The traditional view of drug design is that a single drug should interact with a single molecular target. As science progressed, there was an understanding that most drugs interact with more than one target and that multiple targets may be responsible for either adverse effects or additional therapeutic effects. The idea of polypharmacology, which suggests that the focus of drug design should shift from a single drug that interacts with a single target to a single drug that can have interactions with multiple targets and multiple therapeutic effects, revolutionized the drug discovery process. Discovering new drugs is a long and costly process with years of research and development and clinical trials required before the drugs reach the market for much needed therapeutic applications. By repurposing drugs that are already on the market for a new therapeutic target, the discovery process is accelerated significantly. One such a target disease, for which there is a great need for new effective therapies, is Parkinson’s disease (PD). PD is a progressive neurodegenerative disease that is caused by the death of dopaminergic neurons in the substantia nigra with the resulting loss of dopamine from the striatum. Degeneration in PD leads to varying degrees of motor difficulty and disability, along with other symptoms. Current therapies are focussed on symptomatic management and an improvement of the quality of life of patients, rather than on a cure. There are several therapeutic targets that are currently used in the treatment of PD. One of those targets is the monoamine oxidase (MAO) enzymes, in particular the MAO-B isoform. The MAO enzymes are responsible for the metabolism of amine neurotransmitters, such as dopamine, and inhibition of MAO-B has proven to be an effective strategy to increase the dopamine levels in the brain. Clinically, selective MAO-B inhibitors are administered concurrently with levodopa (a precursor of dopamine) to increase the levels of dopamine derived from levodopa. This approach prolongs the beneficial effects of levodopa. Because MAO-A is responsible for the breakdown of noradrenalin, adrenalin, serotonin and tyramine, non-selective and selective MAO-A inhibitors have therapeutic applications in other neurological and psychiatric disorders such as depression. MAO-A inhibitors, particularly irreversible inhibitors, are also notable from a toxicological point of view. Irreversible MAO-A inhibitors may lead to potentially dangerous effects when combined with serotonergic drugs and certain foods containing tyramine, such as cheeses and processed meats. Selective MAO-B inhibitors and reversible MAO-A inhibitors appear to be free of these interactions. Based on the considerations above, this study aimed to identify clinically used drugs which also inhibit the MAO enzymes as a secondary pharmacological property. Such drugs may, in theory, be repurposed as MAO inhibitors for therapeutic use in the treatment of PD and depression. The identification of potential MAO-A inhibitory properties among clinically used drugs are of further importance since the irreversible inhibition of MAO-A may lead to dangerous effects when combined with certain drugs and foods. To screen clinically used drugs for potential MAO-A and MAO-B inhibitory activities, a pharmacophore approach was followed. A pharmacophore model is a virtual 3D representation of the common steric and electrostatic features of the interaction between an enzyme and a ligand. By identifying hydrogen bond acceptor, hydrogen bond donor and hydrophobic interactions between a reference ligand and an enzyme, a model is created that can search databases for other molecules that would have similar interactions with the enzyme and arguably also act as ligands. This enables the screening of a large amount of molecules in a short amount of time. To assist in the identification of MAO inhibitors, pharmacophore models of the MAO enzymes were constructed using the known crystallographic structures of MAO-A co-crystallized with harmine, and MAO-B cocrystallized with safinamide. The Discovery Studio® software package (Accelrys) was used for this purpose. In this study, virtual libraries of United States Food and Drug Administration (FDA) approved drugs and the United States Environmental Protection Agency (EPA) maximum daily dose databases were screened with pharmacophore models of MAO-A and MAO-B. Among the hits, 26 drugs were selected on the basis of availability and cost, and were subjected to in vitro bio-assays in order to determine their potencies (IC50 values) as inhibitors of recombinant human MAO-A and/or MAO-B. Among the drugs tested, 6 compounds exhibited inhibitory activity towards the MAO enzymes. Of the 6 compounds, pentamidine (IC50 = 0.61 μM for MAO-A and IC50 = 0.22 μM for MAO-B) and phenformin (IC50 = 41 μM for MAO-A) were selected for further analysis. An examination of the recoveries of the enzymatic activities after dilution and dialysis of the enzyme-inhibitor complexes showed that both pentamidine and phenformin interact reversibly with the MAO enzymes. A kinetic analysis suggests that pentamidine acts as a competitive inhibitor with estimated Ki values of 0.41 μM and 0.22 μM for the inhibition of MAO-A and MAO-B, respectively. An analysis of the available pharmacokinetic data and typical therapeutic doses of phenformin and pentamidine suggests that the MAO inhibitory potencies (and reversible mode of action) of phenformin are unlikely to be of pharmacological relevance in humans. Pentamidine, on the other hand, is expected to interact with both MAO-A and MAO-B at typical therapeutic doses. Because of its MAO-A inhibitory activity, pentamidine may thus, in theory, lead to a tyramine-associated hypertensive crisis when combined with tyramine-containing foods. However, pentamidine is unlikely to inhibit central MAO since it does not appear to penetrate the central nervous system to a large degree. In an attempt to gain further insight into the mode of binding to MAO, pentamidine and phenformin were docked into models of the active sites of MAO-A and/or MAO-B. An analysis of the interactions between the enzyme models and the ligands were carried out and the results are discussed in the dissertation. The results of this study show that the pharmacophore model approach may be useful in identifying existing drugs with potential MAO inhibitory effects. The search for new therapeutic MAO inhibitors, that can be used in the treatment of certain neurological disorders, including PD and depression, may be accelerated by employing a virtual screening approach. Such an approach may also be more cost effective than the de novo design of MAO inhibitors. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

The traditional view of drug design is that a single drug should interact with a single molecular target. As science progressed, there was an understanding that most drugs interact with more than one target and that multiple targets may be responsible for either adverse effects or additional therapeutic effects. The idea of polypharmacology, which suggests that the focus of drug design should shift from a single drug that interacts with a single target to a single drug that can have interactions with multiple targets and multiple therapeutic effects, revolutionized the drug discovery process. Discovering new drugs is a long and costly process with years of research and development and clinical trials required before the drugs reach the market for much needed therapeutic applications. By repurposing drugs that are already on the market for a new therapeutic target, the discovery process is accelerated significantly. One such a target disease, for which there is a great need for new effective therapies, is Parkinson’s disease (PD). PD is a progressive neurodegenerative disease that is caused by the death of dopaminergic neurons in the substantia nigra with the resulting loss of dopamine from the striatum. Degeneration in PD leads to varying degrees of motor difficulty and disability, along with other symptoms. Current therapies are focussed on symptomatic management and an improvement of the quality of life of patients, rather than on a cure. There are several therapeutic targets that are currently used in the treatment of PD. One of those targets is the monoamine oxidase (MAO) enzymes, in particular the MAO-B isoform. The MAO enzymes are responsible for the metabolism of amine neurotransmitters, such as dopamine, and inhibition of MAO-B has proven to be an effective strategy to increase the dopamine levels in the brain. Clinically, selective MAO-B inhibitors are administered concurrently with levodopa (a precursor of dopamine) to increase the levels of dopamine derived from levodopa. This approach prolongs the beneficial effects of levodopa. Because MAO-A is responsible for the breakdown of noradrenalin, adrenalin, serotonin and tyramine, non-selective and selective MAO-A inhibitors have therapeutic applications in other neurological and psychiatric disorders such as depression. MAO-A inhibitors, particularly irreversible inhibitors, are also notable from a toxicological point of view. Irreversible MAO-A inhibitors may lead to potentially dangerous effects when combined with serotonergic drugs and certain foods containing tyramine, such as cheeses and processed meats. Selective MAO-B inhibitors and reversible MAO-A inhibitors appear to be free of these interactions. Based on the considerations above, this study aimed to identify clinically used drugs which also inhibit the MAO enzymes as a secondary pharmacological property. Such drugs may, in theory, be repurposed as MAO inhibitors for therapeutic use in the treatment of PD and depression. The identification of potential MAO-A inhibitory properties among clinically used drugs are of further importance since the irreversible inhibition of MAO-A may lead to dangerous effects when combined with certain drugs and foods. To screen clinically used drugs for potential MAO-A and MAO-B inhibitory activities, a pharmacophore approach was followed. A pharmacophore model is a virtual 3D representation of the common steric and electrostatic features of the interaction between an enzyme and a ligand. By identifying hydrogen bond acceptor, hydrogen bond donor and hydrophobic interactions between a reference ligand and an enzyme, a model is created that can search databases for other molecules that would have similar interactions with the enzyme and arguably also act as ligands. This enables the screening of a large amount of molecules in a short amount of time. To assist in the identification of MAO inhibitors, pharmacophore models of the MAO enzymes were constructed using the known crystallographic structures of MAO-A co-crystallized with harmine, and MAO-B cocrystallized with safinamide. The Discovery Studio® software package (Accelrys) was used for this purpose. In this study, virtual libraries of United States Food and Drug Administration (FDA) approved drugs and the United States Environmental Protection Agency (EPA) maximum daily dose databases were screened with pharmacophore models of MAO-A and MAO-B. Among the hits, 26 drugs were selected on the basis of availability and cost, and were subjected to in vitro bio-assays in order to determine their potencies (IC50 values) as inhibitors of recombinant human MAO-A and/or MAO-B. Among the drugs tested, 6 compounds exhibited inhibitory activity towards the MAO enzymes. Of the 6 compounds, pentamidine (IC50 = 0.61 μM for MAO-A and IC50 = 0.22 μM for MAO-B) and phenformin (IC50 = 41 μM for MAO-A) were selected for further analysis. An examination of the recoveries of the enzymatic activities after dilution and dialysis of the enzyme-inhibitor complexes showed that both pentamidine and phenformin interact reversibly with the MAO enzymes. A kinetic analysis suggests that pentamidine acts as a competitive inhibitor with estimated Ki values of 0.41 μM and 0.22 μM for the inhibition of MAO-A and MAO-B, respectively. An analysis of the available pharmacokinetic data and typical therapeutic doses of phenformin and pentamidine suggests that the MAO inhibitory potencies (and reversible mode of action) of phenformin are unlikely to be of pharmacological relevance in humans. Pentamidine, on the other hand, is expected to interact with both MAO-A and MAO-B at typical therapeutic doses. Because of its MAO-A inhibitory activity, pentamidine may thus, in theory, lead to a tyramine-associated hypertensive crisis when combined with tyramine-containing foods. However, pentamidine is unlikely to inhibit central MAO since it does not appear to penetrate the central nervous system to a large degree. In an attempt to gain further insight into the mode of binding to MAO, pentamidine and phenformin were docked into models of the active sites of MAO-A and/or MAO-B. An analysis of the interactions between the enzyme models and the ligands were carried out and the results are discussed in the dissertation. The results of this study show that the pharmacophore model approach may be useful in identifying existing drugs with potential MAO inhibitory effects. The search for new therapeutic MAO inhibitors, that can be used in the treatment of certain neurological disorders, including PD and depression, may be accelerated by employing a virtual screening approach. Such an approach may also be more cost effective than the de novo design of MAO inhibitors. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Genomic clones were isolated and characterized using the human monoamine oxidase A (MAOA) cDNA to screen a phage library, constructed from a human 4X cell line (48, XXXX). The genomic contig derived from overlapping phage clones showed that the size of the MAOA gene is over 80 kb. Exon-containing fragments from these phage clones were subcloned and sequenced. The data from this showed that the MAOA gene consists of 15 exons. A YAC (yeast artificial chromosome) isolated using the MAOA cDNA was characterized. This YAC was found to contain both the MAOA and the MAOB genes. Using PFGE (pulsed-field gel electrophoresis) to investigate the YAC, it was found that the MAOA and the MAOB genes are located within 50 kb and adjacent to each other. The two genes are localized in a 3'-to-3' fashion, suggesting their expression may be regulated independently. The analysis of the homology shown by the two genes clearly demonstrated that they were derived from duplication of a common ancestral gene. A CpG island was discovered to be associated with the 5' end of both genes. A restriction map of -2.5 Mb of genomic DMA around the MAO genes was generated by PFGE. Long-range mapping defined the physical relationship between the marker L1.28 and the MAO genes as L1.28_MAOA_MAOB. A number of genetic diseases have been linked to the Xp11.3 region. Strong linkage was known to exist between the Norrie disease locus and L1.28. Studies showed that some of the Norrie patients have deletions encompassing the region which contains L1.28 as well as the MAO genes. Another YAC isolated by using L1.28 as the probe was also characterized. A phage library was constructed from the L1.28 YAC and the end clones were isolated. Studies on some of the Norrie deletion patients showed that the proximal end clone of the YAC was retained in one of the deletion patients. Previous studies had shown that the Norrie disease locus was also localized proximal to the 5' end of the MAOB gene. The combined information placed the disease locus to an interval of 240 kb within the YAC. More phage clones were characterized in order to define further the region for the Norrie locus which was finally localized within 160 kb. A YAC fragment of 160 kb was isolated and used to screen two human retinal cDNA libraries. Among the cDNAs isolated, one group was found to be deleted in some of the Norrie patients previously without any known deletion, which established their candidacy as the transcripts of the Norrie disease locus. Further characterization of the candidate gene showed that it is conserved across species. The expression of the gene was detected in various tissues. The homology shared between the NDP gene and some of the growth factor binding proteins suggests its role in neural cell proliferation and differentiation.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Ruh Sağlığı ve Hastalıkları Anabilim Dalı, 2006. / Bibliyografya var.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1959. / Typescript. Abstracted in Dissertation abstracts, v. 19 (1959) no. 11, p. 2745-2746. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Research in drug development meets many challenges including lengthy, complex and costly procedures to identify novel pharmacotherapies. In our lab, we developed a method for fast screening of small molecules that interact with monoamine transports – dopamine and serotonin (DAT, SERT). These membrane proteins play important roles in brain neurotransmission responsible for cognition, motion and pleasure. Dysfunction in dopaminergic and serotonergic systems result in neurological disorders such as depression, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and addiction. DAT and SERT are responsible for uptake of dopamine (DA) or serotonin (5HT) into the synapse and they limit neurotransmitter signaling. Drugs that mimic or antagonize actions of endogenous neurotransmitters (DA and 5HT) increase the concentrations of DA and/or 5HT either by blocking the transporter (blockers) or by competing uptake with neurotransmitter (substrate). The uptake of substrates is associated to an inward current that depolarizes the cell membrane. Voltage-gated calcium channels (CaV) can respond to small changes in membrane potential. In our method, we combined permanent cell line expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) (FlpIn TREx expression system) with transient transfection of CaV. This system works as a tightly electrically coupled system. Cells challenged with substrate of the transports produce detectable Ca2+ signal while monoamine transporter blockers can inhibit these Ca2+ signals. The novelty of this method relies on the ability to discriminate between substrate and blockers of monoamine transporters. Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters. Our novel methodology can rapidly assess drug-effect profile on monoamine transporters and benefit development of new psychotherapeutics for treatment of mental illnesses. It can also be used to characterize mechanism of action of emerging drug of abuse, as well as to discover small molecules with novel drug-effect profile useful in basic neuroscience research.

Investigations into the cognitive effects of tobacco smoking have generally focused on nicotine and its effect on nicotinic acetylcholine receptors (nAChRs) in the brain. However, it is now known that chronic smokers exhibit robust inhibition of the monoamine oxidase (MAO) enzyme through the actions of non-nicotine components in tobacco smoke. Therefore, the primary aim of this thesis is to elucidate the effects of nicotine and MAO-inhibition on electroencephalographic (EEG) and event-related potential (ERP) measures of cognition. 24 healthy nonsmoking males were administered 75 mg of moclobemide, and chewed 6 mg nicotine gum, in order to simulate the effects of acute smoking. Four experimental conditions included placebo, nicotine, moclobemide, and a combination of nicotine and moclobemide. Early auditory ERPs were used as measures of cognition, such as the auditory P50 sensory gating paired-stimulus paradigm, the acoustic-change-elicited mismatch-negativity (MMN), the novel sound-elicited P3a, and the target sound-elicited P3b. Three minutes of eyes closed EEG were also recorded. Because these ERPs are often identified as biomarkers for schizophrenia, drug effects were also measured after individuals were stratified for low-baseline amplitude of each ERP measure, as a laboratory model of cognitive deficits in schizophrenia. Overall results showed a synergistic improvement in sensory gating via nicotine combined with moclobemide, accompanied by a reduction in theta band power. Nicotine in the absence of moclobemide increased P3b amplitude, accompanied by an increase in alpha2 band power. Moclobemide in the absence of nicotine increased P3a amplitude, accompanied by a decrease in beta2 power. Stratifying participants by placebo amplitude revealed both nicotine and moclobemide exhibited an inverted-U pattern of effect, i.e. showing greater amplitude increases in individuals with the lowest baseline amplitudes. Overall, this thesis demonstrates how these two components of tobacco smoke affect different facets of auditory processing in different ways, with synergistic effects in some paradigms but antagonizing effects in others. Therefore, chronic smokers and schizophrenia patients who seek transient cognitive improvement through smoking may actually experience cognitive detriments overall, possibly contributing to withdrawal symptoms and/or an exacerbation of already-present psychiatric symptoms.

>Magister Scientiae - MSc / Alzheimer’s Disease (AD) is a neurodegenerative disease which results from the irreversible loss of neurons in the brain. The disease is characterized by progressive cognitive impairment with recurrent short-term memory loss. AD is the leading cause of dementia and 4th leading cause of death in the elderly. Success in the treatment of AD has been limited, with drugs only treating it at a symptomatic level due to its pathology being complex and poorly understood. However, it is known that the cholinesterase and MAO-B enzymes play an important role in the disease through their association with production of amyloid plaques and oxidative stress respectively, two mechanisms associated with cell death and the symptoms seen in AD.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tereza Kalinová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Mentor: Assoc. Prof. Jana Hroudová, Pharm.D., Ph.D. Title of diploma thesis: Effect of cholinesterase inhibitors on monoaminergic system and energy metabolism Cholinesterase (ChE) inhibitors play an essential role in the treatment of Alzheimer's disease (AD). They effect positively cognitive, functional and behavior symptoms of AD. Up to date, donepezil, rivastigmine and galantamine represent the only ChE inhibitors approved for AD treatment. The first ChE inhibitor was tacrine, which was withdrawn from market due to its toxicity and adverse effects. Recently, novel tacrine and 7-methoxytacrine (7-MEOTA) derivatives were synthetized and extensively investigated to find less toxic compounds affecting pathological mechanisms associated with development of AD. There is less known about effects of these drugs on mitochondrial functions and cellular energy metabolism. The aim of this project is to examine in vitro effects of ChE inhibitors on energy metabolism and cellular respiration, specifically on mitochondrial electron transport chain complexes and an enzyme of the citric acid cycle - citrate synthase. Inhibitory effects...