Development of small molecules targeting GPCRs (PAR2 and LPA1) for potential use in cancer diagnosis and therapy

Abstract

Lung cancer continues to be the leading cause of cancer-related mortality for both men and women worldwide. Over the past two decades, advancements in prevention, screening, and treatment have contributed to reducing the overall cancer burden in Canada, though challenges remain. Molecular imaging, particularly PET imaging, employs radionuclide-labeled tracers to non-invasively diagnose diseases by visualizing biochemical processes in vivo. The success of PET imaging depends on radiopharmaceuticals targeting relevant receptors. G protein-coupled receptors (GPCRs) constitute the largest gene family in the human genome, regulating various cellular functions, with their dysregulation implicated in numerous diseases, including cancer. Protease-activated receptor 2 (PAR2) and lysophosphatidic acid receptor 1 (LPA1), both GPCRs, are overexpressed in lung cancer and play critical roles in tumor progression and metastasis, making them promising targets for cancer diagnosis and therapy. This thesis focuses on design, synthesis, and evaluation of small molecules that targeting PAR2 and LPA1 for potential use in cancer diagnosis and therapy. Abnormal activation of PAR2 initiates downstream signaling pathways that promote cancer progression and tumor metastasis in various cancer types. Studies have shown that PAR2 is significantly overexpressed, up to 16-fold, in lung cancer tissues. Chapter 2 presents the design and organic synthesis of novel PAR2 ligands (total of 35 compounds), based on AZ3451, a previously reported potent and selective PAR2 antagonist. Collaborators from Western University evaluated all compounds for functional activity by assessing PAR2-dependent calcium signaling and β-arrestin1/2 recruitment. Based on the functional activity assay, structural modifications of AZ3451 are expected to yield novel biased PAR2 negative allosteric modulators (NAMs). Further assessment of all the compounds for bias in PAR2 mediated mini-G protein recruitment to evaluate all compounds for their therapeutic potential is under way. Among these compounds, a fluorine-containing compound, P1c, was selected as the candidate with an EC50 value of 16.8 nM (n=1) and 1.2nM (n=1), determined through β-arrestin 1 and 2 recruitment in a trypsin-induced assay. To facilitate the development of 18F-radiolabeled compounds targeting PAR2, a precursor SPIAd iodonium (III) ylide was synthesized and characterized. Ongoing efforts focus on investigating the optimal reaction conditions for 18F-radiolabeling. LPA1 has been identified as being overexpressed in various cancers, particularly in lung cancer, where its activation promotes cell migration and invasion. Chapter 3 presents two series of design and synthesis of novel LPA1 antagonists with total 23 compounds. The synthetic scheme of first series is derived from the previously reported potent and selective LPA1 antagonist, RO6842262. The second series is derived from compound 12f, an anti-metastatic agent previously reported by our group. The core structure of this series was identified through scaffold hopping and molecular docking studies. All final compounds have been fully characterized, and their biological activity is currently being evaluated using cAMP and wound healing assays. Notably, compound L22p in the second series exhibited an IC50 value of 1.36 nM (n=1) in cAMP assay, indicating strong antagonistic activity against LPA1. This suggest that the novel scaffold holds promise as a potent LPA1 antagonist. Given the high expression of LPA1 in cancer, the candidate compound described in Chapter 3 will be converted into an iodo-compound, which will then react with SPIAd to generate iodonium (III) ylide precursors. These precursors will subsequently undergo treatment with fluorine-18 ions, followed by hydrolysis, to yield the radiolabeled final compounds.