Activation of Cyclic Phenol Phosphate Analogues as Potential Inhibitors of Autotaxin

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

Zirconia based photocatalysts in degradation of selected herbicides

Hydrothermally synthesized zirconia nanopowders: pure and doped with Si4+ ions were spectroscopically characterized and used as photocatalysts for degradation of herbicides sulcotrione and fluroxypyr. Zirconia is wide band gap ceramic (Eg ~ 5 eV) however, synthesized nanopowders showed unexpected, modest absorbance in visible light range. That fact inspired photocatalytical degradation of herbicides with wide utilization, using solar irradiation (SI) in laboratory conditions. In the scope of this study, degradation of herbicides was only slightly achieved (irradiation time 2h).XV International Conference on Fundamental and Applied Aspects of Physical Chemistry : Proceedings. Vol. 1, September 20-24,2021, Belgrad

Photo-Switchable Control of Membrane Properties of Liposomes and Biochemical Processes

Liposomes are promising agents for drug delivery. They have the ability to encapsulate therapeutic drugs, resulting in decreased toxicity and prolonged circulation time. However, many obstacles to achieving broad utility in liposomal drug delivery still exist, including the ability to control release of therapeutic drugs and modulate surface reactivity. A primary focus of this dissertation involves the development of synthetic photocleavable lipids for controlled release from membranes. Phosphatidylcholine (PC) is a natural lipid that comprises the majority of structural membranes. It contributes heavily to the formation of lipid bilayers in cell membranes, and modifications to the bilayer can induce membrane transitions and changes in permeability. As such, a PC analogue has been developed with a photocleavable 2-nitrobenzyl acyl chain. This lipid (NB-PC) was synthesized in nine steps from 4-(aminomethyl)benzoic acid and lyso-phosphatidylcholine (LPC). This system is designed such that ultraviolet light degrades the fatty acid tail, changing the properties of the liposomes they form and releasing entrapped hydrophobic molecules. This occurred in about half an hour, as determined by a fluorescence assay involving the release of the dye Nile red. Phosphatidylethanolamine (PE), cholesterol, and polyethylene glycol (PEG) were incorporated as additives to examine the versatility of release from liposomes with varying membrane properties. It was found that release remained robust regardless of lipid content. Furthermore, another photocleavable lipid was developed containing an extended conjugated system, 2-nitrobiphenethyl, to enhance photocleavage efficiency and enable two-photon release. This lipid, NBP-PC, was synthesized in seven steps, and UV irradiation reached maximal release within five minutes. This dissertation also describes molecules that have been synthesized or are in progress for other projects. A nitrobenzyl-protected diacylglycerol has been synthesized, which is suitable for in situ binding studies with DAG-binding proteins, such as protein kinase C (PKC). Also synthesized are a biotin–azide linker for anchoring molecules onto streptavidin-coated surfaces and various azobenzene derivatives for studying chiral isomerization

A four-cysteine zinc finger in carboxyltransferase structurally links the functions of enzymatic activity and negative feedback regulation of translation

Acetyl-CoA carboxylase is the first and committed step of de novo fatty acid synthesis in all organisms. In Escherichia coli, the enzyme is expressed as separate proteins for the three functional components: a biotin carboxylase, a biotin carboxyl carrier protein, and a carboxyltransferase. The carboxyltransferase enzyme has an α2β2 heterotetrameric quaternary arrangement. The crystal structure of the β subunit revealed a zinc-binding domain, a feature common among nucleic acid-binding proteins. Carboxyltransferase preferentially binds mRNA coding for its two subunits over other nucleic acids, suggesting a means by which the enzyme can regulate its own expression. In the first study, the role played by the zinc-binding motif in carboxyltransferase is revealed through site-directed mutagenesis of the four coordinating cysteinyl residues. Results indicate that the zinc-binding domain is involved in both enzymatic activity of the enzyme as well as mediating binding of the enzyme to its own subunit mRNA. In this utility, the zinc-binding domain as a structural feature physically links the two functional aspects of the enzyme, possibly as a means to evolutionally conserve the capacity to regulate its own translation. In the second study, the individual interactions of carboxyltransferase with substrate and carboxyltransferase with mRNA are representated by mathematical modeling in an effort to validate these interactions function as a single system in regulating the activity and expression of carboxyltransferase in response to the metabolic state of the cell. Comparison of experimental and simulation results validate the model while also suggesting a more complex mechanism of carboxyltransferase translational regulation not captured by the current model

Reactivity at the membrane interface

Modulation of internal environment and maintenance of cellular structure and stability are basic requirements to ensure cell survival. These cellular functions are provided by the cell outer membrane, a phospholipid bilayer characterised by the fluid mosaic model. Chemical reactivity at the membrane interface has previously been identified between phospholipids and membrane binding species. Observed reactivity, termed intrinsic lipidation, involves non-enzymatic acyl transfer from phospholipids to a nucleophilic membrane bound molecule. Reactivity has been characterised for membrane active peptides and proteins, and been found influential to the structure and function of both the newly modified species, and the bulk membrane. Research presented within this thesis probes fundamental features of observed intrinsic lipidation reactivity at the membrane interface. This work has expanded upon previous intrinsic lipidation research, facilitated by the development of informative and robust analytical techniques for the study of reactivity. Optimised TLC has allowed improved routine high throughput reactivity screening, compared to alternative fluorescence and solution state NMR techniques. Informative analysis and mechanistic understanding of intrinsic lipidation has been achieved through LCMS and solid state NMR analysis. Synthetic protocols for preparation of isotopically labelled 15N small molecules, and 13C phospholipids, has facilitated solid state NMR in particular. Biological relevance of peptide intrinsic lipidation has also been probed to determine the role of reactivity in natural function, and disease induction. Biophysical techniques such as CD and tryptophan fluorescence revealed that solution phase intrinsically lipidated melittin adopts an α-helical structure with central proline kink, in contrast to the random coil of unmodified melittin. Furthermore, at μM concentrations, palmitoylated species were shown to undergo spontaneous micelle formation. Disease related behaviour linked to peptide intrinsic lipidation includes moderate antimicrobial activity, and possible induction of amyloid nucleation. Additionally, this study has identified novel intrinsic lipidation of small molecules in vitro utilising chromatographic and ionisation conditions optimised with synthetically prepared standards. Observed for multiple cationic amphiphilic small molecules, intrinsic lipidation was promoted by primary amines in a hydrophilic environment, due to increased proximity between reactive moieties. Small molecule intrinsic lipidation products were shown to exhibit biological relevance, including spontaneous micelle formation, membrane disruption, and phospholipidosis induction. Pharmaceutical propranolol displayed notable intrinsic lipidation in vitro, and in Hep G2 cell culture. Initial transesterification from membrane phospholipids produced O-acylated propranolol, followed by secondary N-acylated propranolol formation by intramolecular O to N migration. Study of propranolol reactivity has revealed preferential eukaryotic transfer from the sn-1 phospholipid backbone position, and reaction kinetics influenced by temperature, pH, and membrane composition

Analyses and Applications of Metalloprotein Complexes

The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal

Models and analysis of vocal emissions for biomedical applications

This book of Proceedings collects the papers presented at the 4th International Workshop on Models and Analysis of Vocal Emissions for Biomedical Applications, MAVEBA 2005, held 29-31 October 2005, Firenze, Italy. The workshop is organised every two years, and aims to stimulate contacts between specialists active in research and industrial developments, in the area of voice analysis for biomedical applications. The scope of the Workshop includes all aspects of voice modelling and analysis, ranging from fundamental research to all kinds of biomedical applications and related established and advanced technologies

Advances in Condition Monitoring, Optimization and Control for Complex Industrial Processes

The book documents 25 papers collected from the Special Issue “Advances in Condition Monitoring, Optimization and Control for Complex Industrial Processes”, highlighting recent research trends in complex industrial processes. The book aims to stimulate the research field and be of benefit to readers from both academic institutes and industrial sectors