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Inhibition of angiotensin‐converting enzyme‐2 (ACE‐2) activity and radioligand binding of a putative ACE‐2 inhibitor (1067.5)
Author(s) -
Linares Andrea,
Carrera Eduardo,
Speth Robert
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1067.5
Subject(s) - radioligand , chemistry , ace inhibitor , angiotensin converting enzyme , renin–angiotensin system , enzyme , enzyme inhibitor , angiotensin ii , biochemistry , membrane , amino acid , kidney , substrate (aquarium) , stereochemistry , pharmacology , binding site , receptor , endocrinology , biology , ecology , blood pressure
While closely related to angiotensin‐converting enzyme (ACE), ACE‐2 has very different effects on the renin‐angiotensin system. A potent inhibitor of ACE‐2 (MLN‐4760) was developed, but then abandoned when it became clear that ACE‐2 metabolizes angiotensin II (Ang II) to form Ang 1‐7. To develop a novel inhibitor of ACE‐2 to use as a tool to study ACE‐2, we developed an analog of MLN‐4760 (JFS101) in which a leucinyl side‐chain (designed to mimic the carboxy terminal amino acid of Ang I) was replaced with a tyrosyl side‐chain to more closely mimic the carboxy terminal amino acid of Ang II and allow for the inhibitor to be radioiodinated. Both the uniodinated and radioiodinated and the S,S versus the S,R analogs were tested for their ability to inhibit ACE‐2 metabolism of an artificial ACE‐2 substrate (MCA‐APK(Dnp) using recombinant human ACE‐2 (rhACE‐2) and for their ability to bind to ACE‐2 in rat lung and kidney membranes. The S,S isomer of JFS101 inhibited rhACE‐2 in the nanomolar range and was 5‐10% as potent as the S,S, isomer of MLN‐4760, which inhibited rhACE‐2 in the low nanomolar range. The S,R isomer of JFS101 inhibited rhACE‐2 activity in the low micromolar range. Radioligand binding assays using 125 I‐JFS101 (S,S isomer) revealed a high level of binding to lung and kidney membranes 196 and 321 fmoles/mg wet weight at 50 nM 125 I‐JFS101. However, less than 10 % of this binding was displaceable by 1 µM MLN‐4760. In contrast, 2 mM EDTA inhibited ~80 % of total binding at concentrations ranging from 3 ‐35 nM 125 I‐JFS101. The EDTA displaceable 125 I‐JFS101 binding was not saturable suggesting that the K D of 125 I‐JFS101 for lung membranes is >> 35 nM. The inability of MLN4760 combined with the ability of EDTA to inhibit 125 I‐JFS101 binding suggests that 125 I‐JFS101 is not selective for ACE‐2, but that it does bind to another metallopeptidase. Grant Funding Source : NIH‐HL113905