Aromatic amino acids decarboxylase and histidine decarboxylase: deep functional investigations give insights into pathophysiological mechanisms with possible therapeutic implications

Aromatic amino acids decarboxylase and histidine decarboxylase (AADC and HDC) are two homologous enzymes responsible for the synthesis of dopamine/serotonin and histamine, respectively, and other minor signalling aromatic amines. All these molecules are main protagonists or regulators of several physiological pathways, which are fundamental both in central nervous system and in peripheral tissues. Alterations of their homeostasis, indeed, as well as of AADC and HDC functioning or expression, cause and/or participate in the development and progression of several often severe and disabling pathological conditions, such as AADC Deficiency and cholangiocarcinoma. Consequently, AADC and HDC characterization might be useful in the pathophysiological understanding of several diseases and in improving/developing new therapeutic strategies. However, the knowledge of the biochemical features of these two crucial enzymes is still rather limited. Thus, the aim of this thesis is to biochemically characterise human HDC, mostly unknown, and to individuate some possible regulative mechanisms for both HDC and AADC. In addition, a neuronal AADC Deficiency cell model, derived from patient induced pluripotent stem cells (iPSCs), was used to evaluate endogenous AADC features, as well as to research further alterations in dopaminergic pathway. Investigations on human recombinant HDC allowed to discover that, surprisingly, its conformation and catalytic efficiency are influenced by redox state: increasing oxidizing conditions, indeed, favour a more stable and active form of the dimeric enzyme, due to the presence of an intermolecular reversible disulphide bridge involving residue Cys180 of both subunits. Then, in solution analyses of a possible phosphorylation of AADC identified Ser193 as protein kinase A target site, and allowed the detection of an effect on enzyme kinetic parameters, in particular an increased affinity for its substrates. Finally, endogenous AADC levels analyses in dopaminergic neurons derived from AADC Deficiency patients suggested a possible positive feedback mechanism that could tend to increase AADC expression, and the same cell model showed alterations in other cell types besides neurons, in particular glia cells, suggesting that variations in neurons-glia cells Abstract 5 interplay could participate in the pathophysiology mechanisms of AADC Deficiency. Altogether, data and information obtained from the performed experiments have increased AADC and HDC knowledge, as well as paved the way for new hypothesis regarding possible efforts in the development of new disease treatments

Pharmacokinetic Enhancers (Boosters)-Escort for Drugs against Degrading Enzymes and Beyond

Pharmacokinetic enhancers (boosters) are compounds used in combination with a primary therapeutic agent (drug) and are not used for their direct effects on the disease but because they enhance or restore the activity of the primary agent. Hence, in certain cases, they represent an indispensable escort for enzyme-labile drugs. Pharmacokinetic enhancers can exert their activity on different ways. In the most common case, they inhibit enzymes such as human cytochrome P450 enzymes in the liver or other organs and, thereby, block or reduce undesired metabolism and inactivation of the primary drug. In this review, an overview will be given on the therapeutically most important classes of pharmacokinetic enhancers like beta-lactamase inhibitors, inhibitors of CYP (cytochrome P450) enzymes in HIV therapy and hepatitis C, boosters for fluoropyrimidine-type anticancer agents, compounds utilized for enabling therapy of Parkinson's disease with levodopa, and others. Inhibitors of efflux pumps in both pathogenic bacteria and tumor cells will be addresses shortly

Vitamin B6 and Serotonin Metabolism in Neurological Disorders of Childhood

Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6 in man where it functions as a cofactor for more than 140 enzyme catalysed reactions. Several inherited diseases characterised by seizures have been described which result in an intracellular deficiency of PLP; laboratory measurement of B6 forms an important element in the diagnosis and monitoring of these disorders. A review of PLP measured by HPLC in CSF from patients with neurological disorders showed that variance is greater than indicated by previous studies and the age-related reference limit was revised. This thesis also describes the metabolic disorders that may lead to PLP depletion and examines the relationship of CSF PLP to sulphite accumulation, medications and seizures in patient groups. B6 exists as six different vitamers and is catabolised to 4-pyridoxic acid for urinary excretion. An LC-MS/MS method was developed which could measure all vitameric forms in plasma. Its application to children with B6 responsive seizure disorders showed that patients with inborn errors of metabolism have characteristic B6 profiles which allow them to be differentiated from each other and control populations. PLP is the cofactor for aromatic L-amino acid decarboxylase (AADC) which catalyses the final step in serotonin biosynthesis. This thesis tested the hypothesis that hyperserotonaemia observed in some patients with autism is related to an abnormality in this pathway by investigating the relationship between plasma B6 vitamers, AADC activity and whole blood serotonin in a group of patients and controls. Plasma AADC activity was significantly reduced in autistic subjects; this is considered in the context of current biochemical and molecular understanding and its possible relevance to disease mechanisms is discussed

Serotonin and the CNS

Serotonin is an ancient neurotransmitter system involved in various systems and functions in the body and plays an important role in health and disease. The present volume illustrates the broadness of the involvement of serotonergic activity in many processes, focusing particularly on disorders of the brain, including depression, stress and fear, Alzheimer’s disease, aggression, sexual behavior, and neuro-immune disorders. Chapters illustrate techniques and methods used to study the complex role of the serotonergic system in all kinds of processes, present new hypotheses for several brain disorders like sleep and depression, and use mathematical modeling as a tool to advance knowledge of the extremely complex brain and body processes

Decoding Gut Microbial Metabolites through G-Protein Coupled Receptor (GPCR) Activation

The microbiome encodes for a complex web of metabolites of which scientists are just starting to deconvolute. While a lot of focus has been on investigating the implications of the microbial metabolome on health and disease physiologies, we have merely uncovered the tip of the interactome of microbes and host G-Protein Coupled Receptors (GPCRs). Early literature has reported a plethora of short chain fatty acids fermented by dietary fibers acting as GPCR agonists. A few other studies have showcased that gut microbes produced N-acyl amides and secondary bile acids mimicking host ligands and therefore interacting with these GPCRs. Chapter 2 and 3 showcases the different strategies to mine GPCR agonists from the commensal microbiota

Synthesis and biological evaluation of metal chelators of the hydroxypyridinone family as potential treatment of Parkinson’s disease and cancer

A series of 9 hydroxypyridinones (HOPO) metal-based iron chelators (from which 6 of them are novel) have been prepared, characterised and derivatized in a manner to exploit an active transport mechanism; Large neutral Amino Acid Transporter-1 (LAT-1), which is found to be overexpressed in various types of cancer as well as to be presented in the blood-brain barrier (BBB) (Figure i). Figure (i): Structure of HOPO based compounds synthesised. Novel compounds are in the dotted frames. Additionally, it appears that the involvement of iron into metabolic pathways and/or the formation of low levels of reactive oxygen species (ROS) enhances the survival and proliferation of various types of cancer including malignant melanoma. The anticancer capacity of the series of HOPO based metal chelators, have been evaluated in an in vitro model ii consisting of human (eg. A375, VVM1, HS-294T) and rodent (eg. B16F-10) melanoma cells as well as non-melanoma epidermoid carcinoma (eg. A431) and immortalized, non-malignant keratinocyte (eg. HaCaT) cells. The results of this study demonstrated that a single compound a methylated analogue of L-mimosine, can exert anticancer capacity as at the administered concentration it acts as a pro-oxidant triggering the production of high (toxic) levels of ROS, selectively in melanoma cell lines. The accumulation of ROS, drives the cells to apoptosis via activation of a well characterised downstream cascade that includes that activation of the terminal caspase 3/7 via the action of intrinsic (activation of caspase-9 pathway) and extrinsic (activation of caspase-8 pathway). Additionally, the excessive production of oxidative cellular stress and iron misregulation may be substantially involved in the dopaminergic neuron degeneration seen in the brains of Parkinson's disease (PD) patients. Here we evaluated the effectiveness of the synthesised iron chelators, based on the hydroxypyridinone core with the ability to cross the BBB and penetrate the brain. Immortalised human dopaminergic neuronal precursor cells (LUHMES) were treated with the PD-related toxins 6-hydroxydopamine (6-OHDA), which generates superoxide radicals, 1-methyl-4-phenylpyridinium (MPP+), a mitochondrial complex I inhibitor, and the ferroptosis activator, erastin. Extensive cytotoxicological profiling revealed that three (rac-SK-2, rac-SK-3 and L-SK-4) out of the five tested compounds (rac-SK-1, rac-SK-2, rac-SK-3, L-SK-4 and rac-SK-5) rescue dopaminergic neuronal cells without inducing any toxic effects to cells, revealed through multiple cytotoxicological assays. In order to validate which structural features were essential for the transportation and the action of the compound, a series of control compounds (which they were lacking either the amino acid moiety or the coordination unit) have also been designed and screened against both melanoma cancer as well as PD cell lines. These control compounds of the associated molecules supported the rational design behind them according to which, the HOPO core is essential for the metal binding and the amino acid side vector for the transportation across the biological membranes via LAT-1

Other Radiopharmaceuticals for Imaging GEP‐NET

In GEP‐NETs, especially the catecholamine and serotonin biosynthetic pathways are upregulated. Therefore, increased biosynthesis of these specific amines in GEP‐NETs enables imaging with specific amine precursors. For the catecholamine pathway, 6‐18F ‐l‐3,4‐dihydroxyphenylalanine (18F‐DOPA) is available, while for the serotonin pathway, carbon‐11‐labeled 5‐hydroxy‐l‐tryptophan ([11C]‐5‐HTP) is available as tracer. 11C‐5‐HTP PET and 18F‐DOPA PET are excellent functional imaging techniques for evaluating patients with proven pancreatic islet cell tumors and carcinoids. For both tracers, the combination with CT further improves the detection rate of NET, which shows that performing PET scans with these tracers in PET/CT scanners is beneficial for patients.Since well‐differentiated GEP‐NETs generally have a low glucose metabolism, 18F‐fluorodexyglucose (18F‐FDG) PET scanning has limited value for the primary staging of patients with well‐differentiated GEP‐NETs. However, in patients with rapidly progressive disease, dedifferentiation of GEP‐NET tumors can lead to a higher glucose metabolism in tumor cells. In these patients, 18F‐FDG PET can be of benefit for tumor staging. Also, 18F‐FDG PET can be of value when other malignancies are suspected in patients with GEP‐NETs, since these patients experience a higher incidence of these malignancies compared to the general population.Nowadays, (GEP)‐NETs can also be imaged with 68Ga‐labeled analogues of somatostatin, which are also PET tracers. Advantages of 68Ga‐labeled somatostatin analogues are the relatively easy generator‐based synthesis and the possibility to evaluate whether peptide (somatostatin) receptor radionuclide therapy (PRRT) for NETs can be considered

Somatostatin Receptor Scintigraphy in Medullary Thyroid Cancer

Medullary thyroid cancer (MTC) is a neuroendocrine tumor originating from the calcitonin‐secreting C cells. Surgery, consisting of a total thyroidectomy and an extensive lymph node dissection, is the only effective treatment in MTC; however, metastases are frequently found in the regional cervical lymph. The biochemical marker for MTC is calcitonin, and this is frequently used for the detection of persistent/residual/metastatic tumor. The value of 111In‐labeled somatostatin receptor scintigraphy (SRS) in patients with MTC is limited, with sensitivity ranging between 0 and 75%. Other scintigraphic imaging techniques such as 18F‐FDG PET, 18F‐DOPA PET, and PET imaging with 68Ga‐labeled DOTA peptides combined with CT imaging are upcoming. Treatment of patients with metastatic disease with the current available somatostatin analogues, octreotide and lanreotide, does not seem to have an effect on survival but may be considered to control flushing and diarrhea in some patients. Experience with peptide receptor radionuclide therapy is limited in this patient group and disappointing. New therapies in the treatment of metastatic MTC use target tyrosine kinase receptors inhibitors belonging to the same family group of proteins as RET

Neurotransmitter-Related Molecular Modeling Studies

This book focuses of the neurotransmission phenomenon. By definition, neurotransmitters are chemicals that enable communication, i.e., the flow of nerve impulses between nerve cells or between nerve cells and muscles and glands. Recently, one can distinguish excitatory and inhibitory mediators, both of which are endo–exogenous compounds that control the function of the whole organism. From a chemical point of view, neurotransmitters belong to many different structural groups, such as amino acids (such as glycine), peptides (such as substance P, somatostatin), monoamines (such as noradrenaline or dopamine), purine derivatives (such as adenosine), gases (such as nitrogen, NO, carbon monoxide CO), and acetylcholine. From a medical point of view, disturbances in the concentration of neurotransmitters in the body result in the occurrence of mental disorders and diseases (such as depression, schizophrenia, Parkinson’s disease) and contribute to the occurrence of dementia (including Alzheimer’s disease), among other diseases. However, the problem is much wider. These disorders can lead to a number of cardiovascular diseases and can lead to the development of vascular diseases of the brain as well as in many other organs. Therefore, pharmacological intervention is a therapy that tries to interfere with regulatory processes year after year. Such treatments improve survival, reduce the frequency of readmission, and improve patients' quality of life