Abstract
ACE (angiotensin-1-converting enzyme) is a zinc metallopeptidase that plays a prominent role in blood pressure regulation and electrolyte homeostasis. ACE consists of two homologous domains that despite similarities of sequence and topology display differences in substrate processing and inhibitor binding. The design of inhibitors that selectively inhibit the N-domain (N-selective) could be useful in treating conditions of tissue injury and fibrosis due to build-up of N-domain-specific substrate Ac-SDKP (N-acetyl-Ser–Asp–Lys–Pro). Using a receptor-based SHOP (scaffold hopping) approach with N-selective inhibitor RXP407, a shortlist of scaffolds that consisted of modified RXP407 backbones with novel chemotypes was generated. These scaffolds were selected on the basis of enhanced predicted interaction energies with N-domain residues that differed from their C-domain counterparts. One scaffold was synthesized and inhibitory binding tested using a fluorogenic ACE assay. A molecule incorporating a tetrazole moiety in the P2 position (compound 33RE) displayed potent inhibition (Ki=11.21±0.74 nM) and was 927-fold more selective for the N-domain than the C-domain. A crystal structure of compound 33RE in complex with the N-domain revealed its mode of binding through aromatic stacking with His388 and a direct hydrogen bond with the hydroxy group of the N-domain specific Tyr369. This work further elucidates the molecular basis for N-domainselective inhibition and assists in the design of novel N-selective ACE inhibitors that could be employed in treatment of fibrosis disorders.
Highlights
angiotensin-1-converting enzyme (ACE) is a zinc dipeptidyl carboxypeptidase that plays a critical role in blood pressure regulation and electrolyte homoeostasis [1,2]
Summary of results obtained from SHOP methodology R2 is given relative to the crystal structure RXP407 ligand backbone
Given the importance of these two residues in conferring selectivity for both 33RE and parent molecule RXP407, the kinetic results suggest that the actual interaction energies for the two inhibitors with these residues is critical [35]
Summary
ACE (angiotensin-1-converting enzyme; EC 3.4.15.1) is a zinc dipeptidyl carboxypeptidase that plays a critical role in blood pressure regulation and electrolyte homoeostasis [1,2]. Studies involving the generation of mice containing one domain catalytically inactivated have provided important insight into the differing roles of the two domains. In vivo both domains clear the vasodilator peptide bradykinin with approximately equal efficiencies [7,8,9], the C-domain appears to be the prominent site for the production of vasoactive peptide angiotensin II, whereas the N-domain is the primary site for the clearance of tetrapeptide Ac-SDKP (N-acetyl-Ser–Asp–Lys–Pro) [8,9,10]. AcSDKP levels have been shown to increase in patients acutely treated with ACE inhibitors [26] and suggests a possible therapeutic strategy for treating diseases involving fibrosis
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