The care of people with Parkinson’s disease (PD) involves input from different healthcare professionals (HCPs). A literature search identified that the HCPs involved in PD multidisciplinary (MDT) clinics, including interdisciplinary team (IDT), varied both in the number and type of HCPs. None of the studies identified involved pharmacists. Pharmacists have shown benefits when working in MDTs for other long-term conditions (LTCs); however, their role in PD MDTs was identified as a gap in the literature. The aim of the study was to determine the role of pharmacists in a PD IDT. Patients attended a PD IDT clinic comprising PD nurse, physiotherapist, occupational therapist and pharmacist. A mixed methods convergent design was used to collect both qualitative and quantitative data. Qualitative focus group and interview data were analysed using The Framework Method. Quantitative data analysis involved counts of HCP interventions. Pharmacists can support PD IDTs as they have in other LTC MDTs by conducting holistic medication reviews. Three new roles were identified for pharmacists in PD IDTs as: 1) independent prescribing, 2) supporting HCPS in their roles by supporting staff knowledge, and 3) leading the PD IDT clinics. Quantitative data showed the pharmacist made a medication review intervention for all patients. HCPs and patients identified the roles and value of involving pharmacists in PD IDTs. The PD IDT clinics offer a holistic approach to patient care and a greater opportunity for patients to be involved. The findings identified a ‘review-shared care template’ for PD IDTs and recommends development of a ‘pharmacist’s PD competency framework’.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Please see fulltext for abstract / AFRIKAANSE OPSOMMING: Parkinson se siekte ( PD ) is 'n toenemend algemene neurodegeneratiewe siekte wat die progressiewe verlies van dopaminergiese neurone in die substantia nigra pars compacta behels, wat lei tot die ontwrigting van die motororiese senuweestelsel. ’n Neuronale verlies van meer as 50% van die normale vlakke is nodig vir die ontwikkeling van die kliniese simptome, insluitende bewing, spier rigiditeit, bradykinesia, en posturale onstabiliteit. Alhoewel beskikbare medikasies vir PD wel simptome onderdruk, is dit nie in staat om die siekte te voorkom of vordering daarvan te verhoed nie. Een van die mees bedeidende risikofaktore vir PD is toenemende ouderdom. Huidige epidemiologiese studies voorspel ‘n toename in populasie ouderdom wat belangrike implikasies vir die voorkoms van PD kan inhou. Die molekulere meganismes onderliggend aan die neurale agteruitgang in PD is steeds onbekend, maar dit blyk dat ‘n aantal genetiese faktore (in kombinasie met omgewingsfaktore) bydra tot die komplekse patogeniese agteruitgang van die siekte. Agt bevestigde gene is gevind om direk betrokke te wees by die etiologie van PD, naamlik: parkin, PINK1, DJ-1, ATP13A2, SNCA, LRRK2, VPS35, en EIF4G1. Hierdie gene is oorwegend geïdentifiseer en bestudeer in die Europese, Noord-Amerikaanse en Asiatiese bevolkings; terwyl die bestudering van Sub -Sahara- Afrika (SSA) bevolkings, veral dié van Swart Afrika afkoms, onderverteenwoordig bly in genetiese studies. Siende dat resultate verkry vanaf ander bevolkings nie verwant is aan die SSA bevolking nie, en ook nie ‘n voorspelling van wereldwye toename van die siekte kan verteenwoordig nie, is dit van belang dat omvattende genetiese studies uitgevoer word op hierdie onderbestudeerde bevolkings van SSA. Desnieteenstaande, die doel van die studie was om die molekulere etiologie van PD in ‘n groep Swart Suid-Afrikaanse (SA) PD pasiente te ondersoek. ‘n Totaal van 47 pasiente is gewerf vir die studie waarvan 26% ‘n famielie geskiedenis het vir die afwyking. Die gemiddelde ouderdom van aanvang vir die pasiente was 55.3 ± 11.2 jaar. Mutasie volgorderbepaling vir alle bekende PD gene is uitgevoer. Addisionele mutasie volgorderbepaling vir die GBA is ook uitgevoer siende dat heterosigotiese mutasies in die geen dien as ‘n sterk risikofaktor vir die ontwikkeling van PD. ‘n Verskeidenheid mutasie volgordebepalingstegnieke is geinkorporeer vir die studie, naamlik: Sanger volgordebepaling en hoë resolusie smelt tegniek (vir die identifisering van missense mutasies asook kleinskaalse invoegings of weglatings), en multiplex-afbinding afhanklike ondersoek versterkingstoets (vir die opsporing van veranderinge in kopiegetal). Verder is volgende generasie volgordebepaling gebruik vir die sistematiese ondersoek van die bekende PD gene, asook vir 160 kandidaat gene wat vooraf bepaal is. Fluoreserend-geetiketeerde polymerase ketting reaksie primers is gebruik vir genotipering van CAG herhalings uitbreidings in die ataxin-2 (SCA2) en die ataxin-7 (SCA7) gene, gevolg deur elektroforese met behulp van die “ABI 3130x1 Genetic Analyzer”. Mutasie volgordebepaling het voor die lig gebring dat die bekende PD gene klaarblyklik nie ‘n beduidende oorsaaklike rol speel in die patogenese van die siekte in die huidige groep Swart SA pasiente nie, want slegs 2 uit die 47 (43%) pasiente koester mutasies in parkin. Een van die pasiente besit ‘n heterosigotiese duplikasie van ekson 2 en ‘n heterosigotiese weglating van ekson 9; in die ander pasient is ‘n heterosigotiese ekson 4 weglating en ‘n heterosigotiese G430D mutasie geïdentifiseer. Verder is ‘n Q311K verandering in parkin gevind asook vier nuwe variante (I610T, H1758P, N2133S en T2423S) in die LRRK2 geen. Die patogenisiteit van hierdie variante moet egter nog bepaal word. Geen patogeniese herhaalings uitbreidings is gevind in die SCA2 en SCA7 lokusse nie, en die moontlikheid van die twee spinocerebellar ataksie subtipes as genetiese bepalers vir PD is uitgekanselleer. Geen patogeniese mutasies is gevind in enige van die oorblywende bekende PD gene nie, dit is dus waarskeinlik dat die pasiente wel mutasies in nuwe PD-geassosieerde gene besit. Hierdie is die eerste molekulere genetiese studie uitgevoer op uitsluitlik Swart SA PD pasiente, en ook die eerste omvattende ondersoek van al die bekende PD gene in ‘n SSA bevolking. Die algemene patogeniese mutasies in gene, wat voorheen bewys is as siekte veroorsakend in ‘n aantal Europese bevolkings, is nie bespeur in die huidige studie nie en kan dus nie verantwoordelik gehou word vir die voorkoms van PD in hierdie pasiente nie. Maar, siende dat die steekproefgrootte in hierdie studie relatief klein was, kan addisionele volgordebepaling van ‘n groter pasient groep voordelig wees in die bepaal van nuwe siekte-veroorsakende gene wat die potensiaal het om huidige hipoteses, dat die genetiese etiologie van PD duidelik verskil oor verskeie etniese groepe wereldwyd, te weerlê of bevestig. Die voortsetting van genetiese ondersoeke onder hierdie groep pasiente kan bydra tot insig met betrekking tot bevolking-spesifieke genetiese bepalers, en uiteindelik tot die identifisering van nuwe teikens vir medikasies teen die siekte.

Neurodegenerative diseases, such as Parkinson’s disease (PD), likely initiate their pathologies primarily within the brain and later manifest themselves in daily behavioral functions. In patients with PD, the loss of dopaminergic neurons in the basal ganglia results in sensorimotor deficits, including tremor, bradykinesia, olfactory function loss, speech/voice loss, and eating disorders. Although not much is known about the etiology of Parkinson’s disease, extensive studies have focused on correlating different signs of motor degradation with the degree of dopaminergic neuron loss. Despite the fact that different animal models and diverse behavioral methods have been developed to further characterize limb motor function loss, the loss of fine oromotor function, which includes eating/biting disorders and voice/speech loss, has been largely overlooked due to its intrinsic complexity as well as the lack of a precise method for quantitative description. An animal model was developed for the study of oromotor deficits in PD that utilizes the production of ultrasonic vocalization in lab rodents. Parkinsonian animals suffer the same symptoms in their vocalization compared to human PD patients: a significant drop of intensity and pitch variation. Furthermore, a newly developed biting test provided evidence that the animal’s oromotor function have been compromised due to dopamine loss. Overall, these studies show that qualitative analysis of the ultrasonic vocalizations (USVs) of laboratory rats can serve as a sensitive behavioral biomarker for the detection of subtle oromotor deficits in neurodegenerative diseases. / text

The homeobox gene Pitx3 has been implicated as a key regulator for lens development because homozygous mutant aphakia mice, which are hypomorph for Pitx3, fail to develop lenses. One aim of my thesis is to investigate the underlying cellular and molecular mechanism of Pitx3 mediated lens defect by studying knockout mice lacking Pitx3. Chimeric embryos, generated by aggregating the wild type embryos with Pitx3 heterozygous or Pitx3 homozygous mutant ES cells, have been used to analyse lens development. Pitx3 null cells failed to colonise the lens epithelium in Pitx3 null wild type chimeric lens, suggesting that Pitx3 is cell-autonomously required for lens epithelial cells. Further study of Pitx3 null mice revealed an earlier downregulation of the lens epithelial markers PDGFR-alpha and E-cadherin in E11.5 lens epithelium, suggesting the loss of lens epithelial identity in Pitx3 deficient mice. Furthermore, cell cycle inhibitors p27KIP1 and p57KIP2 were ectopically expressed throughout the morphologically normal Pitx3 mutant lens vesicle, suggesting that inactivation of Pitx3 leads to cell cycle exit of epithelial lens cells. In addition, precocious activation of the fibre cell-specific proteins beta- and gamma-crystallins was observed in Pitx3 null lens. Beta-crystallin expression could be observed as early as E10.5 throughout the entire Pitx3 null lens vesicle and gamma-crystallin was detected in the malformed Pitx3 deficient lens at E11.5. RNA in situ hybridisation study revealed that the expression of the transcription factor Foxe3 was lost in Pitx3 null lens at E10.5, suggesting that Pitx3 maintains the lens epithelial cells partly via the regulation of transcription factor Foxe3 during lens development. Accordingly, this study provides the cellular and molecular basis for the lens defect observed in Pitx3 null and Pitx3 hypomorph aphakia mice. Pitx3 is a key transcription factor for the maintenance of lens epithelium and its absence leads to premature activation of fibre cell differentiation programme of lens epithelial cells. In the other part of my PhD, I have further developed the Pitx3-GFP knockin ES cell system with a goal to use this tool for the identification of determinants of midbrain dopaminergic (mDA) neurons, the type of cells lost in Parkinson’s disease (PD) patients. Experimental cell therapy and clinical trials have shown that foetal midbrain tissues, but not tissues from other DA neuron containing regions, can functionally restore the lost mDA neurons when transplanted in Parkinson’s disease patients. Therefore, it is essential to coax mDA properties on stem cell-derived neurons when considering therapeutic development. Within the central nervous system, Pitx3 is expressed exclusively in mDA neurons. Using a Pitx3-GFP knockin mouse line previously generated in the laboratory I have derived heterozygous and homozygous Pitx3-GFP ES cells from mouse blastocysts. In keeping with previous findings in our laboratory, the heterozygous Pitx3-GFP (Pitx3GFP/+) ES cell-derived GFP positive cells of neuronal morphology can be detected after in vitro differentiation using the PA6 coculture system. Furthermore, I have shown that these cells express tyrosine hydroxylase and midbrain markers Engrailed-1 and Nurr-1, demonstrating their midbrain characteristics. I have also generated supertransfectable Pitx3GFP/+ ES cells to offer a rapid and efficient way to express a transgene episomally. The Cre-mediated inducible system of Pitx3-GFP reporter ES cells has also been developed in our laboratory and I have shown that they have high induction efficiency thus allows transgene activation in a temporally controlled manner. The Pitx3 null ES cells showed impaired potential to differentiate into mDA neurons thus they may be used to evaluate candidate Pitx3 downstream target by gain-of-function test. In summary, I have developed a Pitx3-GFP reporter ES cell system to identify mDA regulators functionally by in vitro differentiation.

Oxidative stress is caused when there are more reactive oxygen species (ROS), than antioxidants to scavenge them, resulting in damage to cellular components. It has been implicated as a major player at multiple points in the disease process of Parkinson’s disease (PD) and many other conditions. For example, evidence suggests oxidative damage to the α-synuclein protein may affect its aggregation propensity. In addition, α-synuclein may increase ROS production. However, how this oxidative stress relates to neurodegeneration is not known. Therefore, there is a need for models of α-synucleinopathies and tools to assess the involvement of oxidative stress in the disease process. In order to model α-synucleinopathies, overexpression of the α-synuclein protein was used. A BacMam viral expression system containing human α-synuclein was generated and used to assess toxicity. α-Synuclein overexpression in undifferentiated or differentiated SH-SY5Y cells failed to show toxicity. However, the stability of α-synuclein protein expression and the cell line used may have influenced in the lack of toxicity. The current work provides important guidance for future experimental design. Flavonoids are found in plants and have antioxidant capability. AO-1-530 is a synthetic compound with a flavonoid head group and a long hydrocarbon tail. It is highly cell permeable and localises to the mitochondria. In order to investigate its protective properties, toxin-induced oxidative stress cell assays were established. AO-1-530, in the low micromolar range, was protective against high doses of tert-butyl hydroperoxide (tBHP), whereas natural antioxidants, such as myricetin and quercetin, showed limited protection or required at least 10-fold higher concentrations to achieve similar protection. The ability of AO-1-530 to directly scavenge radicals was assessed cell-free in solution and in a cell-based assay. In solution the mechanism of action was investigated by electron paramagnetic resonance (EPR) spectroscopy. AO-1-530 had similar scavenging ability to myricetin, but was a slightly stronger scavenger than quercetin. The intracellular scavenging ability was quantified by CellROX® Deep Red live imaging. Although the compounds had similar cell-free scavenging abilities, AO-1-530 significantly out-performed both myricetin and quercetin in the intracellular assay, suggesting the mitochondrial localisation is critical to its highly protective properties. AO-1-530 is a powerful, novel tool to study the involvement of oxidative stress in diverse disease models.

Apathy is characterised by a lack of motivation towards goal directed behaviour and is a symptom of various neurodegenerative diseases. There are various tools that can be used to assess apathy but a caveat of these is that they usually assess it as a unidimensional concept. Apathy has been recognised to have a multidimensional substructure. The Dimensional Apathy Scale is the only comprehensive measure designed to quantify neurobiologically-based subtypes, called Executive, Emotional and Initiation apathy. The first aim of this study was to explore multidimensional apathy, and its associations with demographic variables, in healthy, community dwelling adults. Secondly, multidimensional apathy was explored in neurodegenerative diseases, specifically Amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD). For each disease group, the validity and reliability of both the self rated and carer rated DAS were also determined. Finally, the association between specific apathy subtype impairments and executive dysfunction was explored in ALS patients. Four hundred healthy community dwelling adults, eighty-three ALS patients (seventy-five carers), thirty-four PD patients (thirty carers) and forty-nine AD patients (eighty-nine carers) were recruited for the questionnaire study. In the healthy community dwelling adults, Executive apathy decreased with age, whereas Emotional increased with age. Gender differences were also shown with higher apathy in males on Emotional apathy. There were also employment differences, in that Executive apathy was higher in unemployed individuals compared to those who were employed. Emotional apathy showed difference in type of employment, where full time employed individuals were significantly more apathetic than those employed part time. These findings were taken into account in selecting the appropriate control samples to match our patient groups. In the patient groups, ALS patients were found to be significantly more impaired on the Initiation subscale when compared to controls. Furthermore, Initiation apathy was found to be the most frequent impairment above abnormality cut-off on the carer rated DAS. PD patients were significantly more impaired on Executive and Initiation apathy when compared to controls. These two subscales were most frequently above abnormality cut-off in the carer rated DAS. Finally, AD patients were significantly more impaired on all subscales when compared to controls and, on the carer rated DAS, global impairment over all subscales was most often reported as above abnormality cut-off. Additionally in AD, there was a significant disparity between carer and patient ratings on Executive and Initiation apathy, indicating patients’ impaired awareness. When comparing patient groups, there was a significant difference between carer rated apathy subtype impairments for each patient group. Validity and reliability of the DAS was found to be robust when compared to standard measures of apathy and depression. In the experimental study, a sample of ALS patients (and their carers) and healthy controls (and their informants) were recruited to complete a battery of neuropsychological tests, the DAS, other apathy and depression measures. ALS patients were impaired on tasks of executive functioning when compared to controls. Furthermore, apathy subtype deficits were associated with executive dysfunction in ALS. In conclusion, apathy is a multidimensional concept that manifests in different subtype profiles dependent on neurodegenerative disease. This has further implications for understanding and assessment of cognitive dysfunction and neuropsychiatric symptoms, such as apathy, in ALS and other neurodegenerative disease patient groups.

Parkinson’s disease is a clinical syndrome manifesting with slowness and instability. As it is a progressive disease with varying symptoms, repeated assessments are necessary to determine the outcome of treatment changes in the patient. In the recent past, a computer-based method was developed to rate impairment in spiral drawings. The downside of this method is that it cannot separate the bradykinetic and dyskinetic spiral drawings. This work intends to construct the computer method which can overcome this weakness by using the Hilbert-Huang Transform (HHT) of tangential velocity. The work is done under supervised learning, so a target class is used which is acquired from a neurologist using a web interface. After reducing the dimension of HHT features by using PCA, classification is performed. C4.5 classifier is used to perform the classification. Results of the classification are close to random guessing which shows that the computer method is unsuccessful in assessing the cause of drawing impairment in spirals when evaluated against human ratings. One promising reason is that there is no difference between the two classes of spiral drawings. Displaying patients self ratings along with the spirals in the web application is another possible reason for this, as the neurologist may have relied too much on this in his own ratings.

The aim of this thesis is to investigate computerized voice assessment methods to classify between the normal and Dysarthric speech signals. In this proposed system, computerized assessment methods equipped with signal processing and artificial intelligence techniques have been introduced. The sentences used for the measurement of inter-stress intervals (ISI) were read by each subject. These sentences were computed for comparisons between normal and impaired voice. Band pass filter has been used for the preprocessing of speech samples. Speech segmentation is performed using signal energy and spectral centroid to separate voiced and unvoiced areas in speech signal. Acoustic features are extracted from the LPC model and speech segments from each audio signal to find the anomalies. The speech features which have been assessed for classification are Energy Entropy, Zero crossing rate (ZCR), Spectral-Centroid, Mean Fundamental-Frequency (Meanf0), Jitter (RAP), Jitter (PPQ), and Shimmer (APQ). Naïve Bayes (NB) has been used for speech classification. For speech test-1 and test-2, 72% and 80% accuracies of classification between healthy and impaired speech samples have been achieved respectively using the NB. For speech test-3, 64% correct classification is achieved using the NB. The results direct the possibility of speech impairment classification in PD patients based on the clinical rating scale.

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