3.3xl09M), i.e., from approximately 29-fold less active than AIN ligand (above) to about 52-fold less active (here). This supports that notion that hydrolytic cleavage in the assay (i.e., mediated by a specific cell membrane AIN angiotensinase or by another nonspecific peptidases) can convert an Al, AH, or AIII peptides to an AIN ligand, and that low levels of AIN ligand in these preparations accounts for their apparent binding to the AT4 receptor. Additional support for this notion is provided by the results of studies (data not shown) which show that the percentage hydrolysis of AH or AIII in a preparation correlates with the effectiveness of a given preparation as an inhibitor of AIN ligand binding to the AT4 receptor. In this study preparations of AH or AIII were incubated at 37°C for different periods of time and (i.e., x% AIN) the extent of hydrolysis to AIN was determined by reverse phase HPLC (i.e., x% AIN). In every instance 100% of the apparent binding of 125I-AII or 125I-AIII was due to actual binding of 125I-AIV.
As further shown in Table 7, the D-substitution and glycine-substitution data confirms that positions 1-3 of the AIV molecule are critical for determining binding affinity to the receptor. Positions 4-6 are less critical. In fact removal of C-terminal groups appears to enhance binding affinity perhaps by reducing steric constraints. Ligands containing C-N nonpeptide bonds can be produced that possess high affinity. In general, highest affinity is obtainable by dual modifications at bonds between amino acids 1-2 and 3-4. Val(l)Val(3) AIV appears totally resistant to enzymatic degradation upon exposure to rat kidney homogenates. As further shown in Table 7, tripeptides containing straight chain aliphatic amino acids in position 1 exhibit high affinity. To date, the highest affinity is achievable with the Nle-Y-I amide, suggesting that amides are preferable to free acids and that the chain length found in Nle is optimal (both longer and shorter bind with lower affinity). Note that a similar approach to that taken with the N-terminus is useful to characterize the C-terminal. Stereoisomers (e.g., D-Phe), C-terminal extension, and sequential C-terminal deletion, and other peptide analogs can be synthesized and tested for their ability to competitively inhibit with 1251-AIV binding to purified bovine adrenal membranes. If C-terminal extended peptides are found to bind to the receptor, appropriate C-terminal extended peptides can be constructed for use in the affinity chromatography purification of the receptor. Substitutions and modifications of internal amino acids can also be examined for their effects on binding. Once physiological or second messenger systems have been identified (Example 6) as being AlV-dependent events, then they can be used for drug screening, and a second round of synthesis can commence focusing on the development of receptor antagonists.
The materials and methods and experimental assay conditions employed in Example 1 are described below: Materials and Methods: Peptide Synthesis The angiotensin analogs employed in this study were synthesized by the standard Merrifield method utilizing t-Boc protected amino acids and chloromethylated resins on a Vega 250 coupler automated synthesizer. Following synthesis, the crude peptides were purified by preparative reverse-phase HPLC using a 1 h gradient for elution at 9ml min. Initial conditions were 90% H2O, 10% acetonitrile, and 0.1% TFA and the final conditions at the top of the gradient were 65% H2O, 35% acetonitrile and 1% TFA. Purified peptides were amino acid analyzed to determine both peptide and total purity. Typically the peptides produced were greater than 99% pure and contain 20-25% acetate. Tissue Preparation: bovine adrenal cortical tissues
Adrenal cortex was removed from bovine adrenals obtained from a local slaughterhouse. The minced cortex was then homogenized in a Polytron as a 40:1 suspension in assay buffer at 10 sec/ml. The homogenate was then centrifuged at 500g for 10 min. to remove whole cells and nuclei. After a rehomogenization and recentrifugation the combined supernatants were spun at 40,000 x g for 20 min. The pellet was rehomogenized and respun at 40,000 rpm for 30 min. This final pellet was resuspended in assay buffer and layered on a discontinuous sucrose gradient (0.8M/1.2M). After a 100,000 x g spin for 90 min. the purified membranes were located at the density interface and were removed. The sucrose containing membrane suspension was diluted 1 :10 in assay buffer and spun a last time at 40,000 x g for 30 min. The pellet was resuspended in assay buffer at a concentration of lOmg protein/ml and heat treated at 60°C for 30 min. in the presence of 20mM MgC^. The membranes, now devoid of almost all peptidase activity, were ready for use in the binding assay. Binding studies: bovine adrenal cortical membranes
To test the ability of the synthesized analogs to competitively inhibit for 125I-AIV binding a displacement curve was established using purified bovine adrenal cortical membranes. Binding was carried out in 10-75mm siliconized glass culture tubes containing 0.2nM 125I-AIV, 25mg of membrane protein, and the desired analog over a concentration range of 10"12M to lO^M using half-log dilutions. All binding incubations were carried out in duplicate at 37°C for 2 h in a buffer containing: 50mM Tris, 150mM NaCl, 5mM EDTA, lOμM bestatin, 50μM Plummer's Reagent, lOOμM PMSF and 0.2% BSA (assay buffer) in a total volume of 0.25ml. After incubation, the incubates were filtered through GF-B filters soaked in 0.3% polyethyleneimine and washed with four-4ml washes of PBS. The filters were then counted in a Beckman 5500 gamma counter. A typical experiment examines 5 analogs simultaneously and includes a positive control curve in which AIN was used as the displacer. All curves were run in quadruplicate, each with a different tissue preparation. Nonspecific binding was defined as total binding minus binding observed in the presence of lOOmM Sar^I-e^AII or lOOmM AIN. No cross displacement (i.e., of AH binding by AIV or AIV by All) was observed. HPLC analysis of both the bound and free 125I-AII or 125I-ATN ligand indicated that 100% of the "specifically bound" label was either 125I-AII or 125I-AIV, respectively, and the overall hydrolysis of 1 5I-AII under conditions of the assay was less than 2%.
Data were analyzed by the LIGAND program (29) from which Kj values can be obtained. Binding studies: solubilized bovine adrenal cortical receptor
Solubilization and characterization of the receptor from bovine adrenal membranes was accomplished by homogenizing the membranes (above) in hypertonic buffer followed by fractionation of the membranes by sucrose density gradient centrifugation. The membrane preparation was then heat treated at 60°C in the presence of MgC-2 (to inactivate ATI receptors). The heat treatment also reduced endogenous peptidase activity in the preparations by 90-95%. The AT4 receptor in the preparations was then solubilized using 1% zwitterionic detergent 3-[(3-cholamidopropyl) dimethyl ammonio]-l-propanesulfonic acid (CHAPS). Binding studies: rabbit heart P2 membranes from rabbit heart were prepared by homogenization and differential centrifugation at 4°C. Binding was carried out in the presence of 5mM (EDTA), 0.2% heat-treated bovine serum albumin (HTBSA), lOμM Bestatin, 50μM Plummer's inhibitor, lOOμM phenylmethylsulfonylfluoride (PMSF), and 50mM Tris, pH7.4, at 22°C. Binding was initiated by the addition of lOOmg protein and appropriate amounts of labeled ligand. (For kinetic binding studies the samples were incubated for 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, and 220 minutes at 37°C. For equilibrium binding studies the same conditions were used and samples were incubated for 120 min at 37°C.) All incubations were conducted at a final volume of 250ml in 12 x 75mm siliconized (SigmaCote) borosilicate tubes, and they were terminated by rapid vacuum filtration in a Brandel cell harvester through glass fiber filters (Schleicher and Schuell #32) soaked in 0.3% polyethyleneimine. Filters were immediately rinsed with 4 x 4ml 150mM NaCl, 50mM Na2HPO , pH7.2 at room temperature. Filters were allowed to air dry, placed in fresh tubes and counted in a gamma counter. Specific binding was defined as the difference between the absence and presence of 1.OmM displacing ligand.
Dissociation (i.e., of ligand from receptors) experiments were conducted by adding lmM unlabeled AIV ligand competitor to the assay at 120 minutes after initiating binding (at 37°C) with 0.5nM 125I-AIV.
Saturation isotherms for binding were conducted with approximately 25μg of tissue protein incubated with various concentrations of 125I-AIV for 120 min. at 37°C; nonspecific binding was defined in the presence of lmM AIV. Three experiments were conducted resulting in 36 data points for Scatchard analysis.
EXAMPLE 2 Tissue and Species Distribution of the AIV Receptor Species Distribution:
A second major approach to defining separate and distinct binding sites was to examine their relative tissue and species distribution (Table 8). The results presented in Table°8 show the fentamoles of All or AIV bound per milligram of membrane protein in extracts prepared under identical conditions from the tissues and species indicated.
TABLE 8 *Distribution of 125I-SI-AII and 125I-AIV Bindin in Mammalian Tissues. **
Figure imgf000041_0001
*25μg of membrane protein was incubated with 500,000 cpm of label. Specific binding was defined as total binding-nonspecific binding at 100 nM unlabeled peptide.
**n = 2-6; mean ±SE; 125I-SI-AII=125I-Sarι,Ile8-Air, fmol mg protein=femtomoles (i.e. 10"15M) of All ligand bound per mg of total protein in the preparation.
N.Det. = not detectable, i.e., less than 1.8 finol mg protein.
The results show that: a) the human ATV ligand binds AT4 receptors in a wide variety of mammalian species; and b) most mammalian adrenal tissue express an AT4 receptor capable of binding the AIN hexapeptide NYTHPF. Tissue Distribution:
In order to begin to assign physiological functions to the AIN ligand-AT4 receptor interaction, preliminary tissue distribution studies have been conducted in guinea pigs. Guinea pigs were chosen because their adrenal tissues demonstrated high levels of AIN binding (Table 8). The tissue distribution of AT4 receptors was measured by assaying radioligand binding to membrane preparations, i.e., as described in Example 1, above.
TABLE 9
Distribution of 125I Sar Ile8-AII and 125I- AIN Binding sites in the Membranes of Guinea Pi Tissues.*
Figure imgf000042_0001
*n = 4; Binding was carried out as described in Table 6, above.
The combined results presented in Table 9, above, show that the receptor was widely distributed in the tissues. The combined results suggest evolutionary conservation of both the AT4 receptor and the ATV ligand. Additional studies were next conducted to physically compare the receptors in guinea pig brain, rabbit heart, and bovine coronary venule endothelial cells with respect to their binding affinities for AIV ligand. The results of these studies indicate that cells in these different tissues all have AT4 receptors with comparable binding affinity for AIV ligand; each cell type has an AT4 receptor with a K for 125I-AIV of about O.lnM to about 0.5nM. Next, competition studies similar to those presented above in Example 1 were conducted to evaluate the structural requirements for building AIN in different tissues. The results indicate that each AT4 receptor exhibits the same Ν-terminal specificity recorded, above, i.e., for N, I, or K. The results of these combined studies support the notion that a remarkable degree of evolutionary conservation has been maintained for AIN ligand-AT4 receptor system, and this level of conservation is commonly predictive of important, and probably critical, physiological functions.
Surprisingly, it has not been possible to demonstrate binding of 125I-AIV in rat tissues including brain, heart, kidney, aorta, lung, liver, or cultured smooth muscle cells (n=5) (Table 8). At this time, the significance of this finding is uncertain. It is possible that the in vitro assay conditions may not be optimal for this species and that the AT4 receptor or ATV ligand may be rapidly inactivated, e.g., by high levels of peptidases known to be present in rat tissues. In addition, on a technical note, binding of the AIV ligand to the AT4 receptor was inhibited in the presence of Bacitracin (i.e., lOmg/ml; at a final calculated concentration in the assay of 0.07M). Bacitracin is a polypeptide antibiotic with the sequence ICLEIKOIFHDD (i.e., O is ornithine), and it is often included in angiotensin binding assays to inhibit the action of nonspecific proteases (i.e., as an alternative substrate for the proteases). The observation of bacitracin interference is of potential significance for at least two reasons: 1) previous investigators who have included bacitracin in their assay buffers may have inhibited AIV ligand binding to the AT4 receptor; and, 2) inhibition of AIN binding by this polypeptide (notably at a very high molar concentration) may provide an indication of amino acid sequences that may contribute to electrostatic interactions in the AT4 receptor binding site (e.g., R1IR2HR2, where Rj is an amino acid with an aromatic side chain such as OH, SH, or ΝH, and R2 is a polar amino acid).
The results of the tissue distribution studies, above, indicate that the receptor is present in the adrenal tissues in most mammalian species, and can be isolated in P2 membrane preparations from most mammalian species. Receptor Autoradiography in Tissues:
Receptor autoradiography is a useful extension of radioligand binding studies since it provides detailed anatomical information about the location of receptors in tissues and groups of cells in tissues, and thus it facilitates understanding the function of the AIN ligands and AT4 receptors in those sites. Autoradiographic analyses of serial sections of guinea pig brain (20mM thickness) were performed. The autoradiographs showed a pattern of distribution for AH receptors and AT4 receptors in the Habenula (Figure 3), Hippocampus (data not shown) Cerebellum (data not shown), Prefrontal Cortex (data not shown), and Thalamus (data not shown). In each case the receptor distribution in the tissue was determined by binding of 1 5I-Sarι ,Ile -AII, or 125I-AIN, respectively. Specificity of ligand binding in these autoradiographic studies was demonstrated by competing the binding of the specific ligand (i.e., All or AIN) with unlabeled lOOnM Sarj-Heg-AII, or lOOnM AIN, respectively. The data demonstrate that while specific All and AT4 receptors are located at similar sites in the Habenula, Hippocampus, and Cerebellum of guinea pig brain, the two receptors are distinct with regard to exact groups of cells that express the two different receptors. Major differences were observed in the Prefrontal Cortex and Thalamus where AT4 receptors were abundant but All receptors appear to be relatively rare. In the Hippocampus the AT4 receptor is present in the pyramidal cell layer CA1, CA2, and CA3 of the Hippocampus and dentate gyrus. Binding of AIN occurred at a single binding site with high affinity (K =1.29±0.18nM, mean ± SD, Hill Coef = 0.993±0.015) and in a saturable manner (Bmax=449±62 fmol/mg protein). The properties of the hippocampal AIN ligand-receptor system as described in greater detail below (see Example 7). The findings of AT4 receptors in the Hippocampus suggest that the AIN ligand-receptor interactions in the Hippocampus may mediate unique angiotensin-dependent functions including memory enhancement. The AIN ligand-receptor system may provide a link between the Hippocampus and memory. The mutually exclusive cellular distribution of AIN and All receptors is demonstrated in the autoradiograph shown in Figure 3. Panel A reveals intense 125I-AIN binding in the habenula, while Panel D indicates that 125I-Sarι,Ile8-AII binding is localized primarily to fiber tracts including the visual tegmental relay zone and the medial lemniscus. The specificity of ligand binding to receptors in these tissues was illustrated by competing 125I-AIV binding with lOOnM non-labeled ATV (not ATI) [(Figure 3, Panels B and C)]; and, lOOnM non-labeled Sarι,Ile8-AII displaced only the 125I-Sarι,Ile -AII binding [(Figure 3, Panels E and F)]. Some tissues, however, may contain both AH and AT4 receptors.
Quantitative aspects of binding in brain is presented in Example 7, and other tissues data is presented in Example 1, above. The results show that all important cardiovascular tissues in guinea pigs contain the AT4 receptor. This result is not surprising in light of the observation (above) that vascular endothelial cells contain high concentrations of receptors, but this is not responsible for tissue binding of AIN ligand because every vascularized tissue will possess AT4 receptors, i.e., skin and skeletal muscle has low levels of receptor. Materials and Methods:
Autoradiographic analysis of 125I-AIN and 125I-Sarι,Ile8-AII binding in guinea pig tissue was determined as follows. Heart, kidney, brain, and other tissues were cryostat-sectioned into 20mm sections that were mounted on chrome-alum- gelatin-coated slides in multiple sets of seven. The slide-mounted tissue sections were thawed (35°C) and preincubated in assay buffer (150mM ΝaCl, 50mM Tris, 5mM EDTA, 0.1% BSA, lOμM bestatin, 50μM Plummer's inhibitor, lOOμM PMSF, at pH7.4) for 30 min. and then incubated for 1 h in the same buffer with the addition of 225-250pM of 125I-Sar1,He8-AII (for visualizing All receptors) or 125I-AIN (for visualizing AT4 receptors). To define the specificity of the ligand binding, tissue sections were incubated in the radioligand in the presence and absence of lOOnM unlabeled ATI or AIN peptide. After appropriate washing, autoradiograms were prepared by apposing the slide-mounted tissue sections to X-ray film (Hyperfilm, Amersham) for an appropriate exposure time. The amount of radioligand binding in a tissue was quantified using densitometric techniques and 125I standards (Microscales, Amersham, Arlington Hts, IL).
EXAMPLE 3 Receptor Isolation. Purification, and Properties and Production of Monoclonal Antibodies Receptor Isolation and Purification: The AT4 receptor was solubilized in high yield from purified bovine adrenal membranes using the zwitterionic detergent CHAPS (1%) at 4°C over 4 h under conditions where peptidase activity and differential solubilization of the AT4 receptor (but not the ATI receptors) is permitted (see also Example 4, Materials and Methods, below). For example, membranes from a variety of different tissues and cells (e.g., 25mg of P2 membranes, Example 1) were incubated for 4 h in Hepes buffer (20mM, pH7.4) containing 1% CHAPS and a cocktail of protease inhibitors and alternative protease substrates, i.e., lOμM bestatin; 50μM Plummers' inhibitor; 0.2% BSA (bovine serum albumin); and lOOμM PMSF (phenylmethylsulfonyl fluoride).
A most useful component of any AH receptor purification scheme was including a step wherein the solubilized membrane proteins were subjected to a heat treatment at 60°C, e.g., for 20 minutes and in the presence of 20mM Mg"1"*. This step was useful in destroying any residual All receptor leaving the AT4 receptor intact.
The AT4 receptor was stable to chromatofocusing and SDS-PAGE, allowing isoelectric focusing, or one- or two-dimensional PAGE or SDS-PAGE to be used for purification. Due to the slow-off rate of 125I-AIN binding, the receptor was radiolabeled with 125I-AIN ligand to allow ease of identification during purification. As an additional aid to purification, the receptor was successfully cross-linked to a 125I-radiolabeled AIN analog ligand having a C-terminal extension, i.e., from residue 8, with lysine residues (i.e., 125I-Lysu-AIN). The Lysn-AIN analog binds to the AT4 receptor with a K,j that is similar to AIN ligand. Using Bis (sulfosuccinimidly) suberimidate (BS3) as the cross-linking agent, the 125I-Lysι j-AIN analog of AIV was bound to the AT4 receptor and then cross-linked to the AT4 receptor through the e-amino group of Lys. Purification of the AT4 receptor may also be achieved, for example, by ion exchange, lectin chromatography, hydrophobic chromatography with conventional techniques, HPLC, or FPLC. SDS-PAGE analysis of isolated and purified receptor indicated a molecular weight between 130KDa and 150KDa, at about 146KDa for the BS3-cross-linked AT4 receptor from bovine adrenal tissue. The purified, uncrosslinked receptor appears to have a significantly smaller molecular weight, on the order of 60,000KD. Receptor Properties:
Identification of the family to which a receptor belongs commonly permits predictions to be made about possible improvements in purification, useful methods for stabilizing the receptor during purification, cellular sources and assays useful for molecular cloning of the receptor, and identification of novel physiological roles for a receptor. For instance, neurotransmitters and hormones are known to interact with four types of plasma membrane receptors: 1) multisubunit receptors that regulate an intrinsic ion channel; 2) G-protein linked receptors that, via the G-protein, can activate membrane channels and enzymes; 3) guanylate cyclase receptors that possess intrinsic guanylate cyclase activity in a single membrane spanning polypeptide chain; and, 4) protein tyrosine kinase receptors that have intrinsic tyrosine kinase activity capable of phosphorylating multiple protein substrates.
Many common neurotransmitters like acetylcholine, glycine, glutamate, and GABA activate receptor-ion channels. The interaction of the neurotransmitter and receptor results in the opening of an intrinsic ion channel. In all cases these receptors are constructed as heteromultimers and are most likely evolutionarily related. Despite the importance of this receptor class, to our knowledge no known peptide transmitter or hormone acts by such a mechanism. Thus, it is reasoned unlikely that the AT4 receptor is a member of this family of receptors.
Studies have been conducted to determine the receptor family to which the AT4 receptor belongs (see Examples 5 and 7). It has been reported previously that the AH receptor may be a member of the G-protein-linked family of cellular receptors. The majority of known peptide receptors belonging to this family are characterized by seven membrane-spanning alpha-helical regions and when stimulated are capable of activating membrane-bound enzymes like adenylate cyclase, phosphodiesterase, and phospholipase C. (30). Additionally, membrane channel or ion transporter properties can be indirectly modified by the intervening G-protein (31). Although many strategies have been devised to test a particular receptor's linkage with a G-protein, three strategies seem to predominate. In one form or another these include the following approaches: 1) GTP and its analogs are known to alter the binding affinity of agonists to their receptors. Therefore, the ability of GTP or analogs to change agonist-binding affinity is diagnostic of a G-protein-linked receptor. In the presence of GTP, dissociation of the G-protein subunits from the receptor results in a lowered affinity for agonists. This was examined (see Example 5) by the direct assessment of GTP (of GTPγS) effects on agonist binding via changes in dissociation rates or total binding over a range of GTP concentrations, or indirectly by monitoring shifts in IC50 values for agonists during competition for antagonist binding. 2) Another indication of G-protein linkage is the ability of agonists to stimulate the intrinsic GTPase activity of the alpha subunit of G-proteins. This GTPase activity is triggered following receptor occupation and subsequent dissociation of alpha and beta-gamma subunits. 3) A final approach is to determine whether an agonist can facilitate nucleotide cycling. A crucial step in G-protein signal transduction is the agonist-stimulated dissociation of GDP from the alpha-subunit and its replacement with GTP. Changes in cycling are often assessed by comparing the binding of radiolabeled irreversibly bound GTP analogs before and after agonist stimulation.
Studies to date include studies to determine the cellular signal transduction mechanisms activated following binding of AIN ligand to the receptor. The data obtained with isolated AT4 receptor now strongly suggest that although the AT4 receptor may be G-protein linked in certain cells (see Example 5) the AT4 receptor does not belong to the classical family of G-protein-linked receptors for at least three reasons: namely, 1.) Solubilization and stability characteristics of the All receptor (i.e., binding 125I-Sar1,He8.AII) and the AT4 receptors (i.e., binding 125I-AIN) are significantly different which is consistent with: a) large structural differences between the two receptors, and, b) differences in the structural basis of receptor integration into membranes. Thus, it is reasonable to assume that if the ATI/ ATI receptor is a member of the G-protein linked family of receptors, then the AT4 receptor probably is not.
2.) Receptors of the G-protein-linked family of receptors are reportedly susceptible to inhibition at micromolar concentrations of GTPγ S. Studies were therefore conducted to examine 125I-AIN hgand binding to the AT4 receptor in the presence of GTPγ S. The binding of radiolabeled AIN ligand to AT4 receptors isolated from bovine adrenal membranes is not altered by adding GTPγ S to the assay buffer at concentrations ranging from 10"10M - lO^M. (Under these conditions binding of control preparations of AH ligand to AH receptors [i.e., in the same membrane preparations] revealed the typical pattern of a G-protein linked receptor with decreased binding of 125I-Sarι,Ile8.AH at increasing concentrations of GTPγ S; number of experiments = 5). 3.) The AT4 receptor has a demonstrated molecular size of 140 to
150kDa (on SDS-PAGE) for the isolated and purified receptor, and 146KDa for the
BS3 cross-linked bovine adrenal AT4 receptor. These molecular sizes are significantly different from the molecular weights of 55KDa to 65KDa that are commonly associated with members of the G-protein-linked family of receptors.
If the AIN site is not a classical G-protein-linked receptor, then to what family of receptors does it belong? Evidence in recent years indicates the presence of peptide receptors with intrinsic guanylate cyclase activity. These receptors, best exemplified by the mammalian AΝP receptor, consist of a single polypeptide chain with one membrane-spanning region that possesses guanylate cyclase activity that resides near the intracellular C-terminus (32). Since only two such mammalian receptors have been identified (to date), the AΝP and rat intestinal enterotoxin receptor, it is difficult to speculate on the probability that the AT4 receptor is a member of such a family of receptors. Nevertheless, the similarity in the molecular weights and in ion requirements of the ANP and AT4 receptors necessitates the consideration that the AT4 receptor may be a member of such a family.
The final receptor family to which the AT4 receptor may belong is the tyrosine-kinase growth factor family of receptors. These receptors are characterized by a protein kinase activity which preferentially phosphorylates tyrosine residues. Among the substrates of phosphorylation are the receptor itself and phospholipase C, which when phosphorylated initiates the inositol phosphate cascade (33). The tissue response to prototypical peptides which act as tyrosine kinase receptors includes long- term alterations that invariably involve changes in the transcription rate of selective mRNAs. Although often accompanied by acute effects, these peptides appear to play a role in the adaptation of target tissues to chronic changes in the level of a factor. In addition, tyrosine kinase receptors often "cross talk" with other cellular receptor types (34) in response to physiological and chemical stimuli. This type of role is precisely the function envisaged for the ATV ligand-receptor system.
A comparison of the solubilization, physical properties, and functional activities of the AT4 receptor with the cellular biology of members of the tyrosine kinase family of growth factor receptors (e.g., fibroblast growth factor receptor; FGF) suggests a closer relationship of the AT4 receptor to this family of receptors than to the guanylate cyclase family of receptors. For instance, both the AT4 receptor and the FGF receptor have related biochemical characteristics, e.g., the FGF receptor has a molecular weight of about 140-150kDa (35), is relatively heat stable (i.e., at 56°C), and has divalent ion requirements (28). Moreover, as described herein, AT4 receptors appear to have growth factor activity on at least endothelial cells and myocytes. (In the latter case, the tissue distribution and the activities of AT4 receptors are also consistent with a role for AT4 receptors in growth regulation. For instance, as disclosed above, high concentration of AT4 receptors is present in cardiovascular tissues where angiotensins are reported to enhance tissue growth.)
At least three observations are significant in assigning the AT4 receptor to a receptor family. First, the molecular weight of the AT4 receptor is in the range of members of the tyrosine kinase families of receptors. Second, the AT4 receptor, like members of both the tyrosine kinase families of receptors, is characterized by divalent cation binding sites (i.e., Mg"1""1"). And third, the AT4 receptor, like members of the tyrosine kinase and guanylate cyclase families of receptors, is characterized by relatively high heat stability (i.e., 60°C/20minutes). (For comparison, the epidermal growth factor receptor (EGF) is heat stable at 50°C for 30 min., and has specific binding sites for Mn"1"1" and Mg"1-1" [28]). Thus, by at least these criteria the AT4 receptor appears to be a member of the tyrosine kinase family of receptors, and not the G-protein-linked family of receptors. Experimental approaches to validate this vision are presented below, the experiments examine the ability of AIN ligand to stimulate phosphorylation of tyrosine residues in membrane proteins. In addition, the focus of the experiments that follow in Example 4 (below) was directed toward defining the cellular biology of the AIN Ugand receptor interaction, and these studies will also help confirm the classification of the AT4 receptor as a member of the tyrosine kinase family of receptor (e.g., capable of regulating cell growth and intrinsic tyrosine kinase activity of the AT4 receptor). Materials and Methods: Cross-linking to the AT4 receptor
Cross-linking 125I-AIN to the AT4 receptor can be accomplished with Bis (sulfosuccinimidyl) suberimidate (BS3) as discussed above. The cross-linked receptor (approx. mw of 146,000) can then be electroeluted from PAGE gel slices in a substantially pure form for use as an electrophoretic standard. For cross-linking one milligram of total solubilized membrane protein containing AT4 receptor was incubated with 30 x 10-6cpm of 125I-AIN in 50mM Tris, pH7.4 and 150mM ΝaCl containing a cocktail of protease/peptidase inhibitors for 2 hr at 37°C (final volume 0.5ml). Following incubation, the incubate was spun through two successive spin columns packed with 0.8ml of Biogel P-6 extrafine (Biorad) that has been pre-equilibrated with 20mM ΝaP buffer, pH7.4 containing 0.01% CHAPS. The labeled receptor, now in phosphate buffer, was cross-linked with BS3 (final cone. 9mM; added as 90mM in DMSO). The mixture was incubated 30 min. at 0°C. Cross-linking was terminated by the addition of 100ml of IM Tris, pH9.0 with an additional 10 min. incubation at 0°C. The mixture was then spun through a final spin column to remove reactant and free ligand. The centrifugate was now ready for PAGE. Production of Monoclonal Antibodies:
Monoclonal antibodies are useful for purification of receptor, and for identifying the receptor (and fragments thereof) in tissues, cells, and biological fluids. Purified or semi-purified AT4 receptor (preferably nondenatured) can be used as an in vivo or in vitro immunogen. (Those skilled artisans will recognize a variety of options available to them for evoking monoclonal and polyclonal antibodies, e.g., see Harlow, E. and D. Lane, Eds. "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory, 1988). For in vitro immunization antigen can be incubated in picogram quantities with murine, rat, or human lymphocytes. Production of antibodies can be screened by testing for the ability of 125I-AIV-prelabeled receptor to bind to antibodies adsorbed on a polystyrene plastic surface, e.g., in 96 well plates; or, alternatively, by testing the ability of the antibody to inhibit binding of a purified labeled receptor to AIN ligand adsorbed to a solid phase. In either case, antibody producing cells are identified, cultured, and cloned. The monoclonal antibody product of the cloned cell lines can bind the AT4 receptor in ligand-binding and non- binding domains of the AT4 receptor. Νon-binding domains can include structural regions of the molecule as well as enzyme active sites, phosphorylation sites, glycosylation sites, and the like. The presence of antibodies specific for the ligand- binding domain can be assessed directly via the ability of the mono to competitively inhibit in binding assays. As mentioned earlier antibodies are useful for receptor purification and immunohistochemical studies designed to elucidate the cellular location of receptors and also in structure/activity studies designed to map functional domains in the receptor. EXAMPLE 4
AIN Receptor Antagonists and Agonists To test the ability of synthesized AIN ligands to competitively inhibit for 125I-AIN ligand binding to the AT4 receptor, displacement curves were constructed using heat-treated (60°C for 20 min. in 20mM MgCl2) purified bovine adrenal cortical membranes using Methods described in Example 1, above. Effects of AIN analogues on renal blood flow were determined as described in Example 6. The design of AIN analogs followed a question based approach. The unifying question: What are the essential ligand domains for receptor binding and activation? Individual chemical modifications were made to ask specific questions about spacial orientation of molecular surfaces, charge, hydrophobicity and occupancy of space (volume occupied at a specific location). A standardized assay of analog competition was employed to study receptor-ligand binding affinity of the high affinity 125I-AIN- binding receptor in heat treated, sucrose density gradient purified bovine adrenal cortical membranes. Energy minimized, computer generated models ("Macromodel" program run on a Nax mainframe) provided the visual representations of the molecular conformation of highest probability.
Agonist versus antagonist activity was assessed using a laser doppler to monitor renal cortical blood flow following infusion of a test analog into the renal artery (see Example 6). Maximal response was compared to the response (increased in flow) to AIN. (Note that under these conditions All produces a decrease in blood flow in this assay.) Interprestation of physiologic and binding data was based on the precept of a lock and key model of receptor binding and that dynamic change of the receptor upon interaction of the ligand was required for activation (full agonist activity; with second messenger activation).
The following main assumptions were used: 1.) Ligands with the highest affinity, when modeled in an energy minimized conformation offer a visual representation of the receptor binding site field surface (i.e., hydrophobic charge interactions) and charge locations in the "pre-binding state" and "non-activated state" (i.e., just as a clay imprint on a well fitting key represents the interaction surface of the lock). 2.) Specific ligand domains induce changes in the receptor upon binding that produce cellular responses. Ligands that fit the "pre-binding" receptor with high affinity may not activate the receptor, i.e., they may act as antagonists, while structures that induce changes in the conformation of the receptor may be compatible with, and part of, the changes that produce high affinity binding, i.e., they may act as agonists.
The following is a summary of the questions asked and the compounds synthesized to identify antagonists and agonists of the AT4 receptor that interfer with binding of physiological AIV fragments. The inventors believe that mapping the receptor binding site (herein) and understanding of the structure of the receptor and its signal transduction mechanism form the requisite basis for rational design of pharmacologic therapeutic agents that interact with this receptor system in vivo in mammals.
Question #1. What are the absolute AIN ligand amino acid requirements for binding to the AT4 receptor? The approach used to answer this question involved deletion of residues from either the Ν- or C-termius of AIN (i.e., NYHTPF), or from the larger Al sequence of which AIN is a part (i.e., Figure 1). For the most part these studies employed the bovine adrenal cortical AT4 receptor present in membrane preparations prepared as described above in Example 1. The binding assays were also conducted as described in Examples 1 and 2, above, and certain of the results summarized here are also presented above in those Examples.
Deletion of the Ν-terminal Nali residue from NYTHPF to produce YEHPF reduced binding affinity to the bovine adrenal cortical AT4 receptor by 1000-fold. Addition of d-arginine to the Ν-terminal, (i.e., a peptidase analogue of AIII), reduced affinity by 100-fold. Deletion of the C-terminal Phe (i.e., des-Phe6AIN) did not alter binding significantly. Further truncation of the C-terminal PK>5, however, produced a moderate affinity (i.e., 21-fold less than ATV). Fragments containing less than positions #1 through #4 (i.e., Ν-VYIH-C) have Ki*s>500nM. Addition of histidine to the C-terminus, (i.e., AI(3.9), Figure 1), did not alter binding significantly, and further addition of leucine (i.e., AI^.JO) Figure 1) reduced affinity by just 2-fold and resulted in data best plotted in Scatchard analysis to fit a two site binding model.
These results suggest that the binding domain in the AT4 receptor recognizes the Ν-terminus of AIN with a high degree of specificity. The receptor appears to interact less closely with the C-terminal region of AIN, but binding of receptors to this region of AIN may determine the receptor subtype specificity of AT4 receptors in different tissues.
Question #2. Does the binding site that interacts with the #1 residue in AIN (i.e., valine) exhibit any stereospecificity for particular orientations of the Ν-terminal residue? Replacement of the L- valine γ in AIV with D-valinej reduced binding affinity by
1000-fold. This indicates that the domain in the AT4 receptor binding site that interacts with the #1 position amino acid residue in AIV possesses a minimum of "4 non-planar ligand interacting sub-domains that have a fixed spacial orientation" that can be designated by the L-conformation of an L-valine amino acid. Examples of the latter "4 non-planar ligand interacting sub-domains" may be supplied by the side chain residues of 4 amino acids that appear in a requisite 3 -dimensional space within this subdomain of the receptor binding site. (Results discussed in response to Question #4, suggest that one of the 4 non-planar ligand interacting sub-domains interacts with the l°-amine in the N-terminal amino acid.) Compounds that mimic the space filled by L- valine in a hydrophobic environment may mimic the interactions of L- valine with this subdomain of the receptor.
Question #3. Is the hydrophobic nature of the Ri -group (i.e., Vali) in AIV a requirement for receptor binding and agonist activity?
Four analogues were synthesized and tested. Substitution of Valj with Ilej produced a slightly more hydrophobic peptide (i.e., IYTHPF) as determined by retention on reverse phase HPLC, and this peptide exhibited a slight increase in binding to AT4 receptors. Substitution of Valj with Phe greatly increased hydrophobicity but decreased binding affinity to the AT4 receptor by 4-fold. Surprisingly, substitution of Valj with Lys (i.e., KYIHPF) containing a positively charged side chain, greatly decreased hydrophobicity but increased binding affinity to the receptor by more than 45-fold. Substitution of Val ^ with a negatively charged side chain (i.e., Asp) resulted in an analogue (i.e., DYIHPF) with virtually no affinity for the AT4 receptor.
These results indicate that the nature of the Ri -group (i.e., a rigid aromatic ring versus a flexible aliphatic carbon chain having an optional positive charge) dictates the interaction with the binding site in the AT4 receptor, and not the just the degree of hydrophobicity of the amino acid residue. The results presented in Figures 5A and 5B also indicate that Lysj-AIV (i.e., KYIHPF) exhibits full (or increased) agonist activity relative to AIV (i.e., VYTHPF). Figure 5 shows changes in blood flow that result from binding of agonist Lysj-AIV (i.e., KYIHPF) to AT4 receptors in kidney, without changes in systemic blood pressure. Systemic arterial pressure and cortical renal blood flow were measured as described in Example 3, above. (No. of experiments = 10.) Figure 5 A shows no significant changes in arterial blood pressure following adminstration of KYTHPF at 100pmole/25ml min (open circles) or saline control (closed circles). Figure 5B shows changes in renal blood flow following adminstration of KYIHPF at 100pmole/25ml min (open circles) or saline control (closed circles), with the increased blood flow being equal to 38% of the maximum attainable with a strong vasodilatory agent (i.e., bradykinin, as described above).
Question #4. Does the primary (1°) amine in the N-terminal amino acid interact specifically with the Nal j -binding subdomain in the AT4 receptor binding site? As described above in response to Question #2, IleiYIHPF binds to the receptor with nearly the same binding affinity as NYTHPF. Methylation of Ilei in the latter peptide (i.e., to form Ν-methyl-IleiYIHPF) reduced bindng affinity for the AT4 receptor by 67-fold. Substitution of a secondary amine into the R^ position of AIV (i.e., ProiYTHPF) reduced binding affinity to the AT4 receptor by 8-fold. Substitution of Rj with benzoic acid (a partial structural analogue of Phe) or with 6-amino hexanoic acid (a structural analog of Lys) produced peptides with Kj's >lmM. Placement of GABA (gamma-amino-butyric acid) in the Ri position decreased binding by 250-fold, i.e., relative to binding with AIV. This data suggests that the receptor contains a binding site sub-domain that closely interacts with the primary amine function in the Ri residue with respect to absolute space occupancy (volume) and probably a electrostatic charge, i.e., the receptor non-planar ΝH3-binding component of the Ri -binding sub-domain (the same non-planar sub-domain component described in response to Question #1 above), most likely is a negatively charged residue that resides adjacent to the l°-amine when the Rj group is engaging the receptor sub-domain.
Question #5. Is the positive charge of the e-amine in Lysj responsible for the increased binding affinity of KYIHPF to the AT4 receptor, or is this property attributable to the flexible, linear carbon chain? Four different Ri position AIV analogues were synthesized to answer this question: 1) Lysi-substituted AIN (i.e., KYIHPF); 2) norleucine-substituted AIN,
(i.e., ΝLerYIHPF); 3) ornithine-substituted AIN (OrnrYIHPF); and, 4) norvaline- substitubed AIN (i.e., Νvai-YTHPF). The chemical structures of these side chains are shown in Table 10. TABLE 10
Chemical Structures of Aliphatic Carbon Side Chains
Lys Νle Orn Νva
CH CH? CH2 OH?
I I I I
CH2 CH CH2 CH2
I I I I
C i-b CH? ( -t-b CH3
I I I ©
CH2 CH3 ΝH3
I Θ
NH3 NVa-substituted AIV had a 4-fold greater affinity for the AT4 receptor than Orn-substituted AIV. Nle-substituted ATV had a remarkable binding affinity 60-fold higher than Lys-substituted AIV: i.e., NlejYIHPF had a Kj of <1 x 10"12M, a virtually irreversible binding ligand and indicative of partial-agonist activity. To confirm the agonist activity of Nle-substituted AIV, studies were conducted to evaluate the ability of this analogue to stimulate maximal arterial blood flow in rat renal arteries. The studies were conducted as described Example 6, above. Infusion of 0.10 picomoles/minute of Nlei YIHPF into the rat renal artery produced the effect of maximal blood flow, however, the absolute levels of flow stimulated by this analogue were less than the absolute levels produced by ATV or Lysj YIHPF, indicating that Nlei YTHPF is a partial agonist Figures 6A and 6B, described below. Figure 6A shows changes in arterial blood pressure following adminstration of NorLeu YTHPF at 100pmole/25ml/min (open circles), 50pmole/25ml min (open squares) or saline control (closed squares). Figure 6B shows changes in renal blood flow following adminstration of NorLeu YTHPF at 100pmole/25ml min (open circles), 50pmole/25ml/min (open squares) or saline control (closed squares). The infusion of 0.05pmole NorLeui THPF had no effect on mean arterial pressure (Figure 6A) but increased renal blood flow in a dose-dependent manner: a maximum of 19% increase in renal blood flow was observed with infusions of 0.05pmole (Figure 6B); 19% also at O.l pmole (Figure 6B); 21% at lOpmole (Figure 6B); and, lOOpmole NorLeui YTHPF increased renal blood flow by 30% (Figure 6B). (Infusion of 0.15M NaCl in control animals were without any significant effect.)
The data indicates that a flexible, linear carbon chain interacts specifically with the receptor in a high affinity manner; chains having a four carbon atoms bind with a higher affinity than chains with three carbon atoms; a positive charge is deleterious to binding, but does provide an analogue having full agonist activity (i.e., Nlej-AIV). Question #6. What is the specificity of the receptor for the R2 residue? Analogues were prepared with D-tyrosine substitution for L-tyrosine in the R2 position of AIV (i.e., D-Tyr2 AIV). The latter analogues exhibited low binding affinity for the AT4 receptor. Reversal of the positions of the Phe and Tyr residues in AIN (i.e., Phe2Tyr6 AIN; NPIHYF) also resulted in analogues that had very low binding affinity.
These results suggest strict recognition of the R2 Tyr residue, possibly through hydrophobic and hydrogen-bonding interactions. Substitution with Phe, Ala, and beta-alanine is useful to map the nature of the interactions with this sub-domain of the AT4 receptor binding site. Question #7. Will the receptor tolerate the introduction of non-peptide bonds?
Compounds were synthesized with methylene bond isosteres (i.e., (-CH2-NH-) to answer this question. The synthesis was accomplished using the racemate free amino aldehyde synthesis, Schiffs base formation, and reduction with sodium cyanoborohydride. Specifically, synthesis of +H3N-Nal(CH2NH)Tyr-Nal(CH2NH)- His-Pro-Phe-COO" (designated divalinal AIN) was accomplished utilizing standard solid phase protocols with t-Boc protected amino acids and amino aldehydes. The same general protocol is used to produce other AIN ligands with methylene bonds between desired amino acid residues using the appropriate amino acid aldehyde as a reagent. R-group protection was: Tosyl for His and 2,6-dichlorobenzyl for Tyr. Synthesis occurred on a t-Boc-Phe substituted resin (0.76mmol gram of 1% cross- linked divinyl benzene resin from Peninsula).
For amino acid coupling the following protocol was used: methylene chloride wash: 1X1 min; 45% w/v trifluoroacetic acid and 0.08% indole in methylene chloride deprotection: 1X3 min and 1X30 min; methylene chloride wash: 5X1 min; isopropanol wash: 3X1 min; methylene chloride wash: 3X1 min; 10% v/v triethylamine in methylene chloride neutralization: 1X1 min and 1X5 min; methylene chloride wash: 2X1 min; isopropanol wash: 2X1 min; methylene chloride wash: 2X1 min; isopropanol wash: 2X1 min; methylene chloride wash: 3X1 min; amino acid coupling with a 2.5 or 5-fold excess of amino acid and EDC in methylene chloride: reaction times of 1.5 to 3.5 hours; methylene chloride wash: 3X1 min; isopropanol wash: 3X1 min; methylene chloride wash: 3X1 min. The above protocol was repeated for each cycle. Re-links of amino acids repeated all steps beginning with the neutralization. All linkages and deprotections were monitored with the Kaiser ninhydrin test. Acylations less than 94% were repeated.
Nalinal (Ν-t-Boc-L valine aldehyde from Peninsula) was linked to the free amino-terminal of the growing peptide by formation of a Schiffs base intermediate with subsequent bond reduction. For this reaction the above protocol was utilized with the following alterations: prior to coupling, the resin was washed with dimethyl formamide 3X1 min; a 5-fold excess of valinal was added in 1% acetic acid/dimethyl formamide; a 10-fold mole ratio excess of sodium cynoborohydride (Sigma) was dissolved in 3 ml 1% acetic acid dimethyl formamide and added in equal aliquots at 0,3,5,10,15,20,25,30,40 and 50 min with concurrent nitrogen purge; the coupling was allowed to continue for 70 additional min; the resin was washed with dimethyl formamide 3X1 min. Linkage was assessed with the Kaiser test and revealed a slightly reddish color of the beads when greater than 94%.
The finished N-terminal deprotected resin-linked peptide was cleaved from the resin and side chain deprotected with anhydrous HF containing 10% anisole at 0°C for 40 min. The HF and anisole were removed under vacuum and the peptide washed with anhydrous ether. The peptide was extracted with 20% glacial acetic acid and lyophilized. The crude peptide was then purified by preparative reversed phase HPLC in two steps, the first an isocratic method using acetonitrile:triethylamine-phosphate, pH3 followed by a second gradient method using acetonitrile:water (0.1% TFA). The purified product was analyzed by analytical reversed phase HPLC (acetonitrile:triethylamine-phosphate, pH3) gradient method (12-18% over 60 min at 2ml/min).
Replacement of the R1-R2 peptide bond with the methylene bond reduced affinity of binding to the AT4 receptor by 5-fold. Double replacement of both the R1-R2 and the R3-R4 peptide bonds and substitution of the R3 Val with He produced the peptide:
Figure imgf000057_0001
(Divalinal AIV) that had equal or better affinity than ATV for the AT4 receptor. In addition, divalinal AIN has been shown to exhibit enhanced metabolic stability and to be a potent antagonist of AT4 receptor activity. Figure 11 illustrates the comparative stability of 125I-AIN and 125I-Dival AIV following exposure to a membrane fraction prepared from rat kidney. Kidney was chosen as the tissue of study because of its well-known degradative capacity. The metabolish of 125I-ATV and 125I-Dival AIV by rat kidney membranes was determined as follows: Rat membranes (25μg protein) were incubated with .6nM 125I-peptide at room temperature in a buffer containing Tris, 50mM, pH7.4; ΝaCl, 150mM; BSA, 0.1%; EDTA. 5mM; bestatin, 20μM; and Plummer's inhibitor, 50μM. Metabolism was stopped by the addition of acetonitrile (final concentration 50%), and the samples were analyzed by reverse phase (C18) HPLC. As can be seen in Figure 11, ATN is rapidly degraded while Dival AIN remains 100% intact after 4 hr of incubation. In addition, following the procedures of Examples 4 and 6, it has been found that preinfusion with divalinal AIN completely blocks Lys1AIN-induced increases in blood flow, and preinfusion with divalinal AIN actually transforms AIN's effects on blood flow from an increase to a decrease. This effect of divalinal AIN on AIN suggests that AIN also acts at AH receptors, the effects of which are normally masked by AIN's action on AT receptors. Divalinal AIN treatment by itself did not alter blood pressure or renal blood flow (Figure 12A). Additionally, it had no effect on AlV-induced decreases in blood flow (Figure 12B).
It has been further found that AIV potentiates the performance of rats in a passive avoidance task in a dose-dependent manner while All exhibited no specific effect. In this experiment, the mean latency (see ± SEM) for independent groups of rats to reenter the dark compartment following passive avoidance conditioning on Day 1. On Day 1 (5 min prior to testing for retention) the Control Group received 2μl aCSF, angiotensin II (AH), AIV, or divalinal AIV. Each group except the divalinal AIV revealed significant elevations in latency to reenter the dark compartment - comparing Days 1 and 2. In addition, the groups that received lOOpmole or lnmole of AIV indicated a significant elevation in latency to reenter compared with those groups that received aCSF and AH, while these latter groups did not differ from each other. Rats treated with divalinal AIV were not statistically different from preshock controls. Interestingly, treatment of rats with divalinol AIV blocked the typical increase in latency seen in control rats. Responses by rats treated with divalinal AIV were not statistically different than preshock controls. These data indicate that while AIN potently enhanced cognitive function, divalinal AIN acting as an AIN antagonist completely blocks the learning and/or retrieval of the passive avoidance task. Furthermore, these data suggest that endogenous AIN must play a critical role in cognitive function.
These results indicate that the AT4 receptor binding site domain binds analogues in which the peptide bond has been replaced with a non-carbonyl (non- peptidase sensitive) bond that has a similar bond length, and that is non-planar and has a non-rigid carbon-nitrogen bond. Νon-peptide bonds offer pharmacological advantages for a therapeutic composition, i.e., prolonged half-life.
Question #8. What determines agonist versus antgonist activity? Both AIN (i.e., VYIHPF) and LysrAIV (i.e., KYIHPF) exhibit full agnoistic activity, while Νlei-AIN (i.e., ΝleYTHPF) is only a partial agonist. The model capable of explaining this behavior has the following component parts: a) The receptor binding site sub-domain interactions with the side groups (i.e., of Ri) determines receptor activation; b) The interaction at the Rj -sub-domain binding site involves a hydrophobic pocket; c) The space in the latter hydrophobic pocket conforms very closely with the 4 carbon side chain of norleucine; d) Nlej (i.e., in NleiYTHPF) interacts with the hydrophobic pocket without changing the conformation of the pocket; e) Vali (i.e., in VYIHPF) must occupy an "expanded" hydrophobic pocket, i.e., where the receptor hydrophobic pocket is displaced laterally to accomodate the branched carbon side chain in these residues. Lys^ (i.e., in KYIHPF) must similarly occupy an "expanded" hydrophobic pocket because of the charge repulsion from the hydrophobic "walls" of the pocket; and, f) The process of "expanding" the hydrophobic pocket constitutes a molecular trigger for the process transitioning the receptor from the "pre-binding state" to the "binding state".
To study the properties of the "hydrophobic pocket" subdomain of the AT4 receptor binding site it is useful to prepare derivatives of Oπ^ (i.e., Orni YHTPF) at the delta amino group to: a) the charge of the group; b) place a planar, conformationally-fixed bond in the 4 carbon side-chain group that will inhibit binding in the hydrophobic pocket if the "walls" of the pocket are unable to move to accomodate the space required by the conformation; and, c) synthesize conformationally-fixed bonds in carbon side-chains of different length (e.g., 3-5 carbons) to explore the optimal longitudinal dimensions of the flexible wall space in the receptor pocket. Suitable N-delta groups for this exploration are acetate, propionate, benzoic acid, isobutyric acid, and trimethyl acetic acid.
Question #9. Can the shorter peptide AIN(i_4) analogues (e.g., VYTH) be converted to high affinity ligand by norleucine substitution at position Rj?
Answers to this question provide tetrapeptides agonists and antagonists whose interactions with the AT4 receptor are easier to molecularly model, and mimic. The peptides Nle1-ATV(1.5) (i.e., NleYIHP), NlerAIN( ) (i.e., ΝleYIH), and Νleι-ATV(i_3)(i.e., NleYI) may be useful for testing space-filling modifications that can be made to alter binding in the receptor binding site sub-domains. It is considered highly likely that independent modifications that can be made to alter the binding of the latter small Nlei peptides into the AT4 receptor binding site sub-domains will be paralleled when the modification are incorporated into larger AIV ligands.
Question #10. Will substitution of Ilej at position R (e.g., to form VYIHPI, KYEETPI, or NleYIHPI) create antagonist activity?
Three fleg substituted AIV analogues were synthesized (ValιIle6-AIV, Lysιlle6-AIN and Nleilleg-AIN). When tested for in vitro receptor binding activity
Nalιlle6-AIN had a higher binding affinity for the AT4 receptor than AIN (i.e., VYfflPI >VYHTPF); and Lys1Ile6-AIV had a lower affinity than Lys r AIV (i.e., KYIHPI <KYIHPF).
The results suggest that the AT4 receptor binding site is a multi-domain binding site with interactions such that binding in one sub-domain (e.g., within the hydrophobic pocket of the Ri sub-domain) can be excluded by high affinity binding at a distant sub-domain site (e.g., within the subdomain with specificity for the C-terminal Ile6 or Prθ5 residues; i.e., at the R^ subdomain binding site in the receptor). The induced-fit model supplied above in response to Question #8 is compatible with the observed exclusionary binding properties: i.e., binding of R hydrophobic pocket that constitutes the Ri -binding subdomain requires flexibility of expansion in the pocket, and binding of Rg in the R sub-domain binding site confers a rigidity to the receptor that inhibits flexibility in the Rj -binding subdomain. Materials and Methods:
Binding was carried out as described in Example 1, above, in siliconized glass culture tubes containing 0.2nM 125I-AIV, 25 μg of membrane protein, and the desired analogue over a concentration range of 10"12 to lO^M using half-log dilutions. All binding incubations were carried out in duplicate at 37°C for 2 h in a buffer containing: 50mM Tris, 150mM NaCl, 5mM EDTA, lOμM bestatin, 50μM Plummer's Reagent, lOOμM PMSF and 2% BSA (Assay buffer) in a total volume of 0.25ml. After incubation, the incubation mixtures were filtered through glass fiber (GF-B) filters soaked in 0.3% polyethylene-mine and washed with 4-4ml washes of PBS. The filters were then counted on a Beckman 5500 gamma counter. A typical experiment examined 5 analogues simultaneously and included a positive control curve in which non-radiolabeled AIV Ugand was used as the displacer to inhibit binding of 125I-AIV to the AT4 receptor. All samples were run in quadruplicate, each with a different tissue preparation. Data was analyzed by the LIGAND program (29) from which Kj values were obtained. AIN analogues that are peptides were synthesized by the standard Merrifield method utilizing t-Boc protected amino acids and chloromethylated resins on a Nega 250 coupler automated synthesizer (as described in Example 1, above). Following synthesis, the crude peptides were purified by preparative reverse-phase HPLC. The amino acid composition of the purified peptides was determined with respect to both composition and total purity. Typically the peptides used in these studies were greater than 99% pure and contained about 20-25% acetate. EXAMPLE 5
Vascular Effects of the AIV Ligand-AIV Receptor Interactions
In endothelial cells (such as bovine coronary venular endothelial cells), it has been reported previously that these cells may play a critical role in angiogenesis (review, 21). In one study by others angiotensins were reported to be capable of stimulating angiogenesis (22). However, studies in the inventors' laboratory over the past ten years have failed no less than six times to demonstrate detectable levels of
AH receptors in preparations of endothelial cells that were free of smooth muscle contamination (a finding contradictory to one report that ATI receptors may be present on endothelial cells, (23). In addition, All and Sarj leg-AII have been reported to stimulate bovine endothelial cell proliferation (24), but the possible mechanisms were not clear, especially in light of other studies reportedly showing that All and Sarι,Ile8-AII were rapidly metabolized in tissues and biological fluids to smaller metaboUtes. In light of the present disclosure it is now clear, in hindsight, that hydrolysis of All or AHI to AIN can result in binding of AIN to AT4 receptors on endothelial cells with triggering of cell proliferation, and may possibly be involved in the initiation of hyperplastic growth of endothelial cells or vascular smooth muscle cells. AT4 receptors in vascular cells The following study describes the characteristics of a new class of angiotensin binding sites in vascular endothelium that exhibit high specificity and affinity for hexapeptide AIN. Analysis of 125I-AIN binding was performed in membrane fractions of two endothelial cell lines, bovine coronary venular endothelial cells (CVEC) and bovine aortic endothelial cells (BAEC). Kinetic analysis of binding indicated that equilibrium was reached in 60 min. at 37°C, remained stable for at least 4 h, and produced a calculated kinetic K^ of 0.3nM. Saturation equilibrium binding studies analyzed by non-Unear curve fitting suggested the following two site models (mean +/- SEM): CVEC Kdl=14.6 +/- 26.5pM, B^! = 6 +/- lfinol mg protein, K,j2=4.4 +/- 0.8nM, Bmsx2 — 434 +/- 51 fmol/mg protein. Compteition binding curves from CVEC demonstrated high specificity of the receptor for for AIV. The competitive binding affinities of analogues to the receptor showed affinites that (in decreasing order) were ATV >AII(3_7) >AHI >AII >SarιIle8-AII, or AII(4_8) »DUP 753, or CGP42112A. The AT4 receptor in endothelial cells may not be G-protein linked because the non-hydrolyzable GTP analog GTPγS had no effect on 1 5I-AIN binding to receptors in BAEC cells. These data indicate that AIN binds to a site in vascular tissues that is distinct and separate from the classic ATI or AT2 angiotensin receptors. Kinetic binding studies
Kinetic analysis of 125I-AIN binding to AT4 receptors in CVEC membrane revealed that equilibirum was reached in approximately 60 min. and remained stable for at least 4 h. at 37°C (Figure 7A). The kinetic properties of 1 5I-AIV binding to AT4 receptors in membrane fractions of bovine coronary venular endothelial cells (CVEC) at 37°C. are shown in Figures 7 A and 7B. The association (Figure 7 A) and dissociation (Figure 7B) rate constants for binding of 0.6nM 125I-AIV were 9.3 x 107 M^min"1 and 0.028 min"1, respectively. The kinetic Kd calculated from these rate constants is 0.3nM. Calculations were performed by the LIGA D program from a mean of four experiments with duplicate samples. Data presented here represent the results from a single experiment.
Analysis of association data under pseudo first order rate conditions resulted in an observed association rat constant (1^)= 0.084 +/- 0.013. The dissociation rate constatnt (k_ι)= 0.028 +/- 0.005 min-1 was estimated by the addition of lmM undlabeled ligand following incubation of 0.6nM 125I-AIV for 120 min at 37° (Figure 7B) and the actual association rate constant (k ) was calculated to be 9.3 x 107 M^min"1. Based on these rate constants, the apparent kinetic K value is 0.3nM. Less than 10% degradation of the Ugand occurred under the binding conditions used here. Equilibrium binding studies
As shown in Figures 7A and 7B, quilibirum binding of 125I-AIV to AT4 receptors in CVEC and BAEC membranes reached saturation at 37°C in 120 min. Equilibrium saturation binding and Scatchard transformation analysis (insert) for 125I-AIV binding to AT4 receptors in CVEC is shown in Figure 7 A, and BAEC in Figure 7B, membrane fractions after 120 min. at 37°C. The data were best fit by a two site model utilizing the non-linear curve fitting program LIGAND (No. of expts.=4, each with duplicate samples). Scatchard transformation of these data suggested the presence of multiple binding sites in endothelial cell membrane-associated AT4 receptors. The data were best resolved into two componeents corresponding to a high and a low affinity binding site. The final Kd and B-JJ-^. values were as follows:
In CVEV (Figure 7A): site #1 14.6 +/- 26.5pM with 6 +/- lfmol/mg protein; site #2 1.4 +/- 0.2nM with 594 +/- 4fmol/mg protein; and, In BAEC (Figure 7B): site #1 26.9 +/- 9pM with 10 +/- 2fmol/mg protein; site #2 4.4 +/- 0.8nM with 434 +/- 51fmol mg protein.
Values obtained when fitting the data to a single receptor affinity site model were: in CVEC 0.7 +/- O.lnM Kd with Bmax=10 +/- 2fmol/mg protein; and in BAEC 1.0 +/- 0.2nM with Bmax=260 +/- 38fmol/mg protein. The two site model produced a significantly better fit for both cell types as compared with the single site model (i.e., F-test, p<0.001). Competition binding studies
Competition studies were conducted to displace 125I-AIV binding to AT4 receptors in CVEC membrane preparations with specific ligand, i.e., AIV, and other related angiotensin fragments. Figure 8 shows competition displacment curves delineating the abUity of angiotensin fragments to inhibit specific binding of 0.5nM 125I-AIV to AT4 receptors in membrane preparations of bovine coronary venular endothelial cells (CVEC). (No. of exper. =2; each conducted with duplicate samples.)
The rank order affinity of competitive analogues competitively displacing bound AIV from its receptor were as follows: ATV >AIIπ.7) > AIII >AII >Sar1Ile8-Aπ, or Aπ(4.8) »DUP 753, or CGP42112A. (For sequences of All analogues see Figure 1). The results of these studies are summarized in Table 11.
TABLE 11
Competition of 125I-AIN binding to AT4 receptors
Figure imgf000063_0001
* All values represent mean +/- SEM of two experiments with duplicate samples; Kj determined by LIGAND.
G-protein linkage of the AT4 receptor in vascular cells
G-protein interactions with vascular angiotensin receptors are shown in Figure 9, where membrane fractions from rat vascular smooth muscle cells (RVSMC) or bovine aortic endothelial cells (BAEC) were preincubated in various concentrations of a non-hydrolyzable GTP analogue (i.e., GTPγS) for 60 minutes at 22°C prior to use in equilibrium binding assays with 0.5nM 125I-AII (RVSMC) or O.όnM 125I-AIV (BAEC). (No.l of exper. =3; each with duplicate samples.) Data presented here represent results from a single experiment.
Addition of non-hydrolyzable GTP (i.e., GTPγS) to the binding assays did not inhibit (or alter) binding of 125I-AIV to AT4 receptors in BAEC membrane preparations (Figure 9). In constrast, in a positive control GTPγS inhibited 125I-AII binding to ATI receptors in rat vascular smooth muscle cell (RVSMC) membrane preparations in a dose-dependent manner (Figure 9); in agreement with observations reported previously by others. (This property distinguishes AT4 receptors of the invention from ATI and AT2 receptors reported by others previously in vascular tissues.) Discussion
This study is the first to describe a novel angiotensin binding site in vascular endothelium that exhibits high affinity and specificity for the hexapeptide AIV fragment of angiotensin AIL The AT4 receptor is distinct from the ATI or AT2 receptors in vascular tissue. Analysis of the binding characteristics indiactes that the AT4 receptor binds AIV in a saturable and reversible manner, and that 125I-AIV reaches equilibrium in binding to the AT4 receptor in membrane preparations in approximately 60 min. at 37°C. Binding of AIN to its receptor remains stable for at least 4 h (Figure 7 A) with less than 10% degradation of the ligand under these binding conditions. Scatchard analysis of the AT4 receptor binding site by the non-linear curve fitting program LIGAND reveals two components to the binding data. The first component is a high affinity component that exhibits Kd's of 14 and 27pM with Bmax'S of 6 and lOfmol/mg protein for receptors in CVEC and BAEC membrane preparations, respectively. (Because of the extremely low number of these high affinity sites it is unclear at present whether this is a physiologically important state of the receptor; or, is a result of modification of AT4 receptors in the membrane preparations, or changes in receptor binding affinity resulting from co-operative binding of ATV; or alternatively, that this site is an artifact created in the membrane preparations or assay conditions.) The second binding component is a lower affinity component with K^s of 1.4 and 4.4nM (i.e., in CVEC and BAEC, respectively). The second component displays a high concentration of Ugand binding commensurate with large numbers of such receptor sites in the membrane preparations: i.e., these sites bind 594 and 434fmol/mg protein in CVEC and BAEC membrane preparations, respectively. The overall binding affinity (i.e., Kd, single or composite site fit produced by LIGAND) was calculated to be 0.7nM for CVEC and l.OnM for BAEC. These results are in good agreement with the Kd calculated from the results of kinetic binding studies (0.3nM). The pharmacological profile derived from competition displacement of 125I-
AIV bound to these AT4 receptors in vascular tissues is presented in Figure 8 and Table 11, above. This profile reveals a strict structural requirement for the N-terminus of the AIV ligand, i.e., removal of the N-terminus (Vali) of the ATV ligand results in a 200-fold decrease in affinity of the AIN ligand for the AT4 receptor in vascular tissues (i.e., an increase in the Kj). In addition, Ν-terminal extension, i.e., beyond Nali, is detrimental to the binding of AIN ligands to the vascular AT4 receptor as indicated by the inability of All and Sarj -es-AII to competitively inhibit binding of AIN to the AT4 receptor, (i.e., note the 200-fold increase in Kj seen with All and Sar^fleg-AH, when compared with AIN in Table 11). (This property distinguishes AT4 receptors of the invention from ATI and AT2 receptors.) The apparent affinity of AIII for the vascular AT4 receptor (i.e., 20-fold higher Kj than AIN, Table 11) may be an artifact of Ν-terminal metabolism of AIII to form AIN in these membrane preparations. (In previous studies, above, 125I-AHI binding to bovine adrenal AT4 receptors was directly proportional to the amount of Am hydrolyzed to AIN.)
The vascular AT4 receptor appears to exhibit less specificity for the C-terminus than exhibited for the Ν-terminus: i.e., the AIN(i_7) fragment (with the C-terminal Phe8 deleted still bound with reasonable affinity to the receptor (i.e., only a 7-fold increase in Kj over AIN). (These findings are in agreement with the findings above in Example 1 using AT4 receptors in bovine adrenal cortical tissues.)
The vascular AT4 receptors do not apparently bind either DUP 753 or CGP 42112A (i.e., Kj >10"4), but ATI or AT2 receptors are well-known to do so (Timmermans, P. et al. ΗPS 12:55-62, 1991; Whitebread, S. et al. Biochem. Biophys. Res. Comm. 163:284-291, 1989). (This property of failure to bind either DUP 753 or CGP 42112A distinguishes AT4 receptors of the invention from ATI and AT2 receptors.)
Binding of 125I-AIN to vasular endothelial AT4 receptors was not sensitive to inhibition by guanine nucleotides. In contrast, binding of All to ATI and AT2 receptors in membrane preparations of rat vascular smooth muscle cells (RNSMC; Figure 9) was sensitive to inhibition by guanine nucleotides in a dose-dependent manner, i.e., the affinity of the ATI receptor for AH was shifted to a lower value when the receptor was uncoupled from G-proteins by the presence of the GTP analogue GTPγS (Figure 9). This shift in binding affinity in response to gunaine nucleotides is a characteristic of the high affinity form of the ATI receptor (Glossmann, H. et al. J. Biol. Chem. 249:664-666, 1974). The insensitivity of the AT4 receptor to G-protein uncoupling agents was also observed with AT4 receptors in membrane preparations of bovine adrenal cortex. (This property of insensitivity to G-protein uncoupling agents distinguishes AT4 receptors of the invention from ATI and AT2 receptors.)
Despite the inability of AIV to bind to AH receptors, several recent studies have suggested that that ATV-like fragments of All may have unique biological attributes. In cultured chick myocytes, AIN-like fragments of AH have been reported to antagonize the effects of All-induced increases in cytosolic free calcium, protein synthesis, and hypertrophic cell growth while being unable to competitively inhibit for 125I-AII binding (Baker, K.M. et al. Am. J. Physiol. 259:H610-H618, 1990). Topical application of both All and AIN-like fragments of ATI have been reported to mediate endothelium-dependent vasodilation in rabbit brain arterioles. However, in the presence of the amino peptidase inhibitor amastatin, the vascular response to All, but not AINlike fragments, was reportedly blocked (Haberl, R.L. et al. Circ. Res. 68:1621-1627, 1991). AIN-like All fragments and All have also been reportedly applied intracerebroventricularly in the rat where they reportedly are equipotent in enhancing memory and learning (Braszko et al. Brain Res. 542:49-54, 1991). Given the low affinity of AIN for ATI and AT2, disclosed herein, it is most likely that the latter activities previously attributed to binding of All and/or AIN-like fragments at ATI and AT2 sites are, in fact, the result of binding of AIN at the AT4 receptor sites of the invention.
It is likely that the actions evoked by AIN binding to its specific AIN recepotrs may act contrary to the actions of the All and ATI and AT2 receptors. For example, infusion of AIN into rat kidney, as shown above, to stimulate a significant increase in blood flow in the renal cortex, while All binding to ATI and AT2 receptors in these tissues produces the converse effect - a significant decrease in blood flow. Effects on Nascular Tissues:
Assessment of AIN effects on the contractile properties of aorta and inferior vena cava was demonstrated using tissues from rabbits. The presence of numerous AT4 receptors in aortic tissue suggest a possible action of AIN ligand on cerebral vessels. The routine use of rabbit aortic strips or rings in cardiovascular pharmacology dictate that rabbits are suitable for use in such studies. The following protocols are useful for: 1) confirming the vasodilating potential of an AIN ligand, demonstrating that ligand action is dependent on an AT4 receptor, and showing that the action is independent of Al or All receptors; 2) establishing that any observed vasodilation is endothelium dependent; 3) determining whether the mechanism of vasodilation involves prostaglandins, EDRF, or other factors like EDHF as second messengers; and 4) determining the functionality of the many AIN analogues (i.e., such as those synthesized in Example 4) as either AIN ligands or as agonists, antagonists, inhibitors, or promoters of the AIN ligand-receptor interaction. AIN and ATI ligands and various analogues (Example 4) in the presence or absence of angiotensin inhibitors (e.g., SarlsIle8-AII, DUP 753, and CGP42112A) were screened for the vasodilating activity using rabbit aorta and inferior vena cava rings or spiral strips suspended in 20ml organ baths containing Krebs solution at 37°C and continuously gassed with 5% CO2 in oxygen. After a 1 h equilibration period, cumulative dose-response curves were constructed for the analogues over a concentration range of 10"10M to 10"5M. In relaxation studies, aortic strips were pre¬ contracted to 70% of maximum diameter with phenylephrine, and then the test ligand is added and relaxation of the vessel is quantified. Changes in contractile or relaxant response may be calculated for each dose of each different ligand or analogue and subsequently analyzed by analysis of variance. Effects on Endothelial Cells:
The effect of AIN ligand on endothelial cells was examined by measuring growth of bovine endothelial cells. Cells were grown at 37°C in 35mm culture plates CO2/air under 5% CO2/95% air in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5 μg/ml insulin and 10% (v/v) newborn bovine serum (ΝBBS). The test medium was supplemented with 3H-thymidine and either All ligand (50nM) or AIN ligand (50nM) or lOng/ml acidic or basic FGF (as a positive control). Negative controls were also included using ethidium bromide (lmM). The cells were harvested at various times, and cellular lysates were prepared for scintillation counting by lysing and washing the cells on glass fiber filters. Materials and Methods: Reagents
ATV (VYIHPF), AII(3_7) (VYHTP), and AII(4.8) (YIHPF) were synthesized as described in Example 1, above. All reagents and other peptides were obtained from Sigma Chemical Co., with the exception of: Plummer's inihibitor (Calbiochem); bestatin (Peninsula Biochem); DUP 753 was a gift from Dr. Ron Smith of Dupont Merck and CGP 42112A was a gift from Dr. Marc de Gasparo of Ciba- Geigy. Angiotensin fragments numbering was based on the sequence of All (Figure 1). Cell Culture Bovine coronary venular endothelial cells (CVEC) were isolated by a bead- perfusion technique and characterized as described previously (Schelling, M.E. et al. Am. T. Physiol. 254:H1211-H1217, 1988). Bovine aortic endothelial cells (BAEC) were a gift from Dr. Stephen Schwartz (University of Washington). Cells were grown in 100mm tissue culture plates (Falcon, Becton Dickinson Co.) coated with 1.5% gelatin in PBS (per liter of distilled water: 8.12g NaCL, 1.14g Na2HPO4, 0.28g NaH2PO4) in Dulbecco's modified Eagle's medium (DMEM; FLOW Labs) supplemented with 2mM sodium pyruvate, 2mM L-glutamine, lOOmg/ml heparin, lOOmg/ml Penicillin-G, 50mg/ml Streptomycin, 44mM NaHCO3, and 10% fetal bovine serum (GIBCO). Cells were passaged 1:3 by tryptic digestion (0.05% trypsin, 0.025% EDTA in
Figure imgf000068_0001
PBS, pH7.4 at 37°C). All data collected in this study was from cell lines passaged between passage 5 and passage 9. Tissue preparation
Cells were grown to confluence in 100mm culture dishes. Dishes were washed once in Ca++/Mg++-free PBS, pH7.4 at 37°C follwed by the addition of 2ml of cold isotonic assay buffer (150mM NaCl, 50mM Tris, ImM PMSF, lOμM bestatin, 50μM Plummer's inhibitor, pH7.4 at 4°C). Cells were then removed from the plates with a rubber policeman and homogenized in 5ml assay buffer for approximately 10 sec (Polytron, Brinkman Inst. Co.). Cell extracts were centrifuged at 40,000 x g for 20 min at 4°C, the supernatant was discarded and the pellet was rehomogenized in assay buffer and centrifiigation was repeated for a total of two high speed centrifugation steps. The final pellet was resuspended in assay buffer to a working concentration of approximately 5mg/ml as determined by the method of Lowry (J. Biol. Chem. 193:265-267, 1951). Iodination of AIV AIV (and other peptides) were iodinated using an immobilized lactoperoxidase-glucose oxidase system (Enzymobeads, Biorad Laboratories) to a specific activity of 2176Ci mmole. 125I-AIV was separated from unlabeled peptide by HPLC (Beckman) using a reverse phase Cι8 column (5mm x 250mm; Adsorbosphere, Alltech, Associates). Receptor binding assays
Binding assays were performed at 37°C in a total volume of 250ml (isotonic buffer, pH7.4 at 37°C). Bound and free ligand were separated at the conclusion of each experiment by the addition of ice-cold PBS (pH7.4), and separation of bound from free was achieved by 4 vacuum filtration washes with 4ml of this buffer (Schleicher and Schuell #32, Brandel Cell Harvester). Radioactivity retained by the filters was determined using a Tracor Analytic gamma counter, model #1185 having 68% counting efficiency. Nonspecific binding was ascertained in the presence of ImM unlabeled AIV. Kinetic binding experiments (N=3) were performed at 37°C over a time course of 240 min with 11 time points and duplicate samples. The apparent pseudo-first order association rate constant k^ was deterimined by the non-linear curve fitting program LIGAND. Dissociation experiments (N=4) were conducted at 37°C by preincubating cell extracts for 120 min with 0.5nM radiolabeled Ugand followed by the addition of ImM unlabeled Ugand (final cone). Binding was determined for duplicate samples representing 10 time points over 180 min. The apparent dissociation rate constant, k.j, was determined by LIGAND. The apparent association rate constant, ki, was then calculated from the equation - k_ι)/[L], where [L] is the radioUgand concentration, and the apparent kinetic equilibrium dissociation constant, Kd, was derived from the equation Kd = k_ι/kj.
Saturation equilibrium binding and competition displacement studies (with CVEC, N=4 expts., 46 total data points; BAEC, N=3, 34 data points) were conducted over 120 min. of incubation in the presence of increasing concentrations of radioUgand or competing Ugands, respectively. Saturation data were analyzed by LIGAND for the determinations of maximum number of binding sites (Bmax) and Kd.
For determining the Unkage of G-proteins to the AT4 receptor, membrane preparations were first preincubated in GTP assay buffer (50mM Tris, 150mM NaCl,
5mM MgCl2 ImM EGTA, ImM PMSF, 50μM Plummer's inhibitor, lOμM bestatin, pH7.4) at 22°C for 60 min in solutions of GTPγS calculated to produce a final concentration in the assay of lOOmM, lOmM, lOnM and 0 GTPγS. The rat vascular smooth muscle cell line WKY IN passage #17, was included as a positive control for G-protein linkage to ATI receptors. All data are presented as the mean +/- SEM, standard error of the mean. Endothelial cell growth and the effects of AIN ligand on EDRF production Bovine aortic endothelial cells were grown at 37°C in 35mm culture plates under 5% CO2 in air in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5μg/ml insulin and 10% (v/v) newborn bovine serum (NBBS). The medium was aspirated 10-12 hours after seeding and replaced with serum-free medium. The medium was again aspirated 10-12 hours later and replaced with either test or control medium. Control medium was DMEM with 5g/ml insulin and 2%, 5%, or 10% (v/v) NBBS as indicated. The test medium was supplemented with either All or AIN ligand ± the antagonist Sarι,He8-AH at various concentrations. The medium was changed every 48 h (i.e., with supportive DMEM medium for the remainder of the experiment).
For measurements to determine the effects of AIV in stimulating an increase in endothelial cell numbers, cells can be harvested on various days during the culture period by washing the plates with calcium free medium (CMF) two times for 5 min. followed by incubation in 0.1% trypsin in CMF for 5 min. The cells can then be washed free from the plate and aspirated by Pasteur pipet into 15ml centrifuge tubes containing 3 ml DMEM with 20% (v/v) ΝBBS. The plates can be washed with an additional 1ml DMEM 20% ΝBBS which was transferred to the appropriate centrifuge tube and spun at 300 x g for 10 min. Excess medium was aspirated and the pellet resuspended in a final volume of 1ml of the control medium. Aliquots can then be counted using a hemocytometer and cell number expressed as cells/plate.
As an adjunct to the determination of cell numbers, thymidine incorporation was measured. For quantitation of DΝA synthesis [methyl-3H]thymidine (60Ci/mmol, lOmCi per plate) was added to cultures 12 h after addition of the All or AIN. Twelve h later, medium was removed and 1ml of a 1% aqueous solution of Triton X-100 was added. The cells were incubated with this solution for 5 min. and the entire contents of the plate transferred to 10ml of absolute ethanol. This material was then filtered under vacuum through 2.4cm glass fiber filters (GF/A, Whatman), and the filters were washed twice with 10ml of absolute ethanol and assayed for radioactivity by scintillation counting.
EXAMPLE 6 Physiological Function of Angiotensin IN Receptor and Ligand Angiotensins Al, AH, and Am are reported to have a wide variety of effects on target issues, some of which are acute while others appear more long-term. All reportedly has a cellular effect of increasing c-fos levels in cultured vascular smooth muscle cells (17), and c-fos is reported to be one common pathway for triggering cell growth. Considering the widespread distribution of AT4 receptors in many organs and tissues (EXAMPLES 1 and 2, above), it is Ukely that AIN has multiple functions, including long-term effects on cells by triggering increased expression of c-fos, i.e., activities previously mistakenly attributed to All and AHI.
The following studies focus on the role that the AIN ligand-receptor system may play in three organs enriched in AT4 receptors: blood vessels, kidney, and adrenal glands. (Other organs such as brain or heart which also possess high levels of specifically localized AT4 receptors can be studied in a similar manner.) Renal Blood Flow: The AIN Receptor and All Receptor Have Physiologically Distinct and Opposing Activities:
Physiological studies, described below, investigated the involvement of AIN ligand in the regulation of renal blood flow. The rationale for initially choosing to examine the kidney was at least two-fold. First, the AT4 receptor is found in high concentrations in kidney and endothelial cells (Example 1 and 2, above). Second, vascular endothelial cells are reported to regulate vascular tone and to play a role in the control of renal blood flow. Superficial blood flow in the rat kidney was assessed using laser doppler methods in anesthetized rats following direct infusion of a test substance into the renal artery. The results are presented in Figure 4 which depicts the percentage change in cortical renal blood flow following infusion into the renal artery of 25μl min of a 0.15M ΝaCl solution containing 100pmol 25μl AIN (closed circles; number of experiments (n)=13); 0.15M saline (open circles; n=9); 100pmol 25μl of AIN lacking the Ν-terminal Nalj residue (i.e., YTHPF; D-Nali; closed squares; n=9); and 100pmol/25μl of AH (open squares; n=8). The infusion of experimental compounds and saline had no effect on systemic arterial blood pressure (see results in Example 4). The infusion of AIN (closed circles) show that AIN ligand infused at lOOpmol min. stimulates a profound and long-lasting increase in blood flow. In contrast, infusion of AH (also at lOOpmol/min.; open squares Figure 4) produced a dramatic decrease in renal blood flow. The AIN analogue d-Nali-AIN (i.e., lacking the Ν-terminal valine and lacking binding activity for the AT4 receptor, see Example 1, above) had no effect on renal blood flow (closed squares; Figure 4). The experimental protocols employed in these studies is detailed in the
Materials and Methods, below. Materials and Methods: Experimental Protocol #1 :
For comparison of AH, AIN, d-Na^ AIN and saline infusion on renal blood flow, the respective agents were infused into the renal artery at lOOpmol/min. for 10 min. at 25μl/min. Saline and the AIN analogue d-Nalj AIN were included as controls, i.e., the number of experiments= 8; average standard error of the mean (SE) = ± 3% change blood in flow. As expected, saline and d-Nalj AIV had no effect on renal blood flow. Also, as expected, AH produced a dramatic decrease in flow followed by an autoregulatory return toward baseline. AIV produced an equally dramatic increase in flow that showed little autoregulation.
Consistent with the involvement of different receptors in the mediation of AH and AIV effects, the specific AH antagonist Sarj.Ileg-AII (lnmol min - 10 min. pretreatment) completely blocked the AH effect while having no effect on AIV. The decrease in blood flow witnessed with AIN was dose dependent and was not accompanied by alterations in mean arterial pressure, suggesting that the effects of the AIN ligand-receptor system may be limited to selective vascular beds or that compensatory changes in cardiac output occurred during AIN infusion. Experimental Protocol #2:
The AIN-induced increase in renal blood flow was not blocked preinfusing AIL Sarι,Ile8-AII was infused over the 10 min. immediately prior to infusion at lnmol/min., and a comparison was made with the change in blood flow that occurred when AIV ligand was infused without the All preinfusion. In 8 experiments an average change in AIN-induced blood flow of <3% was recorded with the All preinfusion, which was within the standard error of the experiments, i.e., SE = ± 3%. Thus, as predicted from the competition binding studies conducted above (Example 1), Sarι,Ile8-AII was unable to block the vasodilatory effect of AIN ligand. When tested in control experiments for the ability of AIN Ugand to block All- mediated decrease in blood flow (i.e., in the same type of preinfusion experiment, but using AIN preinfusion instead of AH). AIN Ugand completely blocked the constrictive action of AH. Therefore, the results support the notion that AIN may antagonize certain of the actions of AH.
Effects of ATN-Ligand-Receptor Interactions on Renal Functions Results presented above demonstrate that intravenous application of AIN ligand can dramatically increase renal blood flow and urine flow in a dose-dependent fashion. This effect appears to be mediated by the AT4 receptor and not by nonspecific, nonreceptor-dependent processes. Neither All nor d-Vali AIV (a nonbinding ATV analogue) could reproduce the effects of AIN ligand, and the specific All antagonist Sarl5Ile -AII was unable to block the action of AIN ligand.
Another assessment of the AIN ligand-receptor effects on renal functions was provided by analyzing distribution of radio-labeled insulin and p-aminohippicuric acid; in combination with measurements of urine flow, urine osmolality, urine Νa+ and K+, and hematocrit. The effects of AIV ligand, All, and other AIV analogues were determined, i.e., a) on renal blood flow, b) glomerular filtration rate, c) osmolal clearance, d) filtration fraction, and e) tubular function. Dose-response curves for AIV ligand and AH ligand were constructed in the presence and absence of the AH antagonist Sar^Iles-AII. In addition, AIV analogues with special in vitro properties (e.g., AIV antagonists, AIV superagonists, or metabolically resistant analogues of AIV) were tested in a similar manner (above) to determine their effects on renal function. Studies were carried out as acute preparations in anesthetized rabbits and using jugular and urethral catheters. EXAMPLE 7
Neurological Effects of the AIV- AIV Ligand-Receptor Interaction Local Effects:
Given the presence of AT4 receptors in the brain (Example 2, above; Figures 6-10) and most likely in cognitive and motor memory and learning centers (i.e., hippocampus, frontal cortex, cerebellum, and thalamus), and in areas within the hindbrain cardiovascular nuclei involving the tractus solitarious, it is reasonable to suspect that at least in some tissues AIV Ugand is produced locally in neural tissues, i.e., by synthesis of Al and conversion to ATV. Two scenarios of local production can be envisioned. In the first, ATV ligand is produced locally from precursors synthesized in the tissue. In the second, circulating ATV precursors (e.g., AT, All or AHI) are converted locally to AIV ligand. Whether the first or second scenario is an operative mechanism in a particular tissue can be determined by introducing radiolabeled precursors (i.e., 125I-AI) into the bodily fluid bathing the tissue (e.g., plasma or CNS fluid), and by then collecting samples of the fluid at different times and assaying by reverse-phase HPLC to determine if the AIV precursor has been converted to ATV ligand in the fluid. If it has been converted, the second scenario is operative; if it has not been converted a second series of experiments is conducted. In the second series of experiments biosynthesis of ATV precursors is evaluated (i.e., with radiolabeled amino acids) and conversion of the precursor into AIV ligand is examined in pulse-chase type experiments. If biosynthetically radiolabeled AIN precursor chases into AIN ligand, then the first scenario is operative in the tissue. Changes in the AIN-Ligand-Receptor System in Response to Neurological Effects:
A representative experimental protocols for showing changes in the AIN- ligand-receptor system in response to neurological and physiological effects is described in the Materials and Methods, below. AT4 receptors in brain:
A comparison was made of the binding affinities (under equilibrium binding conditions) of AT4 receptors in different regions of guinea pig brain. The results of Scatchard analysis of binding data (conducted in the manner described above in Example 1) are summarized in Table 12, below.
TABLE 12 Bindin of AIN in Regions of Braina
Figure imgf000074_0001
a.) mean +/- SD; no. of experiments =4 b.) HSTA= hypothalamus, thalamus, septum, antereoventral third ventricular area. The Hippocampal AIN Ligand-Receptor System:
Hippocampal AT4 receptors identified in tissues by receptor autoradiography in Example 2, above, were evaluated further by isolating hippocampal membranes (i.e., including hypothalamus, thalamus, septum, anteroventral third ventricular area, HSTA, above) and then solubilizing the receptor. (A similar approach may be employed with AT4 receptors in other tissues.) The results presented below show that the guinea pig hippocampal AT4 receptor binds AIN ligand with a high affinity (Kd = 1.29 ± 0.18nM, mean ±SD, Hill Coeff. = 0.993 ± 0.015) and in a saturable manner ( ma_ - 449 ± 62fmol/mg protein). (It is noteworthy that the guinea pig hippocampal AT4 receptor binds AIN Ugand with approximately the same binding affinity as the bovine adrenal AT4 receptor described in Example 1, above.) The density of the AT4 receptors in hippocampal cells and tissues was considerably higher than reported in brain for AH receptors (43,44). In the present studies no AH receptors could be detected in Hippocampus by binding of 125I-Sar1,Ile8-AII (data not shown). The Ν-terminal structure of the binding AIN ligand is paramount in determining the binding affinity. The C-terminal requirements seem less stringent as evidenced by the binding affinity of AII(3.7) (Kd = 20.9 ± 2. InM). Neither AH, AIII, Sarι,Ile8-AH, Dup 753 nor CGP42112A appear to bind indicating that this binding site is neither the ATI nor AT2 sites described for AH/AIII. Autoradiographic analysis of Hippocampus binding confirms the inability of Sarι,Ile -AH to competitively inhibit for 125I-AIN binding. Conversely AIN was unable to displace 1 5I-Sar1,Ile8-AII binding at this site. The finding of AT4 receptors in the Hippocampus suggests that AIN ligand-receptor interactions may mediate unique central angiotensin-dependent functions including memory enhancement and provide a link between the Hippocampus and memory.
Saturation isotherms and corresponding Rosenthal plot for 125I-AIN binding to AT4 receptors in guinea pig hippocampal membranes show specific binding of 125I-AIN ligand to isolated hippocampal membrane AT4 receptors purified from guinea pig brain. Nonspecific binding was defined in the presence of non-labeled competitor, i.e., lOOnM AIN. The experiment was carried out 5 times (n=5); 125I-AIN bound saturably and ligand analysis of the binding data indicated the presence of a single high affinity binding site (Kd = 1.29 ± 0.18nM), Bmax = 449 ± 62 femtomol mg protein; Hill Coef = .993 ± .015; mean ± SD.
Structural characteristics of AIN Ugands that determine binding to the hippocampal AT4 receptor were determined in competition binding studies, i.e., similar to those described above in Example 1. The results of these competition studies are presented in Table 13.
TABLE 13 Competition of 125I-AIN Bindin to Guinea Pi Hi ocam us Membranes *
Figure imgf000075_0001
*n = 2, mean ± SD; 25mg of total membrane protein was incubated with O.όnM 125I-AIN plus a variable concentration of unlabeled angiotensin as a competitor. The results of these studies confirm those presented above in Example 1 with bovine adrenal AT4 receptors. The Ν-terminal of the AIV ligand (e.g., valine) is a major determinant of binding affinity. In agreement with the saturation isotherm data, AIN exhibited a high specificity for AIN (Table 13). Ν-terminal extended peptides including Sari, Ile8-AII, AH, and AIII had significantly reduced affinities for the AT4 receptor while
Figure imgf000076_0001
which has the N-terminal L-Nal removed, did not bind. (The low, but apparent ability of AIII to bind, may (as above) be due to conversion of AIII to AIV. The C-terminal specificity of the hippocampal AT4 receptor appears less.
Removal of Phe from the C-terminal of AIN ligand diminishes, but does not eliminate binding (Table 13), while removal of Phe, Pro, and He eliminates binding. As seen in
Table 7 neither Dup 753 nor CGP42112A competitively inhibited for the binding of
125I-AIN to the AT4 receptor. In addition, the peptides listed in Table 14, failed to bind to the HEN receptor in guinea pig brain as evidenced by their inability to significantly alter binding of AIN to receptors in this tissue.
TABLE 14
Figure imgf000076_0002
a.) Peptides that fail to bind to guinea pig brain tissues as evidenced by Kd >10_6M.
This study demonstrates the existence of a unique angiotensin binding site in guinea pig Hippocampus which is specific for the Ν-terminal deleted All hexapeptide, AIN. The location of this specific binding site in the Hippocampus supports the hypothesis that the AT4 receptor is the receptor that mediates angiotensin-dependent cognitive effects in the brain. It is clear from the autoradiographic sections shown in Figures 6-10, above, that the 125I-AT4 receptor is not restricted to the Hippocampus. The localization of 125I-AIN binding sites in other brain regions detailed in Table 15 presents an opportunity to expand the realm of angiotensin AIN-related actions. TABLE 15 Autoradio ra hic Quantitation of AIN Rece tors in Brain
Figure imgf000077_0001
a.) n=4 experiments; *n=3 experiments; **n=2 experiments; b.) displacement of 125I-AIN by Sar,He-AII Cognitive effects of the AIN ligand-receptor system
Learning: The results presented in Figure 10A show the mean latency (sec +/- SEM) for independent groups of rats to re-enter the dark compartment on Days 2-4 following passive avoidance conditioning on Day 1. One minute prior to the shock trial on Day 1, members of each group received aCSF (2ml), or lOOpmol in a total volume of 2ml aCSF of AH or AIN. On subsequent test days each animal was placed back into the lighted compartment and latency to enter the dark compartment was measured. Members of the group that received AIN on Day 1 showed significantly elevated latency times to re-enter the dark side on Day 2, as compared with the mean results from animals in the aCSF and All test groups. On day 1 artificial cerebrospinal fluid (aCSF), AH, or AIN was administered by intracerebroventribular (icv) injection into rat brains one minute prior to training. Training was conditioned (as desribed above) to avoid a dark compartment. On Days 2,3, and 4 of the experiment the animals were tested for the latency of time before they would re-enter the dark compartment. Enhancement of memory retrieval was observed on days 2 and 3 after learning of the reponse (Figure 10B). As can be seen from the results presented in Figure 10A the effect diminished with time after the learning of the response.
Memory Retrieval: The effects of AIN ligand on learning and memory were tested in rats by measuring the passive avoidance response, i.e., the mean latency period (time in seconds) for which the animal avoided a dark compartment. Training was conditioned to avoid the dark compartment by administering a 0.25mA foot shock over a period of 2 seconds with the door to a lighted compartment closed. On day 2 retrieval of the cognitive memory was tested 5 minutes after intracerebroventribular (icv) injection of AH or AIN. The results presented in Figure 12A show that AIV has a positive effect on memory retrieval at lnmol and lOOpmol, i.e., the AIN test animals avoided the dark side for a longer latency period than ATI-injected animals, or CSF-iηjected control animals. Materials and Methods: Hippocampal AT4 receptor studies: Hippocampus was from 4-month old male guinea pigs following decapitation.
The tissue was homogenized in 40 volumes of hypotonic buffer containing 50mM Tris, pH7.4 and 5mM EDTA, and spun at lOOOg for 10 min. The supernatant was removed and recentrifuged at 40,000g for 30 min. The pellet was rehomogenized in hypotonic buffer and recentrifuged. The 40,000g pellet was homogenized in isotonic buffer (50mM Tris, pH7.4, 5mM EDTA, 150mM ΝaCl, 20mM bestatin, 50mM Plummer's inhibitor, lOOmM PMSF, and 0.1% heat treated BSA) and recentrifuged a final time at 40,000 x g. The pellet was resuspended at a concentration of 2.5mg protein/ml as determined by the Lowry protein assay. Binding assays, which totaled 250ml, contained 10ml 125I-AIN ligand (sp. act-2176 Ci mmol), 10ml tissue homogenate, 10ml unlabeled peptide (if employed), and the remainder isotonic buffer. Incubations were carried out for 2 h at 37°C. Preliminary experiments demonstrated that incubation for 1 h at 37°C was necessary for equilibrium to be reached and that binding was stable for at least 4 h. At that time less than 10% of the 125I-AIN was shown to be by HPLC analysis. Saturation isotherms were developed using 12 concentrations of 125I-AIN in duplicate and included total and nonspecific binding [+100nM AIN]. Competition curves were developed using 500,000cpm/tube (0.6nM) of 125I-AIN and varying unlabeled peptide (lO^M to 10-nM) in half-log dilutions (Dup 753), CGP42112A: lO^M to 10-nM). Autoradiographic studies: Autoradiographic analysis of Hippocampus binding was carried out using
20mM tissue sections mounted on slides. Slices were initially preincubated in isotonic buffer for 30 min at room temperature, then incubated in labeled ligand (O.όnM) for 2 h, rinsed, dried, and exposed to X-ray film as previously described.
EXAMPLE 8 Isolation. Purification, and Characterization of the AIN Angiotensinase Enzyme AIN Angiotensinase:
The results of studies conduced in Examples 1-3, above, with bovine adrenal cortex indicate that a high affinity peptidase (Km =3nM) is present in these preparations that is capable of catalyzing hydrolysis of AH or AIII to AIN. Hydrolytic conversion of All (or AIII) to AIN may result from the action of an AIN- specific aminoendopeptidase, capable of hydrolyzing an arginyl-valinyl peptide bond (between positions #2 and #3 in AH; Figure 1) in an angiotensin termed herein AlV-angiotensinase. (Alternatively, the conversion of AIII to AIV may result from the action of nonspecific proteases but these enzymes may also cleave all angiotensins at sites other than the All R2-N3. and are not termed herein AIV angiotensinase.). In either case, cleavage of the AH Arg2-Val3 peptide bond in Al, AH, or AIII generates AIV.
Considering the important evolutionary conservation of the AT4 receptor and AIV ligand, and their most significant physiological roles, it is most likely that certain tissues and ceUs possess a specific ATV angiotensinase enzyme(s), i.e., that cleaves Al, All, and AHI in an efficient manner to permit regulatable formation of AIN. The AIN angiotensinase enzyme may be identified, isolated, and purified using the experimental approaches described below, in the Materials and Methods, in combination with the assays described in the Examples above (see Example 1). Data presented herein indicate that AH and Am are exceUent and specific inhibitors of 125I-AIN formation from 125I-AI. Materials and Methods: Experiment #1. Formation of AIN Ligand From AIN Precursors in Circulation.
125I-labeled angiotensins (107 dpm) - Al, All, or AHI, and tetradecapeptide can be injected into the carotid artery of a guinea pig and blood samples (50μl) can be collected at 30-sec or 1-min. intervals from a second cannula in the femoral artery into lOOμl of 20% TCA for 10 min. Samples may be analyzed by reverse-phase HPLC utilizing methods that have been reported previously (47). The data are analyzed to determine the rate of formation of AIN Ugand from potential AIN precursors. Experiment #2: Formation of AIN from Precursors via Action of Adrenal Enzymes. Guinea pig adrenals were excised and homogenized in a Krebs-Ringer buffer containing the full complement of ions (as above in Example 1). After a low speed spin at 500g for 10 min. to remove whole cells and nuclei, the supernatant is centrifuged at 40,000g for 30 min. The supernatant is recentrifuged at 100,000g for 90 min. yielding both a soluble (100,000g supernatant) and a microsomal (100,000g pellet) fraction. The 40,000 x g pellet is rehomogenized and fractionated on a discontinuous sucrose fractionated gradient (0.4M-1.2M sucrose, in 0.2M steps). The membranes at the 0.8M to l.M and IM to 1.2M interfaces can be collected and combined, resuspended in a 10X excess of Krebs buffer. The membranes were then centrifuged at 40,000 x g for 30 min. After a final resuspension in Krebs buffer and centrifugation at 40,000 x g for 30 min., the final plasma membrane fraction is ready for the assay. Soluble, membrane, and microsomal fractions may be incubated at various protein concentrations and times at 37°C with 106 cpm of 125I-AI, AH, AIII, and tetradecapeptide. Conditions were chosen (as above) to yield less than 10% total precursor hydrolysis thus assuring that comparisons of conversion rates is carried out under initial rate conditions. The reaction is terminated with 20% TCA and the products were evaluated by reverse-phase HPLC. The assay may also be useful for identifying AIV angiotensinase enzyme in chromatographic and other SDS-PAGE fractions isolated from adrenal, plasma, neural, and other tissues and bodily fluids. Experiment #3 : Characterization of AlV-Specific Angiotensinase.
If guinea pig adrenal tissue (as expected) possesses an AIN angiotensinase, the specificity of the enzyme(s), its activity on various substrates, and metal ion requirements can be established by incubating preparations of the isolated enzyme with angiotensins (e.g., in the presence of inhibitors of nonspecific proteases), and followed by examination of the hydrolytic products on reverse-phase HPLC. The sequence of the hydrolytic products may be determined by automated amino acid sequencing. Incubation conditions with varying concentrations of the angiotensin substrate were used to develop data for double reciprocal plots thus allowing the affinity of enzyme(s) for the different angiotensins to be determined. Next, competition studies can be undertaken using various angiotensin analogues and unrelated peptides in order to establish the structural requirements of the AIV angiotensinase enzyme(s). Finally, the ability of numerous divalent ions to activate AIN angiotensinase can be monitored. These experiments can be carried out with AIN angiotensinase enzymes that have been EDTA-stripped and the EDTA/Me"1"* removed by dialysis. CITATIONS
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SEQUENCE LISTING (l)GENERAL INFORMATION:
(i) APPLICANT:Harding, J.W.
(ii)TITLE OF INVENTION: "Angiotensin IV Peptides and Receptor" (iii)NUMBER OF SEQUENCES:6
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(A)NAME:Sundsmo,John,S. (B)REGISTRAΗON NUMBER:34,446 (C)REFERENCE/DOCKET NUMBER:WSUR-l-6263 (ix)TELECOMMUNICATION INFORMAΗON (A)TELEPHONE: 1-206-682-8100; 1 -206-224-0727 (direct)
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(2)INFORMATION FOR SEQ ID NO:l: (i)SEQUENCE CHARACTERISTICS :
(A)LENGTH:14 amino acids (B)TYPE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPE:peptide (A)DESCRIPTION:Angiotensinogen
(ix)SEQUENCE DESCRIPTION: SEQ ID NO:l: Asp Arg Val Tyr He His Pro Phe His Leu Val He His Ser 1 10
(3)INFORMATION FOR SEQ ID NO:2 :
(i)SEQUENCE CHARACTERISTICS: (A)LENGTH:10 amino acids (B)TYPE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear
(ii)MOLECULE TYPE:peptide
(A)DESCRIPTION:Angiotensin I (ix)SEQUENCE DESCRIPΗON: SEQ ID NO:2: Asp Arg Val Tyr He His Pro Phe His Leu 1 10
(4)INFORMATION FOR SEQ ID NO:3:
(i)SEQUENCE CHARACTERISTICS: (A)LENGTH:9 amino acids (B)TYPE:amino acid
(C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPE:peptide
(A)DESCRIPTION: [des-Asp] Angiotensin I (ix)SEQUENCE DESCRIPΗON: SEQ ID NO:3:
Arg Val Tyr He His Pro Phe His Leu 1 9
(5)INFORMAΗON FOR SEQ ID NO:4: (i)SEQUENCE CHARACTERISTICS:
(A)LENGTH:8 amino acids (B)TYPE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPE:peptide (A)DESCRIPTION:Angiotensin II
(ix)SEQUENCE DESCRIPΗON: SEQ ID NO:4: Asp Arg Val Tyr He His Pro Phe 1 8
(6)INFORMATION FOR SEQ ID NO:5:
(i)SEQUENCE CHARACTERISTICS: (A)LENGTH:7 amino acids (B)TYPE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear
(ii)MOLECULE TYPE:peptide
(A)DESCRJPTION:Ang-otensin m (ix)SEQUENCE DESCRIPΗON: SEQ ID NO:5: Arg Val Tyr He His Pro Phe 1 7
(7)INFORMATION FOR SEQ ID NO:6:
(i)SEQUENCE CHARACTERISTICS: (A)LENGTH:6 amino acids (B)TYPE:amino acid
(C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPE:peptide
(A)DESCRIPΗON:Angiotensin IV (ix)SEQUENCE DESCRIPTION: SEQ ID NO:6:
Val Tyr He His Pro Phe 1 6

Claims

What is claimed is:
1. A substantially purified angiotensin AT4 receptor, or fragment thereof having a binding affinity with a Kd of below about 3 x 10"6M for an AIN ligand having the sequence NYTHPF and having a binding affinity with a Kd greater than l x lO"6M for AII or Am.
2. An AT4 receptor or fragment thereof of Claim 1 which comprises a polypeptide having a molecular size of about 60kD to about 200kD on SDS-PAGE.
3. An AT4 receptor or fragment thereof of Claim 1 , wherein the receptor exhibits a binding affinity to VYTHPF with a K-j less than about 3 x 10"8M.
4. An AT4 receptor binding site polypeptide comprising a first subdomain capable of binding an AIN ligand having a penultimate Ν-terminal norleucine residue, and a second subdomain capable of binding a C-terminal region of said AIN ligand, wherein the first subdomain comprises both a hydrophobic pocket conforming closely to the space filled by norleucine and a negatively charged amino acid side chain capable of electrostatic interaction with the primary amine group of the norleucine, and wherein binding of an amino acid at the second subdomain alters the binding affinity at the first subdomain.
5. An AIN ligand capable of binding the AT4 receptor of Claim 1 with a binding affinity having a Kd of below about 3 x 10"6M, said ligand comprising a compound of the formula:
Figure imgf000087_0001
wherein R is a substituted or unsubstituted amino acid residue having a neutral or positively charged aliphatic side chain Z1} said amino acid being selected from among N, I, L, A, G, F, P, M, K, norvaline, norleucine, and ornithine,
R2 is a substituted or unsubstituted neutral nonpolar amino acid residue selected from among Y, W, Ν, Q, F or C,
R3 is a substituted or unsubstituted neutral polar amino acid residue selected from among G, A, N, I, L, F, P, or M, and
X is nothing, R4, R4-R5, or ^-Rs-Rg, wherein R4 is a substituted or unsubstituted basic amino acid residue selected from the group consisting of K, R and H, R5 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, N, I, L, F, P, and M, and R^ is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, N, I, L, F, P, M, and polyamino acid residues containing one or amino acid residues which do not prevent binding of the AIN Ugand with the AT4 receptor.
6. An AIN ligand of Claim 5 wherein Zj comprises an aliphatic chain of 4 carbon atoms in length.
7. An AIN ligand of Claim 5 wherein the amino acid residues are linked by peptidic linkages.
8. An AIN ligand of Claim 5 which comprises one or more non-peptidic linkages between adjacent amino acid residues.
9. An AIN ligand of Claim 5 which in which one or more of R4, R5, and -^comprises a D-amino acid residue.
10. An AIN ligand of Claim 5 comprising an Ν-terminal sequence of NYHTPF, NYIHP, NYIH, NYI, KYIHPF, KYIHP, KYIH or KYI.
11. An AIN Ugand of Claim 5 comprising a first Ν-terminal L-amino acid residue having a flexible aliphatic carbon side chain and a primary amine, and a second L-amino acid residue having a phenolic side chain, wherein the first and the second amino acid residues are chemically bonded through a carbon nitrogen bond that comprises a planar or non-planar rigid or non-rigid bond having a bond length substantially equivalent to a carbonyl bond.
12. An AIN Ugand of Claim 11, wherein the flexible aliphatic side chain comprises
Figure imgf000088_0001
or CH3(CH2)y-, wherein x and y are integers from 1 to 10.
13. An AIN Ugand of Claim 11, wherein x or y is 3 or 4.
14. An AIN ligand of Claim 11, wherein R^ is selected from the group consisting of norleucine, norvaline, ornithine, lysine.
15. An AIN ligand of Claim 11 , wherein R2 is tyrosine.
16. An AIN ligand of Claim 12, selected from the group consisting of NorLeu YIHPF, ΝorNalYIHPF, OrnYIHPF, KYIHPF, VYIHPF.
17. An AIV antagonist or agonist capable of binding the receptor or fragment of Claim 1 with a Kd of below about 3 x 10"6M.
18. A method of inhibiting proliferation of a vascular smooth muscle cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
19. A method of inducing proliferation of an endothelial cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
20. A method of inducing increased production of an endothelial cell relaxing factor in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
21. A method of increasing renal blood flow in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
22. A method of inhibiting an activity induced by All or Am in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
23. A method of enhancing AIV activity in a host in need thereof, comprising administering to the host a therapeutically effective dosage of an AIV agonist ligand of Claim 5.
24. A method of enhancing memory or learning in a host in need thereof comprising administering to the host a therapeutically effective dosage of an AIV agonist ligand of Claim 5.
25. A method of inhibiting AIV activity in a host in need thereof, comprising administering to the host a therapeutically effective dosage of an AIV antagonist ligand of Claim 5.
26. A method of inhibiting All-mediated aldosterone release from an adrenal cortical cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
27. A method of altering catecholamine release from an adrenal medullary cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
28. A method of potentiating cardiocyte growth in an animal host in need thereof, comprising comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.
29. A method of identifying the presence of an inhibitor of AIV ligand binding to an AT4 receptor in a biological fluid, comprising the steps of: a) adding an amount of an AIV ligand effective to produce measurable receptor binding to a first cell culture comprising an AT4 receptor to form a control mixture; b) adding said AIV ligand and a sample of said biological fluid or fraction thereof to a second cell culture comprising AT4 receptor; c) measuring the level of binding of said AIV ligand to the cells in the first and second cultures; and d) determining the presence of an inhibitor of AIV ligand to the AT4 receptor when the level of binding in the second culture is significantly lower than in the first culture.
30. An antibody capable of specifically binding to AIV, but not to AH or
AΠI.
31. A method of determining the presence or amount of AIV in a sample, comprising contacting the sample with an antibody of Claim 30 and then determining the amount AIV bound or unbound to the antibody as an indication of the presence or amount of AIN in the sample.
32. A method of isolating and substantially purifying an AT4 receptor to remove ATI and AT2 receptors, comprising the steps of: a) selecting cells expressing an AT4 receptor; b) preparing a membrane preparation of said cells in the presence of protease inhibitors, wherein said protease inhibitors are capable of inhibiting greater than 90% of angiotensin hydrolysis in the membrane preparation; c) solubilizing said AT4 receptor in said membrane preparation with a zwitterionic detergent under conditions that favor solubilization of said AIN receptor but not an ATj or AT2 angiotensin receptor; d) heat-treating said solubilized preparation under conditions suitable for destroying said ATj and AT2 receptors; and, e) fractionating said solubiUzed AT4 receptor preparation and identifying fractions capable of binding said AIN ligand, but not said Al or AH ligands.
PCT/US1993/006038 1992-06-24 1993-06-24 Angiotensin iv peptides and receptor WO1994000492A1 (en)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997003093A1 (en) * 1995-07-07 1997-01-30 Sagami Chemical Research Center Peptide derivatives and angiotensin iv receptor agonist
US7238358B2 (en) 1995-09-01 2007-07-03 Corixa Corporation Compounds and methods for immunotherapy and diagnosis of tuberculosis
WO1997016201A1 (en) * 1995-10-30 1997-05-09 Brigham & Women's Hospital Angiotensin iv and analogs as regulators of fibrinolysis
WO1998005624A1 (en) * 1996-08-06 1998-02-12 Taisho Pharmaceutical Co., Ltd. Amino compounds and angiotensin iv receptor agonists
DE19825494A1 (en) * 1998-06-08 1999-12-09 Hoechst Marion Roussel De Gmbh New angiotensin receptor, production and use of the same
US6177407B1 (en) 1998-08-13 2001-01-23 University Of Southern California Methods to increase blood flow to ischemic tissue
WO2000009144A1 (en) * 1998-08-13 2000-02-24 University Of Southern California Methods to increase blood flow to ischemic tissue
WO2001055176A2 (en) * 2000-01-27 2001-08-02 University Of Southern California Methods for inhibiting smooth muscle cell proliferation
WO2001055176A3 (en) * 2000-01-27 2002-07-25 Univ Southern California Methods for inhibiting smooth muscle cell proliferation
WO2003011304A1 (en) 2001-08-02 2003-02-13 Howard Florey Institute Of Experimental Physiology And Medicine Modulation of insulin-regulated aminopeptidase (irap)/angiotensin iv (at4) receptor activity
US7910555B2 (en) 2006-07-07 2011-03-22 Washington State University Research Foundation C-Met receptor regulation by angiotensin IV (AT4 ) receptor ligands
US8236761B2 (en) 2006-07-07 2012-08-07 Washington State University Research Foundation C-Met receptor regulation by angiotensin IV (AT4) receptor ligands
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds

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AU4649293A (en) 1994-01-24

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