Abstract
The objective this study was to test the efficacy of a novel angiotensin-converting enzyme-2 (ACE-2) inhibitor JFS101. 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. We developed a radioiodinizable analog of MLN-4760 (JFS101) that would more closely mimic Ang II. Both the uniodinated and monoradioiodinated as well as 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. In the results, the S,S isomer of JFS101 inhibited rhACE-2 in the nanomolar range and was 5-10% as potent as MLN-4760. The S,R isomer inhibited rhACE-2 activity in the low micromolar range. Radioligand binding assays using 125I-JFS101 (S,S isomer) revealed a high level of binding to lung and kidney membranes; however, less than 10 % of this binding was displaceable by 1 μM MLN4760. In contrast, 2 mM EDTA inhibited ~80 % of total binding at 3 -35 nM 125I-JFS101. In conclusion, the EDTA displaceable 125I-JFS101 binding was not saturable, suggesting that the KD of 125I-JFS101 for lung membranes is >> 35 nM. The inability of MLN-4760 combined with the ability of EDTA to inhibit 125I-JFS101 binding suggests that 125I-JFS101 is not selective for ACE-2, but rather binds to other metallopeptidases that may be identified in future research.
Faculty Sponsors
Dr. Robert Speth
Project Type
Event
Location
Alvin Sherman Library
Start Date
4-4-2014 1:00 PM
End Date
4-4-2014 5:30 PM
Inhibition of Angiotensin-Converting Enzyme-2 (ACE-2) Activity and Radioligand Binding of a Putative ACE-2 Inhibitor
Alvin Sherman Library
The objective this study was to test the efficacy of a novel angiotensin-converting enzyme-2 (ACE-2) inhibitor JFS101. 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. We developed a radioiodinizable analog of MLN-4760 (JFS101) that would more closely mimic Ang II. Both the uniodinated and monoradioiodinated as well as 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. In the results, the S,S isomer of JFS101 inhibited rhACE-2 in the nanomolar range and was 5-10% as potent as MLN-4760. The S,R isomer inhibited rhACE-2 activity in the low micromolar range. Radioligand binding assays using 125I-JFS101 (S,S isomer) revealed a high level of binding to lung and kidney membranes; however, less than 10 % of this binding was displaceable by 1 μM MLN4760. In contrast, 2 mM EDTA inhibited ~80 % of total binding at 3 -35 nM 125I-JFS101. In conclusion, the EDTA displaceable 125I-JFS101 binding was not saturable, suggesting that the KD of 125I-JFS101 for lung membranes is >> 35 nM. The inability of MLN-4760 combined with the ability of EDTA to inhibit 125I-JFS101 binding suggests that 125I-JFS101 is not selective for ACE-2, but rather binds to other metallopeptidases that may be identified in future research.
