Modulation of cyclic adenosine monophosphate (cAMP) signalling and its therapeutic potential: pharmacological characterisation of PDE inhibitors
The alteration of intracellular cyclic adenosine monophosphate (cAMP) levels plays important regulatory roles in both physiological and pathological conditions, such as cancer. As the most aggressive form of brain tumour, glioblastoma is currently incurable due to limited treatment modalities. Low level of intracellular cAMP levels has been reported to be a feature of brain tumours. Thus, it is hypothesised that increasing cAMP concentrations by targeting regulatory proteins involved in the cAMP signalling pathways may offer advantages in preventing or treating glioblastoma. The overarching goal of this study, therefore, is to determine the dynamic effects of cAMP modulation on glioblastoma cell proliferation. The efficacies of compounds targeting various proteins involved in cAMP pathways were first investigated. The mechanisms explored were elevation of cAMP level through phosphodiesterases (PDEs) inhibition, adenylyl cyclase (AC) activation, as well as modulation via β-adrenoceptor (β-AR) and G proteins. A series of compounds were evaluated applying various assaying techniques and pharmacological tools using cAMP accumulation assays, cell proliferation, caspase-3/7 activation, and flow cytometry to determine cell cycle. It was demonstrated that increasing cAMP levels by multiple PDE inhibition or AC stimulation resulted in cell growth suppression on both rat and human glioblastoma models. The study was also extended to identify the role of possible crosstalk between calcium through SOCE (store-operated calcium entry) and cAMP pathways, which both were found to contribute to cell growth modulation. The effect of the elevation of intracellular cAMP on cell proliferation was further explored through the direct activation of adenosine A2A receptor (A2AR) and inhibition of cAMP degradation via PDE10A. Previous computational studies revealed that the triazoloquinazoline-based compounds (compound 1-6), initially known as PDE10A inhibitors, are bound at the orthosteric site of A2AR. To validate the computational results, these compounds were characterised using NanoBRET-based ligand binding studies with HEK293T expressing Nluc-A2AR and functional assays in lung cancer cell lines and glioma/glioblastoma cell models, which both cell models expressed endogenous levels of PDE10A and A2AR. The study highlighted that compounds 1 and 5 were dual-target ligands to A2AR and PDE10A, whereas compound 3 appeared to be a pan-agonist of adenosine receptors (ARs), and compound 4 was more potent when A2BR was expressed. Compound 2 seems to possess toxic effects that may be independent of action to A2AR or PDE10A. Lastly, preliminary studies were conducted to investigate the possibility of biased signalling by RAMPs on protease-activated receptor 4 (PAR4) and calcitonin-like receptor (CLR). Using PAR4 transiently transfected HEK293T cells, both cognate ligand and agonist peptide were used to profile PAR4 signalling including RAMPs-trafficking to the plasma membrane, promoting intracellular calcium release and recruiting β-arrestins. The effects of RAMPs were also investigated in HUVECs and cardiomyocytes focusing on the effect of endogenous ligands on cell growth. Whilst RAMPs altered PAR4 initial signalling events in promoting β-arrestin recruitment, the study on heterodimer complex of RAMPs and CLR on cell growth further corroborated that signalling bias can be translated into physiological responses in HUVECs and cardiomyocytes. To conclude, these studies provided evidence on how the alterations of intracellular cAMP levels affected cell proliferation in numerous cancer models, and that the cAMPmediated anti-proliferative effect was cell-line dependent. Targeting multiple PDEs suppressed cell growth in cancer-derived cells, therefore providing a viable target to reduce tumour progression. Given the critical role of PAR4 in platelet aggregation and pro-proliferative of calcitonin peptide family, this research may have important implications for the role of RAMPs in cardiovascular pathologies.
cAMP, phosphodiesterase, glioblastoma, GPCR, proliferation
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