Structure-based drug design efforts traditionally focused on development of enzyme inhibitors and receptor blockers. This strategy has been successfully applied to inhibit a number of enzymes relevant to hypertension including Angiotensin Converting enzyme (ACE) and Angiotensin receptor (AT). Despite the usefulness of frequently prescribed ACE inhibitors and AT blockers in reducing high blood pressure, these compounds are much less effective in preventing and reverting hypertension-induced endorgan damage while side effects and development of resistance remain serious limitations to their use.

Discovery of Angiotensin Converting Enzyme 2 (ACE2), a new regulator of Renin-Angiotensin System, provided a valuable alternative target for treatment of hyper-tension. ACE2 is a zinc-dependent metallopeptidase that plays a protective role in the early stages of heart failure. ACE2 can cleave a number of peptides implicated in high blood pressure, thus pharmacological enhancement of ACE2 activity could be beneficial in therapy. Structural information about ACE2 mechanism was used in the design of its inhibitors; however there were only a few reports on rational enzyme enhancement for therapeutic purposes to date. Recently, we identified a novel structural pocket in the hinge-bending region of ACE2 and demonstrated that targeting of small molecules to this site could selectively enhance ACE2 activity and reduce hypertension in vivo.

In this study we aimed to get further insight on the mechanism of ACE2 enhancement with the ultimate goal of finding better, more potent activators of ACE2. Since stabilization of the open conformation may facilitate faster product release and accelerate the overall rate, we used structural analysis to select the regions of highest intrinsic flexibility. Comparison of the crystal structures revealed three major structural hotspots controlling the conformational equilibrium in ACE2. Site 1 located in the largest hinge between the two subdomains was predicted to contribute the most to the conformational shuffling and was selected to be probed with a chemical library of FDA-approved compounds. We used molecular docking of small molecule library into Site 1 followed by functional testing of the top-scoring compounds and identified four FDA-approved drugs capable of enhancing ACE2 activity. One of these compounds, aromatic diamidine diminazene, acts as a potent activator of ACE2 in vitro and reduces high blood pressure in vivo. We aimed to further probe stereochemical preferences of ACE2 and test our strategy by using additional compound libraries. We took the ligand-based approach combined with principles of combinatorial chemistry to design and synthesize a small library of novel, more efficient drug-like derivatives of diminazene.

This dissertation describes the work that has been done to achieve these goals as well as three novel chemical entities with improved biological properties and stronger effects on ACE2 than the lead compound diminazene that were created in our laboratory. This study offers a new conformation-based selection method that utilizes existing structural and dynamic information on enzyme behavior to design and develop novel small molecule activators for highly flexible enzymes like ACE2 as a basis for alternative pharmacotherapy.