Development of N-domain selective Angiotensin-I Converting Enzyme (ACE) inhibitors using Computer Aided Drug Discovery (CADD)

Angiotensin-I (Ang-I) converting enzyme (ACE) is a zinc metalloprotease that plays a vital role in the Renin Angiotensin Aldosterone System (RAAS) and is a key antihypertensive drug target. In addition to Ang-I, ACE cleaves many other physiological substrates, thus extending its function beyond the regulation of blood pressure. Somatic ACE (sACE) consists of two structurally homologous yet distinct catalytic sites termed the N- and C-domains. The two catalytic domains of ACE have distinct substrate affinities and play different regulatory roles. The antifibrotic tetrapeptide Ac-SDKP is hydrolysed solely by the N-domain and thus is a potential target for interactions between the ligand and unique residues within the active site of the N- and C-domains, which need to be exploited to effect either N- or Cdomain selectivity. N-domain selective ACE inhibition has been demonstrated with peptides while crystallographic studies have shown that the N-domain to C-domain substitution of Arg381 with Glu403 within the S₂ subsite is integral to N-domain selective ACE inhibition. Three computer aided drug discovery (CADD) approaches were pursued to design N-domain selective drug-like ACE inhibitors (ACEi) with an acidic P₂ functional group that would confer N-domain selectivity via an interaction with Arg381 in the S₂ subsite. Firstly, a fragment-based screening protocol was performed by running a set of chemical filters on 16 000 drug fragment compounds (MW < 350), all of which contained a metal chelating group. 60 Ligands capable of binding to both the zinc metal and Arg381 in the S₂ subsite of the N-domain were tested for ACE inhibition against the two domains of ACE. Two of the fragments identified in this screen showed a modest ACE inhibition (IC₅₀ +/- 200 μM), but no domain selectivity. Secondly, a combinatorial library was created to explore the P₂ structure activity relationship (SAR) of a scaffold based on the core structure of the clinical ACEi, Enalaprilat. Over 400 variants were created to generate a combinatorial library. These compounds were docked against the two domains of ACE and a synthetic scheme was developed to synthesise compounds from this library. Using this scheme, one Enalaprilat analogue, SF07 was synthesised as a mixture of diastereomers. SF07 exhibited low micromolar N-domain inhibition with no C-domain inhibition observable below 100 μM. For the third approach, 25 000 compounds containing biological data pertaining to ACE were extracted from the GVK BIO GOSTAR database. These compounds were filtered for drug-like properties and manually inspected for promising P₂ functionality. The N-domain selectivity of these compounds was then assessed via molecular docking against the two domains of ACE. This screen identified a series of diprolyl compounds with varied groups in the P₂ position. These compounds were subsequently synthesised and tested in vitro for inhibition against both domains. The most N-domain selective compound from the series proved to be SG6, a diprolyl compound with an Asp group in the P₂ position. SG6 displayed potent inhibition (Kᵢ = 12 nM) and was 83-fold more selective towards the N-domain than the C-domain. This study has demonstrated the N-domain selective inhibition of ACE by drug-like peptidomimetics. Two promising leads on drug-like N-domain selective ACE inhibitors, SG6 and SF07, have been identified. These two compounds have the potential to pave the way for clinical N-domain selective ACEis and a novel treatment for cardiac and pulmonary fibrosis