Stimulating the Activity of NEP and ACE2: A Unique Approach to Prevent Dementia

Abstract

Introduction An overactive renin-angiotensin system (RAS) contributes to the pathogenesis of dementia. Angiotensin-converting enzyme 2 (ACE2) and neprilysin (NEP) converts angiotensin II to angiotensin 1–7, while NEP also breaks down amyloid-beta (Aβ) peptide. We hypothesise that activating the neuroprotective arm of RAS can attenuate neuroinflammation and subsequent dementia. The objective of the study was to determine the ability of a new peptide drug lead ‘2A’ (provisional patent number 2020904375, Australia) to stimulate ACE2 and NEP. Methods High-throughput plate reader based assays: The effect of 2A (9 – 26 µM) and alanine scan analogues of 2A (9 µM) on rhNEP and rhACE2 activity was determined using a quenched fluorescent substrate-based assay. Specific enzyme activity was calculated using a standard curve of 7-methoxy-coumarin-4-acetic acid. Further validation of stimulatory effects of 2A using natural substrates Aβ1–40, Aβ1–42 and angiotensin II: Liquid chromatography-mass spectrometry (LCMS) was used to determine the effect of 2A (9 µM) on the breakdown of Aβ1–40, Aβ1–42 and angiotensin II by NEP. Tissue biodistribution of 2A in C57BL/6J mice: 2A (1 mg/kg) was administered via the intranasal route to 12-week old male C57BL/6J mice. LCMS-MS was used to determine the levels of 2A in several tissues including the brain. Results 2A increased the activity of NEP and ACE2 in a concentration-dependent manner. The Vmax of NEP (0.035 ± 0.002 µmols of substrate cleaved/min) in the presence of 2A was significantly higher compared with enzyme alone (0.008 ± 0.001 µmols of substrate cleaved/min; P<0.001; N=10). Alanine scan of 2A revealed that the present primary structure of 2A was important for stimulating ACE2 and NEP activity. 2A significantly increased NEP-mediated formation of Aβ1–40 (P<0.03), Aβ1–42 (P<0.03) and angiotensin II (P≤0.008) breakdown products compared with respective control. Aβ1–40 levels (8 ± 0.7 as % of initial) was less with NEP + 2A compared to with NEP + scrambled control (42 ± 11%; P=0.02; N=3-4). Levels of 2A detected in brain tissue of C57BL/6J mice was significantly higher compared to 2A levels detected in heart, spleen, kidney and spinal cord tissue when administered through the intranasal route (P<0.03; N=5). Conclusions Our results indicate that 2A stimulates ACE2 and NEP activity, as reflected by the enhanced breakdown of angiotensin II and Aβ. Our findings also indicate that intranasal administration delivers significantly higher levels of 2A to the brain compared to systemic organs, thus suggesting that this route of delivery is highly suitable for pre-clinical studies.