Methods of identifying agonists or antagonists of angiotensin IV
First Claim
1. A method for screening for an agent that is an agonist or an antagonist of the interaction between an angiotensin IV ligand and an angiotensin IV receptor, comprising:
- (a) adding an angiotensin IV ligand to a first sample comprising an angiotensin IV receptor to form a control mixture, said angotensin IV ligand being a compound of the formula;
space="preserve" listing-type="equation">R.sub.1 R.sub.2 R.sub.3 X,wherein R1 is a substituted or unsubstituted amino acid residue having neutral or positively charged aliphatic side chain Z1, said amino acid being selected from among V, I, L, A, G, F, P, M, K, norvaline, norleucine, and omithine,R2 is a substituted or unsubstituted neutral polar amino acid residue selected from among Y, W, N, O, F or C,R3 is a substituted or substituted neutral polar amino acid residue selected from among G, A, V, I, L, F, P, or M, andX is nothing, R1, R4 -R5, or R1 -R5 -R6, wherein R1 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, V, I, L, F, P, and M, and R6 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, V, I, L, F, P, M, and polvamio acid residues containing one or amino acid residues which do not prevent binding of the AIV ligand with the AT4 receptor;
with the proviso that R1 can not be V when R2 is Y, R3 is I, R4 is H, R5 is P and R6 is F;
(b) adding the angiotensin IV ligand and a putative agent to a second sample comprising an angiotensin IV receptor to form a test mixture;
(c) determining the rate, amount or affinity of binding of the angiotensin IV ligand to the angiotensin IV receptors in the control mixture and the test mixture; and
(d) determining that the putative agent is an angiotensin IV agonist if the rate, amount or affinity of binding of the angiotensin IV ligand to the angiotensin IV receptor is greater in the test mixture than in the control mixture, or is an angiotensin IV antagonist if the rate, amount or affinity of binding of the angiotensin IV ligand to the angiotensin IV receptor is less in the test mixture than in the control mixture.
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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 ; SEQ. ID. NO. 1) 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; SEQ. ID. NO. 8) 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 (SEQ. ID. NO. 1) angiotensin AIV N-terminal peptide but not an angiotensin AII or AIII N-terminal peptide, i.e., DRVYIHPF (SEQ. ID. NO. 2) or RVYIHPF (SEQ. ID. NO. 3), respectively. Also disclosed are processes for isolating angiotensin AT4 receptor and AIV angioteninase, identifying angiotensin AIV agonists and antagonists, and constructing diagnostic assays to specifically measure AIV and AI-specific angiotensinase in biological fluids.
53 Citations
7 Claims
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1. A method for screening for an agent that is an agonist or an antagonist of the interaction between an angiotensin IV ligand and an angiotensin IV receptor, comprising:
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(a) adding an angiotensin IV ligand to a first sample comprising an angiotensin IV receptor to form a control mixture, said angotensin IV ligand being a compound of the formula;
space="preserve" listing-type="equation">R.sub.1 R.sub.2 R.sub.3 X,wherein R1 is a substituted or unsubstituted amino acid residue having neutral or positively charged aliphatic side chain Z1, said amino acid being selected from among V, I, L, A, G, F, P, M, K, norvaline, norleucine, and omithine, R2 is a substituted or unsubstituted neutral polar amino acid residue selected from among Y, W, N, O, F or C, R3 is a substituted or substituted neutral polar amino acid residue selected from among G, A, V, I, L, F, P, or M, and X is nothing, R1, R4 -R5, or R1 -R5 -R6, wherein R1 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, V, I, L, F, P, and M, and R6 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, V, I, L, F, P, M, and polvamio acid residues containing one or amino acid residues which do not prevent binding of the AIV ligand with the AT4 receptor; with the proviso that R1 can not be V when R2 is Y, R3 is I, R4 is H, R5 is P and R6 is F; (b) adding the angiotensin IV ligand and a putative agent to a second sample comprising an angiotensin IV receptor to form a test mixture; (c) determining the rate, amount or affinity of binding of the angiotensin IV ligand to the angiotensin IV receptors in the control mixture and the test mixture; and (d) determining that the putative agent is an angiotensin IV agonist if the rate, amount or affinity of binding of the angiotensin IV ligand to the angiotensin IV receptor is greater in the test mixture than in the control mixture, or is an angiotensin IV antagonist if the rate, amount or affinity of binding of the angiotensin IV ligand to the angiotensin IV receptor is less in the test mixture than in the control mixture. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of identifying the presence of an inhibitor of angiotensin IV ligand binding to an angiotensin IV receptor in a biological fluid, comprising the steps of:
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(a) adding an amount of an angiotensin IV ligand effective to produce measurable receptor binding to a first cell culture comprising an angiotensin IV receptor to form a control mixture, said angiotensin IV ligand being a compound of the formula;
space="preserve" listing-type="equation">R.sub.1 R.sub.2 R.sub.3 X,wherein R1 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 V, I, L, A, G, F, P, M, K, norvaline, norleucine, and omithine, R2 is a substituted or unsubstituted neutral nonpolar amino acid residue selected from among Y, W, N, O, F or C, R3 is a substituted or unsubstituted neural polar amino acid residue selected from among G, A, V, I, L, F, P, or M, and X is nothing, R1, R1 -R5, or R4 -R5 -R6, wherein R1 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, V, I, L, F, P, and M, and R6 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consist of G, A, V, I, L, F, P, M, and polvamino acid residues containing one or amino acid residues which do not prevent binding of the AIV ligand with the AT4 receptor; with the proviso that R1 can not be V when R2 is Y, R3 is I, R1 is H, R5 is P and R6 is F; (b) adding said angiotensin IV ligand and a sample of said biological fluid or fraction thereof to a second cell culture comprising angiotensin IV receptor; (c) measuring the level of binding of said angiotensin IV ligand to the cells in the first and second cultures; and (d) determining the presence of an inhibitor of angiotensin IV ligand to the angioterisin IV receptor when the level of binding in the second culture is significantly lower than in the first culture.
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Specification