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Activation of Cyclic Phenol Phosphate Analogues as Potential Inhibitors of Autotaxin



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ABSTRACT Autotaxin (ATX) is a member of the nucleotide pyrophosphatase/phosphodiesterase family of ectoenzymes (NPP/ENPP). ATX is mostly present in blood, but it is also expressed at high levels in the brain, kidney, lymphoid organs, ovary, lung, and intestine. ATX has lysophospholipase D activity that catalyzes the hydrolysis of lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline. LPA is a bioactive lipid mediator that facilitates many physiological and pathological processes including cell survival, proliferation, and migration. ATX/LPA signalling has been associated with in a number of human diseases including obesity, diabetes, rheumatoid arthritis, multiple sclerosis, neuropathic pain, Alzheimer’s disease, and cancer. Elevated ATX expression is found in various tumours and has been associated with tumour growth. Because most LPA is produced by ATX activity, an inhibitor of ATX would block subsequent LPA signalling, which is a target for anticancer drug development. Therefore, ATX has become an attractive drug target for developing new anticancer therapies. Our objective for this project is to prepare and assess a series of novel cyclic phenol phosphate analogues for their ability to function as irreversible ATX inhibitors in vitro. In order to investigate the ability of the cyclic phenol phosphate analogues to inhibit ATX, the following aims were outlined: (i) the development of synthetic methodologies for the preparation of a series of cyclic phenol phosphate analogues as potential inhibitors of ATX; (ii) an assessment of the aqueous stability of these analogues over 6 h in 50 mM TRIS buffer at 37 °C and pH 8.0 using high performance liquid chromatography (HPLC) for analysis; (iii) the determination of the ability of these compounds to inactivate ATX in vitro. In order to investigate the ability of the cyclic phenol phosphate analogues to inhibit ATX, we proposed and designed strategies to synthesize a series of acyl and alkyl ether derivatives of cyclic phenol phosphate analogues. Our initial strategy to synthesize acyl derivatives utilized the dealkylation of 1 (4-methoxy-1,3-bezenedimethanol); however, this approach was unsuccessful despite attempts to optimize reaction conditions and introduction of alcohol protecting groups. These unsuccessful reactions were likely the result of multiple competing reactions. A second strategy to synthesize alkyl ether derivatives analogues was developed, and we successfully synthesized two model analogues; A1 (unsubstituted cyclic phenol phosphate) and A2 (methoxy-substituted cyclic phenol phosphate). We accomplished this through a multi-step synthetic procedure using the salicylaldehyde and its derivatives as our starting materials. We also synthesized an alcohol starting material with a saturated fourteen-carbon ether linkage by modified literature procedures, but incorporation of the cyclic phosphate moiety was unsuccessful. We also evaluated the stability of the synthesized analogues under specific conditions. Analogues A1 and A2 were incubated in 50 mM TRIS buffer at 37 °C and pH 8.0 over six hours and determined to be stable under these conditions, suggesting any ATX inhibitory activity would be the result of the parent compound rather than any decomposition products. Finally, these compounds have been submitted to our colleagues at the University of Memphis for assessment of their in vitro ATX inhibitory ability; however, they have not been able to carry out the ATX inhibition assay yet. Also, we carried out the preliminary in silico docking studies. Our proposed and synthesized analogues of both acyl and alkyl ether derivatives of cyclic phosphate analogues were docked to the active site of the ATX (PDB ID: 3NKM) to assess their binding affinity. The results of the docking experiment revealed no major differences. However, both A1 and A2 show the highest values (i.e. lowest affinity), which indicate that the long side chains of the other ligands play a role in binding to ATX. Thus, the proposed cyclic phenol phosphates have not been previously prepared and can therefore represent a new orientation for the study of ATX inhibitor compounds and may lead to the development of new treatments for ATX-related human diseases in the future, including cancer.



Autotaxin (ATX), lysophosphatidic acid (LPA), inhbitors, cancer



Master of Science (M.Sc.)


Pharmacy and Nutrition




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