Angiotensin-1-converting enzyme (ACE) is a zinc metalloprotease that plays a major role in blood pressure regulation via the renin-angiotensin-aldosterone system. ACE consists of two domains with differences in inhibitor binding affinities despite their 90% active site identity. While the C-domain primarily controls blood pressure, the N-domain is highly selective for cleavage of the antifibrotic N-acetyl-Ser–Asp–Lys–Pro. Inhibitors, such as 33RE, that selectively bind to the N-domain thus show potential for treating fibrosis without affecting blood pressure. The aim of this study was to elucidate the molecular mechanism of this selectivity using in vitro and in silico techniques.
ACE inhibition by 33RE was characterized using a continuous kinetic assay with fluorogenic substrate. The N-domain displayed nanomolar(Ki=11.21±0.74 nM) and the C-domain micromolar (Ki=11278±410 nM)inhibition, thus 1000-fold selectivity. Residues predicted to contribute to selectivity based on the N-domain-33RE crystal structure were mutated to their C-domain counterparts. S2 subsite mutation drastically decreased affinity (Ki=2794±156 nM) due to loss of hydrogen bonds, yet did not entirely account for selectivity. Additional substitution of all unique S2’ residues completely abolished selectivity (Ki=10009±157 nM). Interestingly, these residues do not directly interact with 33RE. All six mutants were therefore subjected to molecular dynamics simulations in the presence and absence of 33RE. Trajectory analyses highlighted the importance of S2’ subsite residues in formation of a favourable contact face between the two ACE subdomains and thus a stable, closed, ligand-bound complex.
This study provides a molecular basis for the inter-subsite synergism responsible for 33RE’s 1000-fold N-selectivity and aids the future design of novel inhibitors for fibrosis treatment.