Proteins as Therapeutic Targets in Ovulation, Cancer and Antibiotic Resistance

Abstract

The research undertaken in this thesis presents studies on the design, synthesis and evaluation of potential inhibitors, aiming to achieve a therapeutic response in ovulation (Chapters 2 and 3), cancer and diet-induced diabetes (Chapter 4) and antibiotic resistance (Chapter 5). Proteins play an important role in every aspect of human life and as such, are ideal targets to interrupt disease progression, a concept introduced in Chapter 1. Furthermore, Chapter 1 presents three proteins with relevance in ovulation (N-cadherin), cancer and diabetes (mammalian target of rapamycin) and antibiotic resistance (D-Alanine-D-Alanine Ligase) as biological targets for the development of new therapeutic agents. Strategies to progress drug development towards the effective inhibition of these proteins are described, as well as techniques to evaluate the activity of potential inhibitors against such proteins in vitro and in vivo. Chapter 2 details the effect of known N-cadherin inhibitors CRS-066 and LCRF-0006, and analogues thereof, on endocrine response, and oocyte maturation in ovarian follicles and ovulation. Analysis of these inhibitors in xCELLigence real-time adhesion assays revealed CRS-066 inhibited the adhesive capacity of ovulating cumulus oocyte complexes (COCs) in low micromolar drug concentrations. In spheroid formation assays, using cells which overexpress extracellular N-cadherin, CRS-066 reduced formation of cell spheroids. Further analysis of CRS-066 and LCRF-0006 revealed an ability to disrupt cumulus expansion and oocyte meiotic maturation in vitro. Finally, in vivo evaluation in mouse models showed that CRS-066 effectively blocks ovulation. These combined results emphasise the importance of N-cadherin function in ovarian granulosa cell differentiation and ovulation, highlighting the potential of N-cadherin inhibitors for development of non-hormonal contraceptives. The further development of CRS-066 toward this application is discussed in Chapter 3. Chapter 3 presents the design and synthesis of a series of analogues of the known N-cadherin inhibitor CRS-066. The 4-aminopiperidine core was replaced with a selection of bioisoteric heterocycles to give 15 analogues, to investigate structure activity relationships and improve drug potency towards the inhibition of N-cadherin. Of these analogues, five displayed an ability to inhibit spheroid formation with similar potency (0.3-1.0 μM), to CRS-066 (0.3 μM) in N-cadherin-specific spheroid formation assays. Further biological evaluation revealed the ability of the 1,4-diazepane-containing analogue to completely inhibit COC expansion and cause 100% arrest of oocyte maturation at the germinal vesical stage at 1 μM concentration. Analysis of this most active analogue in xCELLigence real-time adhesion assays showed decreased N-cadherin-mediated adhesiveness of preovulatory COCs with an IC50 of 5.3 μM, equipotent to CRS-066 (IC50 = 4.0 μM). Collectively, these data identify 1,4-diazepane as an important structural motif for the development of N-cadherin inhibitors targeting ovulation. Strategies to achieve a therapeutic response in cancer and diabetes are addressed in Chapter 4. The presented research explores the use of bone-targeting conjugates to achieve tissuespecific inhibition of mTOR activity in osteoclasts and breast cancer cells, for the treatment of breast cancer-induced osteolysis and diabetes. The design and synthesis of two conjugates containing literature mTOR inhibitors (Everolimus and AZD8055) bound to bone-homing polyaspartic acid oligopeptides via a 3-maleimidopropionic acid linker is described. The active inhibitor was designed to be liberated from the inactive conjugate by esterase activity on the bone surface, to allow systemic administration without showing dose-limiting off-target effects. The initial inactivity of the intact Everolimus- (EVAC6) and AZD8055-conjugates (AZAC6), as well as the activity of the mTOR inhibitors after time-dependent liberation from the conjugate over 6 h, was demonstrated by Western blot analysis. AZAC6 was further analysed in cell proliferation assays, where the conjugate showed similar anti-proliferative activity as AZD8055 after 24 h. Evaluation of AZAC6 in mouse serum stability assays revealed 29% of the intact conjugate recovered from serum after 6 h, verifying sufficient conjugate stability to enable further in vivo investigation. An osteolytic breast cancer mouse model revealed increased activity of AZAC6 compared to AZD8055, however both the unmodified inhibitor and the conjugate failed to significantly reduce tumour burden and osteolytic bone damage over a 10- day treatment compared to vehicle-only control mice. Chapter 5 presents the structure-guided design, synthesis and evaluation of acylsulfamide bisubstrate inhibitors targeting the D-Ala-D-Ala ligase from Staphylococcus aureus (SaDdl), as potential new antibiotic agents. An X-ray co-crystal structure of ATP and D-Ala-D-Ala dipeptide bound to SaDdl is presented, to aid a structure-guided inhibitor design through molecular docking. A versatile 4-step synthesis gave 14 bisubstrate inhibitors. The most active compound showed moderate activity against SaDdl (70% remaining enzyme activity; IC50 = 1.7 mM), compared to the only FDA-approved Ddl inhibitor (D-cycloserin, 35% remaining enzyme activity; IC50 = 0.1 mM)). This paves the way for the development of linked bisubstrate SaDdl inhibitors with increased potency.