Publication
The renin-angiotensin system (RAS) is one of the major regulators of physiological and pathophysiological processes in the cardiovascular system. The RAS comprises two major axes: one deleterious, composed of the angiotensin II peptide activating the AT1 receptor, and another protective, composed of the angiotensin-(1-7) (Ang-(1-7)) peptide targeting the Mas receptor. While AT1 activation is associated with vasoconstriction, fibrosis and inflammation, activation of Mas leads to vasodilation, anti-fibrotic and anti-inflammatory effects. Numerous pre-clinical studies point out Mas as a promising pharmacological target for the treatment of various cardiovascular disorders. Additionally, activation of the beneficial Ang-(1-7)/Mas axis has recently emerged as a novel strategy for treating erectile dysfunction (ED) associated with diabetes, hypertension and atherosclerosis. Aging is a well-established factor for ED development; however, little is known about local regulation of the RAS in this condition. Thus, the aim of this thesis was to assess regulation of the RAS in aging-associated ED rat model and evaluate possible options for disease management through pharmacological modulation of the RAS. In the first part of the thesis, strong upregulation of AT1 and Mas receptors was revealed in aged cavernosal tissue, which also exhibited excessive collagen deposition. At the same time, penile sections from Mas-knockout (Mas-KO) mice (FVB/N background) were similarly characterized by increased collagen content, which indicated the protective role of Mas in cavernosal fibrosis. The ability of Ang-(1-7) to inhibit TGFbeta-induced myofibroblast transition of primary cavernosal fibroblasts further suggested Mas as a promising pharmacological target to treat fibrosis-associated ED. However, the use of Ang-(1-7) as a drug is complicated by pharmacokinetic limitations, such as low bioavailability and short half-life. Development of a small-molecule Mas agonist, able to produce effects inherent to Ang-(1-7), could overcome these issues. Thus, in the second part of the thesis, in order to facilitate the discovery of novel Mas modulators by computational tools, homology modeling and molecular docking were applied to reveal the structure of the Mas-Ang-(1-7) complex. Site-directed mutagenesis studies were further performed to validate the generated model and identify crucial interaction points. In the third part of the thesis, the validated Mas model was used for virtual screening of available compound libraries (68000 compounds in total). Top-ranked candidates (120 compounds) were further tested in a cell-based assay. Obtained positive hits (40 compounds) were afterwards submitted for ex vivo testing, based on the ability of Ang-(1-7) to relax isolated mouse aortic rings, and 20 compounds among them evoked vasodilation. Finally, in order to evaluate selectivity of the vasoactive compounds towards the Mas receptor, their activity was tested using aortic rings isolate