Methods for identifying agents for treating cardiovascular disease using inhibition of myeloperoxidase binding
The present invention provides methods and agents for treating subjects who have or are at risk of developing or having cardiovascular disease. Such agents inhibit binding of myeloperoxidase (MPO) to a molecule comprising the MPO binding site of apolipoprotein A-1 (apoA-1) and include a peptide fragment of apoA-1 comprising at least 4 contiguous amino acids in SEQ ID. NO: 2, a modified form of the apo-1 fragment comprising one or more D amino acids, a retro-inverso form of the apoA-1 peptide fragment, an organo-mimetic of the apoA-1 peptide fragment, a peptide-mimetic of the apoA1 peptide fragment, or a nucleic acid encoding the apo A-1 peptide fragment. The present invention also provides methods of identifying or screening test agents for treating subjects having or at risk of having or developing CVD. The method comprises incubating one or more test agents and MPO with a molecule comprising the MPO binding site of apoA-1 under conditions which permit binding of MPO to the MPO binding site and determining whether one or more of the agents inhibit such binding.
1. A method for screening a test agent for cardiovascular therapy, comprising:
a) contacting MPO with a molecule comprising the helix 8 domain of apoA-1 in the presence of the test agent and taking a first measurement of a signal that indicates the extent of binding of MPO to the molecule;
b) contacting MPO with the molecule in the absence of the test agent and taking a second measurement of the signal that indicates the extent of binding of MPO to the molecule;
c) comparing the first and second measurements; and
d) selecting a test agent that inhibits MPO binding to the molecule.
2. The method of claim 1 , wherein the test agent is a peptide, a peptide mimetic, a peptide-like molecule, or a synthetic, semi-synthetic or natural chemical compound having a molecular weight of less than 10 kDa.
3. A method of identifying a test agent for treating cardiovascular disease, comprising screening a plurality of test agents in a competitive binding assay that comprises MPO and HDL, MPO and apo A-1, or MPO and an apoA-1 peptide fragment that comprises the MPO binding site of apoA-1, and selecting the test agent that inhibits binding of MPO to amino acids A190-L203 of apoA-1.
4. The method of claim 3 , wherein the test agents are peptides, peptide mimetics, or small organic molecules.
5. The method of claim 3 , further comprising comparing the inhibitory activity of the test agent with the inhibitory activity of a peptide that is 4 to 14 amino acids in length, and comprises, respectively, 4-14 contiguous amino acids in SEQ ID NO: 2 or SEQ ID NO: 3.