2A: A Novel Angiotensin Converting Enzyme 2 Stimulator

Abstract

Introduction Chronic activation of the renin angiotensin system (RAS) contributes to the pathogenesis of cardiorenal disease. Angiotensin converting enzyme 2 (ACE2) converts angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) which has cardio and reno-protective effects. Currently, there are no ACE2 stimulators which have clinical translational potential. Although putative ACE2 stimulators, xanthenone and diminazene aceturate, were identified over a decade ago, their effects were later attributed to pharmacological mechanisms independent of ACE2 and the mechanism of action of these compounds remains controversial.1 We have developed the world's first ACE2 stimulator referred to as ‘2A’ which is a 10 amino acid peptide derived from a snake venom (Provisional Patent Number 2020904375, Australia). Hypothesis We hypothesised that 2A will stimulate ACE2 activity and reduce Ang II-dependent inflammation and fibrosis. Methods A high throughput assay was used to measure the effects of 2A (2.6μM) on ACE2 activity in vitro. Liquid chromatography-mass spectrometry was used to monitor the effects of 2A (1.7µM) on ACE2-mediated conversion of Ang II to Ang 1-7. Expression of collagen type III and interleukin (IL)-6 in EA.hy926 cells was determined in the presence of (i) Ang II (10nM; 24-h) alone, and (ii) Ang II (10 nM; 24-h) + 2A (26µM; 24-h). Western blots were used to measure collagen type III and IL-6 in EA.hy926 cells. Streptozotocin (STZ)-induced diabetic mice (C57BL/6J) were treated with either 2A (1mg/kg; n=7) or saline vehicle (n=3) for 4 weeks via subcutaneous osmotic mini-pumps. At the study end, effects of 2A on expression of renal inflammatory and fibrotic markers were determined via western blots. Results The Vmax of ACE2 alone (0.05±0.001μmol substrate cleaved/min) was significantly less compared with ACE2+2A (0.09±0.002μmol substrate cleaved/min; P≤0.001; n=4-5). Consistent with this, incubation of 2A with ACE2 (24-h) reduced the levels of Ang II by 83% (P=0.008; n=3-4) and increased the levels of Ang 1-7 by 65% (P=0.003; n=3-4), in comparison to respective levels of Ang II and Ang 1-7 observed with ACE2 alone. The expression of collagen type III and IL-6 in EA.hy926 cells treated with Ang II + 2A was 86 and 63% less respectively, compared to cells treated with Ang II alone (P≤0.02; n=3). Chronic 2A administration did not cause any adverse effects in STZ-induced diabetic mice (n=7). Renal expression of IL-6, collagen I and collagen III tended to be less in diabetic mice treated with 2A than those treated with vehicle. Conclusion Our data indicate that 2A stimulates ACE2 activity and enhances the production of Ang 1-7. Consistent with this, 2A reduced Ang II-mediated expression of collagen type III and IL-6 expression in cultured endothelial cells. 2A tended to reduce renal expression of IL-6, collagen I and collagen III in diabetic mice. Together, these data indicate that 2A increases ACE2 activity and has the potential to reduce kidney fibrosis and inflammation in the setting of diabetes.