Thesis (doctoral)--Umeå Universitet, 1986. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 197-208). Available also in a digital version from Dissertation Abstracts.

BACKGROUND AND RATIONALE Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disorder affecting the central nervous system, primarily, the substantia nigra. It is characterized by loss of dopaminergic neurons in the nigro-striatal pathway, and ultimately patients with Parkinson’s disease may lose up to 80% of their dopamine-producing cells in the brain. Symptoms include bradykinesia, muscle rigidity, resting tremor and impaired postural balance. Symptomatic relief is obtained by using levodopa and various adjunct therapy including dopamine agonists, catechol-O-methyltransferase inhibitors and monoamine oxidase B inhibitors. Levodopa is used as the gold-standard for treatment of this disease. It effectively controls motor symptoms, however, motor complications that impair the quality of life develop with continued levodopa use. No treatments currently available can halt disease progression, therefore novel drugs that can slow down or stop disease progression are urgently required. The monoamine oxidase (MAO) A and B enzymes are flavoenzymes that play an important role in the oxidative degradation of amine neurotransmitters such as dopamine, serotonin and epinephrine. Early attempts to block dopamine metabolism in the brain using nonselective MAO inhibitors was effective but led to side effects such as hypertensive crisis, thus they lost favor. The MAO-B enzyme is of particular importance in Parkinson’s disease because it is more active than MAO-A in the basal ganglia, and is thus primarily responsible for the catabolism of dopamine in the brain. Selegiline and rasagiline, both irreversible, selective MAO-B inhibitors have proven efficacy in symptomatic treatment of Parkinson’s disease, but due to the irreversible nature of their binding, it can take several weeks after treatment termination for the enzyme to recover. Use of reversible inhibitors such as lazabemide and safinamide do not have this disadvantage, and have safer side effect profiles. Unfortunately, clinical trials for lazabemide use in Parkinson’s disease have been discontinued. Therefore, due to the lack of disease modifying agents for Parkinson’s disease, as well as safety concerns of current PD therapy, an urgent need exists for novel, safe and efficient MAO inhibitors. Current research is thus aimed at designing selective or non-selective reversible inhibitors that bind competitively to the enzyme. The MAO inhibitory potential of chromone derivatives has been illustrated previously. Evaluation of C6- and C7-alkyloxy substituted chromones, for example revealed that these compounds were potent, selective and reversible MAO-B inhibitors. It has further been shown that chromone 3-carboxylic acid is a potent selective, irreversible MAO-B inhibitor. Phenylcarboxamide substitution in position 3 of chromone 3-carboxylic acid also results in potent, selective MAO-B inhibitory activity. Therefore, further evaluation of the effect of substitution with flexible side chains in the 3-position to evaluate MAO-B inhibition is of importance. The chromone ring system is thus a privileged scaffold for the design of inhibitors that are selective for MAO-B and has the additional advantages of generally exhibiting low mammalian toxicity and ease of synthesis. AIM The aim of this study was to design, synthesize and evaluate novel chromone derivatives as inhibitors of monoamine oxidase. RESULTS Design and Synthesis 3-Aminomethylene-2,4-chromandiones and ester chromone derivatives were synthesized by coupling several aromatic and aliphatic amines and alcohols, to chromone 3-carboxylic acid, in the presence of CDI (carbonyldiimidazole). 15 Compounds were successfully synthesized and characterized by using NMR and IR spectroscopy, as well as mass spectrometry. X-ray crystallography was used to obtain a crystal structure for the 3-aminomethylene-2,4- chromandione derivative, 46, in a bid to verify the structures of the synthesized compounds. Melting points of all compounds were determined, and the purity determined using HPLC techniques. MAO inhibition studies A fluorometric assay was employed using kynuramine as substrate, to determine the IC50 (50% inhibition concentration) values and SI (selectivity index) of the synthesized compounds. Generally, the esters exhibited weak MAO-A and MAO-B inhibition, while the 3- aminomethylene-2,4-chromandione derivatives showed promise as selective MAO-B inhibitors, with IC50 values in the micromolar range. Compound 38, 3- [(benzylamino)methylidene]-3,4-dihydro-2H-1-benzopyran-2,4-dione, was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM and a SI of 122 for MAO-B inhibition. Interesting trends were revealed through analysis of the structure activity relationships, for example, for the 3-aminomethylene-2,4-chromandione derivatives, the presence of a chlorine moiety in the side chains of the compounds resulted in a decrease of MAO-B inhibition activity. Chain elongation further also resulted in weakening the MAO-B inhibition activity, while chain elongation in the ester derivatives led to a slight increase in MAO-B inhibition activity. Reversibility studies The reversibility of binding of the most potent compound in the 3-aminomethylene-2,4- chromandione series, 38, was evaluated. None of the synthesized inhibitors were potent MAO-A inhibitors, therefore reversibility of MAO-A inhibition was not examined. Recovery of enzyme activity was determined after dialysis of the enzyme-inhibitor complexes. Analysis of the kinetic data obtained showed that MAO-B catalytic activity was recovered to 115% of the control value. This suggests that compound 38 is a reversible inhibitor of MAO-B. Mode of inhibition A set of Lineweaver-Burk plots were constructed to determine mode of inhibition of compound 38. The results show linear lines that intersect at a single point just to the left on the y-axis. This indicates that compound 38 interacts competitively with the MAO-B enzyme. In conclusion, chromone derivatives were synthesized and evaluated as inhibitors of MAO. Compound 38 was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM. The effect of chain elongation and introduction of flexible substituents in position 3 of the chromone 3-carboxylic acid nucleus was explored and the results showed that 3- aminomethylene-2,4-chromandione substitution is preferable over ester substitution. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

BACKGROUND AND RATIONALE Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disorder affecting the central nervous system, primarily, the substantia nigra. It is characterized by loss of dopaminergic neurons in the nigro-striatal pathway, and ultimately patients with Parkinson’s disease may lose up to 80% of their dopamine-producing cells in the brain. Symptoms include bradykinesia, muscle rigidity, resting tremor and impaired postural balance. Symptomatic relief is obtained by using levodopa and various adjunct therapy including dopamine agonists, catechol-O-methyltransferase inhibitors and monoamine oxidase B inhibitors. Levodopa is used as the gold-standard for treatment of this disease. It effectively controls motor symptoms, however, motor complications that impair the quality of life develop with continued levodopa use. No treatments currently available can halt disease progression, therefore novel drugs that can slow down or stop disease progression are urgently required. The monoamine oxidase (MAO) A and B enzymes are flavoenzymes that play an important role in the oxidative degradation of amine neurotransmitters such as dopamine, serotonin and epinephrine. Early attempts to block dopamine metabolism in the brain using nonselective MAO inhibitors was effective but led to side effects such as hypertensive crisis, thus they lost favor. The MAO-B enzyme is of particular importance in Parkinson’s disease because it is more active than MAO-A in the basal ganglia, and is thus primarily responsible for the catabolism of dopamine in the brain. Selegiline and rasagiline, both irreversible, selective MAO-B inhibitors have proven efficacy in symptomatic treatment of Parkinson’s disease, but due to the irreversible nature of their binding, it can take several weeks after treatment termination for the enzyme to recover. Use of reversible inhibitors such as lazabemide and safinamide do not have this disadvantage, and have safer side effect profiles. Unfortunately, clinical trials for lazabemide use in Parkinson’s disease have been discontinued. Therefore, due to the lack of disease modifying agents for Parkinson’s disease, as well as safety concerns of current PD therapy, an urgent need exists for novel, safe and efficient MAO inhibitors. Current research is thus aimed at designing selective or non-selective reversible inhibitors that bind competitively to the enzyme. The MAO inhibitory potential of chromone derivatives has been illustrated previously. Evaluation of C6- and C7-alkyloxy substituted chromones, for example revealed that these compounds were potent, selective and reversible MAO-B inhibitors. It has further been shown that chromone 3-carboxylic acid is a potent selective, irreversible MAO-B inhibitor. Phenylcarboxamide substitution in position 3 of chromone 3-carboxylic acid also results in potent, selective MAO-B inhibitory activity. Therefore, further evaluation of the effect of substitution with flexible side chains in the 3-position to evaluate MAO-B inhibition is of importance. The chromone ring system is thus a privileged scaffold for the design of inhibitors that are selective for MAO-B and has the additional advantages of generally exhibiting low mammalian toxicity and ease of synthesis. AIM The aim of this study was to design, synthesize and evaluate novel chromone derivatives as inhibitors of monoamine oxidase. RESULTS Design and Synthesis 3-Aminomethylene-2,4-chromandiones and ester chromone derivatives were synthesized by coupling several aromatic and aliphatic amines and alcohols, to chromone 3-carboxylic acid, in the presence of CDI (carbonyldiimidazole). 15 Compounds were successfully synthesized and characterized by using NMR and IR spectroscopy, as well as mass spectrometry. X-ray crystallography was used to obtain a crystal structure for the 3-aminomethylene-2,4- chromandione derivative, 46, in a bid to verify the structures of the synthesized compounds. Melting points of all compounds were determined, and the purity determined using HPLC techniques. MAO inhibition studies A fluorometric assay was employed using kynuramine as substrate, to determine the IC50 (50% inhibition concentration) values and SI (selectivity index) of the synthesized compounds. Generally, the esters exhibited weak MAO-A and MAO-B inhibition, while the 3- aminomethylene-2,4-chromandione derivatives showed promise as selective MAO-B inhibitors, with IC50 values in the micromolar range. Compound 38, 3- [(benzylamino)methylidene]-3,4-dihydro-2H-1-benzopyran-2,4-dione, was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM and a SI of 122 for MAO-B inhibition. Interesting trends were revealed through analysis of the structure activity relationships, for example, for the 3-aminomethylene-2,4-chromandione derivatives, the presence of a chlorine moiety in the side chains of the compounds resulted in a decrease of MAO-B inhibition activity. Chain elongation further also resulted in weakening the MAO-B inhibition activity, while chain elongation in the ester derivatives led to a slight increase in MAO-B inhibition activity. Reversibility studies The reversibility of binding of the most potent compound in the 3-aminomethylene-2,4- chromandione series, 38, was evaluated. None of the synthesized inhibitors were potent MAO-A inhibitors, therefore reversibility of MAO-A inhibition was not examined. Recovery of enzyme activity was determined after dialysis of the enzyme-inhibitor complexes. Analysis of the kinetic data obtained showed that MAO-B catalytic activity was recovered to 115% of the control value. This suggests that compound 38 is a reversible inhibitor of MAO-B. Mode of inhibition A set of Lineweaver-Burk plots were constructed to determine mode of inhibition of compound 38. The results show linear lines that intersect at a single point just to the left on the y-axis. This indicates that compound 38 interacts competitively with the MAO-B enzyme. In conclusion, chromone derivatives were synthesized and evaluated as inhibitors of MAO. Compound 38 was the most potent MAO-B inhibitor with an IC50 value of 0.638 μM. The effect of chain elongation and introduction of flexible substituents in position 3 of the chromone 3-carboxylic acid nucleus was explored and the results showed that 3- aminomethylene-2,4-chromandione substitution is preferable over ester substitution. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Dysregulation of dopaminergic homeostasis has been established as the primary source of numerous neurological disorders including Parkinson’s and drug addiction. A tonic increase of dopamine (DA) in the nucleus accumbens is required for associating everyday events and behaviors with rewards. Yet many addictive exogenous compounds such as amphetamine (AMPH) and cocaine (COC) produce a much greater augmentation of synaptic DA levels that are linked to euphoria and a shift in behavior towards drug seeking. The protein responsible for maintaining extracellular levels of DA is the dopamine transporter (DAT). It is primarily located in the perisynaptic area at terminals of pre-synaptic neurons where its main function is to sequester DA from the extracellular space and to transport it back into the cell, a process that is electrogenic. AMPH and COC directly interact with DAT and alter its ionic currents. Not much is known about the effect of psychostimulant-induced DAT currents on neuronal excitability and neurotransmitter release. We use synthetic chemistry, molecular biology, and biophysics in heterologous expression systems to decipher the actions of drugs of abuse on DAT. Furthermore we demonstrate drug-induced DAT currents can activate Ca2+ channels associated with dopaminergic excitability. Lastly, we focused on investigating drug effects on excitability in a human midbrain dopaminergic cell line. Understanding how psychostimulants interact with DAT to produce the dysfunctional states of the transporter may facilitate the development of unique therapeutic strategies to treat psychostimulant dependence.

No description available.

Fluorescent analogs of biomolecules have been known as useful probes to study the structure, conformations and dynamics of cellular processes. These probes are more ideal than fluorescent labeled probes, as fluorescent analog probes retain the shape, size, conformation, and recognition element of the natural substrate, while giving useful intracellular information about detection and dynamics of biomolecules. The monoamine neurotransmitters control the central and periphery nervous systems. Serotonin (5-HT), in particular, is a versatile chemical messenger responsible for a multitude of biological processes, such as regulation of emotion, vasoconstriction, and bone metabolism. The study of serotonergic complex pathways is vital and essential in drug discovery for the diseases that result from the depletion and deregulation of serotonin in synapse. The extracellular concentration of serotonin is controlled by several transporters, most preferably the serotonin transporter (SERT). Selective serotonin reuptake inhibitors (SSRIs), along with dual- and triple-acting inhibitors, affect SERT and hence 5-HT in depression and related diseases. In this present investigation, firstly, a set of fluorescent analogs of neurotransmitter probes based on ethylamino-functionalized substrates were successfully designed and these fluorescent probes were synthesized by convenient synthetic methods. Secondly, optical properties of these fluorescent probes were investigated in organic medium, in order to test their suitability for screening and imaging the biological cells. Finally, their uptake was examined in the murine osteocytic cell line, MLO-Y4, platelets of blood sample and HEK-293 cells expressing the dopamine transporter (DAT), norepinephrine transporter (NET) or SERT. The fluorescent probes targeting bone-derived cell line expressing 5-HTT provide useful information in understanding the dynamics of 5-HT regulation with respect to SSRI treatment. A novel fluorescent analog of 5-HT probe was developed that may be utilized to study 5-HTT function in the context of 5-HT uptake or regulation in cell culture, tissue explants, or even in vivo.

Presenilin-1 (PS-1) is the catalytic core of the ã-secretase complex, which is best known for its role in the generation of the Alzheimer disease (AD)-related â-amyloid peptide. Mutated forms of PS-1 are known to be associated with particularly aggressive forms of AD. Changes in monoaminergic neurotransmitter systems, including the serotonin and norepinephrine systems, have long been associated with some of the earliest events in AD, whereas changes in the availability of these same monoamines have historically been associated with clinical depression. Therefore, it is not surprising that depression has now been proposed as a risk factor for developing AD and that pre-demented carriers of mutated forms of PS-1 are more prone to developing depression. MAO-A is historically associated with depression and is also a known risk factor for AD. Given this, I hypothesized that MAO-A represents a neurochemical link between depression and AD, and I chose to examine the influence of PS-1 mutations on MAO-A function in vivo/ex vivo and in vitro.<p> I first focused on the PS-1(M146V) knock-in mouse model of AD-related PS-1/ã-secretase function. I used a radioenzymatic assay to estimate MAO-A catalytic activity and western blot analysis to determine MAO-A protein expression, and found that MAO-A activity does not correlate with MAO-A expression in the cortex and cerebellum of the PS-1(M146V) mice. Furthermore, the potency of the MAO-A inhibitor clorgyline (CLG) is greater in both the cortex and cerebellum of the PS-1(M146V) mice compared to the potency of CLG in wildtype littermates. CLG dose-response curves suggest that there might be a change in cooperativity in the MAO-A protein from PS-1(M146V) cortex (which would suggest a change in conformation and/or access of the substrate to the catalytic pocket in MAO-A). High-pressure liquid chromatography was used to analyze monoamine levels in these same regions. The levels of monoamines (i.e. serotonin, dopamine and norepinephrine) suggest that PS-1 (M146V) inhibits MAO-A function in the cortex, but not in the cerebellum. Furthermore, CLG has no significant effect on amine levels in cortex, but tends to increase their accumulation in cerebellum.<p> The overexpression of PS-1 (M146V) in neuronal cultures reveals that this protein affects MAO-A activity and, more importantly, the PS-1(M146V) protein co-precipitates with MAO-A, thus suggesting a possibility for a direct protein-protein interaction. This is supported by the observation that MAO-A activity is increased in cell extracts incubated with the PS-1 substrate-competitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Preliminary studies have been undertaken to determine the motif in MAO-A that could be acting as a binding site/target site for PS-1.<p> These combined results support the hypothesis that PS-1 proteins can influence MAO-A function and, furthermore, that MAO-A is a novel interactor for PS-1/ã-secretase. This could well explain some of the ambiguous literature associated with both of these proteins in disorders as diverse as depression and AD.

The enzyme monoamine oxidase (MAO) is linked to mental disorders such as depression and neurodegenerative diseases. Our laboratory has recently demonstrated that increases in calcium (Ca2+) can enhance MAO activity and that this might contribute to Alzheimer disease (AD). AD has been linked to gain-of-function mutations in the presenilin-1 (PS-1) protein that not only promote the generation of the toxic amyloid-â peptide, but that also alter intracellular Ca2+ availability. <p>Radioenzymatic MAO assays were used to demonstrate that over-expression of different AD-related PS-1 mutant proteins, i.e. Y115H, ÄEx9 and M146V, in hippocampal-derived HT-22 cells alter either basal and/or Ca2+-sensitive MAO-A activity. The effects of PS-1 mutant proteins on the availability of intracellular Ca2+ are not consistent suggesting that this may not be the primary means of regulating MAO activity. The sensitivity of MAO to Ca2+ was also demonstrated in cortical (both MAO-A and MAO-B responded to Ca2+) and cerebellar (only MAO-A responded to Ca2+) samples from transgenic mice overexpressing the PS-1 (M146V) mutation. These changes in MAO coincided with changes in the availability of the neurotransmitters dopamine, noradrenaline and serotonin in the cerebellum, but not in the cortex, and reflect the known regional differences in neurotransmitter regulation. Immunoprecipitation studies and the observed increase in MAO-A activity following in vitro chemical inhibition of the ã-secretase complex (comprising several proteins including PS-1) support the notion that PS-1 constitutively associates with MAO-A. These effects on Ca2+-sensitive MAO function could contribute to AD-related pathology and could also contribute to the depression often associated with AD.

Monoamine oxidase-A (MAO-A) is an enzyme that has historically been linked to major depressive disorder (MDD). The prevalence of MDD among breast cancer patients is almost 25%, but realistically it is underdiagnosed within this patient population. Most breast cancer is deemed estrogen receptor positive [ER(+)] and is commonly treated with the anti-estrogenic chemotherapeutic compound tamoxifen. Resistance to tamoxifen has been associated with a paradoxical activation of the stress-associated kinase, p38(MAPK) (normally associated with cell death). Our research group has recently demonstrated that p38(MAPK) can regulate the function of MAO-A in glial cells. Taken together, MAO-A, depression and p38(MAPK) are all associated with a poor prognosis in breast cancer patients, particularly those with an ER(+) status. Several mechanisms have been proposed in each respect and we hope to further elucidate this relationship by focussing on the interaction between MAO-A and p38(MAPK) in the context of breast cancer. The hypothesis states that a functional interaction between the p38(MAPK) and MAO-A systems alters breast cancer cells in an ER-dependent manner. The proposed objectives of this project are to determine what might be influencing MAO-A function in breast cancer cells, and how changes in MAO-A function affect cell phenotype. Using pharmacological approaches (i.e. antidepressant drugs), we investigated the role of MAO-A and p38(MAPK) on selected characteristics of ER(+) (e.g. MCF-7) and ER(-) (e.g. MDA-MB-231) breast cancer cells under four treatment conditions, which include clorgyline (CLG), an antidepressant MAO-A inhibitor, and SB203580, an inhibitor of p38(MAPK). Our results indicate that the very high MAO-A activity in MDA-MB-231 (MB-231) cells was partly dependent on p38(MAPK) activity. The tumourigenic properties (e.g. anchorage-independent growth, migration) of MB-231 cells depended on both MAO-A and p38(MAPK) functions, although the effects were not additive suggesting that both inhibitors were exerting their respective effects via common signalling targets. The role of MAO-A and p38(MAPK) on MB-231 mitochondrial function and cell growth was negligible. In contrast, MAO-A and p38(MAPK) only influenced mitochondrial function in MCF-7 cells and did not affect any of the other tumourigenic properties measured. Immunocytochemical methods, supported by Western blotting, revealed an increase in E-cadherin expression in both cell lines. This suggested that MAO-A and p38(MAPK) could be influencing transitions between epithelial and mesenchymal phenotypes. Our in vitro findings suggest that MAO-A and p38(MAPK) might contribute to a common mechanism in breast cancer cell lines, but that their influence on cell phenotype is less dependent on the respective cell's ER status and perhaps more so dependent on the cell's metastatic potential. If this is the case, then the contribution of MAO-A and p38(MAPK) to [clinical] metastatic breast cancer should be duly considered. Our ongoing investigations are focussing on the influence of clinically relevant antidepressants on breast cancer cell phenotype in vitro.