Is Diminazene an Angiotensin-Converting Enzyme 2 (ACE2) Activator? Experimental Evidence and Implications.

Abstract

Antiprotozoal veterinary drug diminazene aceturate (DIZE) has been proposed to be an angiotensin-converting enzyme 2 (ACE2) activator. Since then, DIZE was used in dozens of experimental studies, but its mechanism of action attributed to ACE2 activation and enhanced formation of angiontensin-(1-7) [Ang-(1-7)] from Ang II was not carefully verified. The aim of this study was to confirm the effect of DIZE on catalytic activity of ACE2 and extend it to other peptidases involved in formation and degradation of Ang-(1-7). Concentration-dependent effect of DIZE on the initial rate of a fluorogenic substrate hydrolysis by human and mouse recombinant ACE2 was measured at assay conditions imitating that of the original report, but no activation of ACE2 was documented. Similar results were obtained with a more physiologically relevant assay buffer. In addition, DIZE did not affect activity of recombinant neprilysin, neurolysin, thimet oligopeptidase, and ACE. Efficiency of the fluorogenic substrate hydrolysis (Vmax/Km value) by ACE2 in response to different concentrations of DIZE was also measured, but no substantial effects were documented. Likewise, DIZE failed to enhance the hydrolysis of ACE2 endogenous substrate Ang II. Identity of the commercial recombinant ACE2 variants used in these experiments was confirmed by inhibition with two well characterized inhibitors (DX600 and MLN4760), activation by NaCl, and Western Blotting using validated antibodies. These observations challenge the widely accepted notion about the molecular mechanism of DIZE action and call for not ascribing this molecule as an ACE2 activator. SIGNIFICANCE STATEMENT: DIZE has been proposed and widely used in experimental studies as an ACE2 activator. The detailed in vitro pharmacological studies failed to confirm that DIZE is an ACE2 activator. In addition, DIZE did not substantially affect the activity of other peptidases involved in formation and degradation of angiotensin-(1-7). Researchers should refrain from calling DIZE an ACE2 activator. Other mechanisms are responsible for the therapeutic benefits attributed to DIZE.