No description available.

Voltage-gated Ca2+ (Cav) channels are activated upon depolarization. They specifically allow Ca2+ ions to come into the cell. These Ca2+ ions are bi-functional because they not only control cell excitability but also couple electrical activity to complex downstream signaling events, such as excitation-contraction coupling in muscles and neurotransmitter release in neurons. In the brain, Cav channels are expressed in the pre- or post-synaptic membrane of most excitable cells, neurons. In the past few years, their expression and function have also been characterized in many nonexcitable cells such as astrocytes. This dissertation focuses on the regulation of one subtype of postsynaptic Cav channels, Cav1.2, in neurons. In the first part of chapter I, I provide a literature overview of Cav channels in terms of their subtypes, localizations, physiological functions, and biophysical properties. For years, Cav channels were studied as single entities. But now, based on multiple proteomic studies, we know that these channels actually do not live alone. They interact with numerous proteins depending on the physiological conditions. Such interactions can anchor the channels to optimal sites of action, and tether Cav channels to their modulatory molecules. Therefore, it is crucial to understand how Cav channels are regulated by their macromolecular assembly. Among these protein partners, our lab studied the regulation of Cav channels by a subset of PDZ-domain containing proteins. Because these proteins play an important role in scaffolding and they colocalize with both pre- and post-synaptic Cav channels. Indeed, previous studies from our lab and other groups have revealed that PDZ proteins participate in a multitude of Cav regulation. The second part of chapter I introduces the diverse modulation of neuronal Cav channels by numerous PDZ proteins. In neurons, Cav1.2 channels regulate neuronal excitability and synaptic plasticity. Their functions have been implicated in learning, memory, and mood regulation. A study published in the journal Lancet showed that the gene encoding Cav1.2 is a common risk factor for five major psychiatric disorders. A PDZ protein, densin-180 (densin) is an excitatory synapse protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin exhibit similar behavioral phenotypes that closely match those in mice lacking Cav1.2. In chapter II and III, we investigated the functional impact of densin on Cav1.2 channels and their auxiliary subunit β2a. Besides the regulation of Cav channels by their interactome, we have also known for a long time that Ca2+ currents undergo a negative feedback regulation. This regulation is called Ca2+-dependent inactivation (CDI) and it is mediated by Ca2+ that directly traverses the pore. CDI has been described for Cav channels in multiple cell types. In the heart, CDI prevents excessively long cardiac action potentials, which in turn can prevent activity-dependent arrhythmia. In neurons, CDI may be neuroprotective by preventing excitotoxic Ca2+ overloads. In the last 18 years, two essential components have been revealed in the mechanism of CDI. One is the protein calmodulin (CaM). CaM interacts directly with sites on the C-terminus of Cav channels. It binds to the incoming Ca2+ ions and produces a mysterious conformational change that determines the conductance of the channel. The other molecular player is Cavβ protein family. Cavβ comprises four subfamilies β1 through β4, which generally enhance the channel inactivation, except β2a. In chapter IV, Xiaohan Wang from Roger Colbran’s lab in Vanderbilt University, and I identified a new molecular determinant for Cav1.2 CDI. The α2δ subunit is an extracellular component of the Cav channel complex. Similar to Cavβ subunits, α2δ subunits are essential for the biophysical properties, surface level, and trafficking of Cavα1 subunits. There are four isoforms of α2δ subunits (α2δ1 to α2δ4). They display distinct tissue distributions. Although the roles of α2δ subunits in Cav channel regulation were studied extensively, studies have proposed that the function of α2δ subunits may be in part or entirely independent of Cav channel complex, such as synaptogenesis. Considering the important role of α2δ in physiology and pathology, it is imperative to identify the factors that regulate the properties of α2δ. In chapter V, I explored the trafficking dynamics of α2δ1 and revealed a potential regulator of α2δ1 for its protein stability and localization. One beauty of doing research is that it always motivates us to think and ask more questions on our journey of demystifying nature. While looking at the evidence that I find, I realize how much more we could do in the future. In chapter VI, I conclude the findings of each chapter and share my perspectives on the future direction for these research projects.

Philosophiae Doctor - PhD / Neurodegenerative disorders are characterised by progressive loss of the brain’s physiological functions as a result of gradual degeneration of neurons in the central nervous system. Even though they are classified as diseases of the elderly, occurrence earlier in life is possible, but that would suggest the influence of genetic and/or environmental factors. Due to the continuous rise in modernisation and industrialisation over the years, there has been an increase in incidence and prevalence of neurodegenerative disorders. With the advances in technology and life expectancy, the rates of the common forms (Alzheimer’s disease and Parkinson’s disease), are expected to increase exponentially by 2050. Unfortunately, there is still no clinically approved treatment or therapy to slow down or halt the degenerative process as most registered drugs only offer symptomatic relief. Confounding this issue is the lack of definite mechanism of neurodegeneration, which is still poorly defined and not completely understood. Nonetheless, the pathology of most neurodegenerative disorders is believed to be a combination of interrelated processes that eventually leads to neuronal cell death. Among the postulated processes, the impact of excitotoxicity mediated by NMDA receptor over-activation is prominent and it is implicated in virtually all neurodegenerative disorders. With this basic insight, it is believed that molecules capable of inhibiting NMDA receptors and associated calcium channels, without affecting the normal physiological functions of the brain, could potentially serve as good neuroprotective drugs. Competitive and uncompetitive blockers (MK-801 and ketamine) have been explored, but none were clinically accepted due to undesirable side effects such as hallucinations, sedation and depression. However, NGP1-01, a polycyclic cage molecule, has been shown to be neuroprotective through modulation of NMDA receptors and voltage gated calcium channels and attenuation of MPP+ -induced toxicity. A similar approach could be useful in the design and development of new neuroprotective drugs. The aim of this study was to synthesise a series of open and rearranged cage-like molecules and explore their neuroprotective potential in neuroblastoma SH-SY5Y cells. The proposed structures, with norbornane scaffolds that contained different moieties, were designed to structurally resemble NGP1-01 and MK-801. Once synthesised, the compounds were purified and characterised, and were evaluated for their biological activities. Compounds were first screened for cytotoxicity at different concentrations. Thereafter, they were evaluated for neuroprotective effects against MPP+ -induced excitotoxicity and for calcium flux modulatory effects on NMDA receptor and voltage gated calcium channels. The norbornane derivatives were synthesised and characterised, and all final products were afforded in sufficient yields. All compounds with the exception of two compounds displayed good cytotoxic profiles towards the SH-SY5Y neuroblastoma cells at 10 µM, 50 µM and 100 µM concentrations as they demonstrated percentage cell viabilities close to 100% (control treated cells). Only two compounds showed percentage cell viability of 51% and 59% at 100 µM. Utilising the same cell line, all compounds, tested at 10 µM, attenuated MPP+ -induced toxicity after 24 hours of exposure to a neurotoxin. This was evident in the 23% to 53% enhancement (significant with p < 0.05) in cell viability when compared to the MPP+ only treated cells. In comparison to known NMDA receptor and/or voltage gated calcium channel blockers (MK-801, NGP1-01 or nimodipine), the synthesised compounds demonstrated mono or dual inhibition of calcium channels as they effectively attenuated calcium influx by blocking NMDA receptors and/or voltage gated calcium channels expressed in neuroblastoma SHSY5Y cells. This group of compounds were found to be more potent NMDA receptor inhibitors, probably due to similarities with MK-801 and memantine, than voltage gated calcium channel inhibitors. All compounds demonstrated moderate to good calcium inhibitory effects at NMDA receptors in the range of 23% to 70% while a selected few displayed very little or no activity at the voltage gated calcium channels. In conclusion, 27 compounds with norbornane scaffolds were successfully synthesised and evaluated for cytotoxicity and neuroprotection. The abilities of the synthesised compounds to protect neurons from the neurotoxin MPP+ and reduce calcium flux into neuronal cells were successfully demonstrated. These characteristics are essential in neuroprotection as they may prove significant in halting or slowing down the disease progression. The compounds showing a good cytotoxicity profile, neuroprotective effects and ability to reduce calcium overload, could potentially act as neuroprotective agents with good safety profiles or contribute as lead structures to the development and design of structurally related molecules that could clinically benefit people with neurodegenerative disorders.

No description available.

Amphetamine (AMPH) and its derivatives embody a remarkable breadth of pharmacology. These molecules exert their effects, both therapeutic and pathological, at the human monoamine transporters, which tune synaptic dynamics by evacuating monoamine neuromodulators from the synapse subsequent to neuronal impulses. These transporters are electrogenic, and the transporter-mediated current can be correlated to a surrogate measure of the change in membrane voltage: Ca++ currents from co-transfected L-type Ca++ channels. The present work makes use of this assay, with which it is possible to derive pharmacodynamic metrics from both substrates and inhibitors. This work presents data on a heretofore-unstudied class of amphetamine analogs: the enantiomers of N-Me 4-Me AMPH and N-Et 4-Me AMPH. Remarkably, while both enantiomers of the N-Me version of this compound function as substrates at hDAT, both enantiomers of the N-Et version are inhibitors. This switch does not occur at hNET, where all enantiomers of both N-Me and N-Et 4-Me AMPH function as substrates. Further, (S)-N-Et 4-Me AMPH is a substrate at dDAT. EC50 and IC50 values for all drugs at both transporters are presented. I present the results of super-resolution microscopic co-localization studies on the plasmalemmal spatial relation of the human dopamine transporter and voltage gated calcium channel, L-type 1.2 (CaV1.2). I discuss future aims toward a unified understanding of the mechanisms of monoamine transporter function, with an emphasis on what amphetamine can illuminate in this regard.

Calmodulin (CaM) is a ubiquitous calcium-binding protein responsible for the binding and activation of a vast number of enzymes and signaling pathways. It contains two lobes that bind two calcium ions each, separated by a flexible central linker. This structural flexibility allows CaM to bind and regulate a large number of diverse protein targets within the cell in response to Ca2+ gradients. Voltage gated calcium channels (CaVs), as main sources of extracellular Ca2+, are crucial for a number of physiological processes, from muscle contraction to neurotransmission and endocrine function. These large transmembrane proteins open in response to membrane depolarization and allow gated entry of Ca2+ ions into the cytoplasm. Their regulation is currently the subject of intense investigation due to its pharmacological and scientific importance. CaM has been previously shown to pre-associate and act as a potent inhibitor of one class of high-voltage activated (HVA) channels called L-type channels via its interaction with their C-terminal cytoplasmic region. This interaction is primarily mediated by a conserved CaM-binding motif called the ‘IQ’ motif (for conserved isoleucine and glutamine residues), although the exact molecular details of its involvement in inactivation are currently unclear. Elucidation of these details was the primary objective of this dissertation. Recently, a novel sequence motif within this channel called ‘NSCaTE’ (N-terminal spatial calcium transforming element) has been described as an important contributor to calcium-dependent inactivation (CDI) of L-type channels. It was presumed to be unique to vertebrates, but we also show its conservation in a distantly related L-type channel homolog of Lymnaea stagnalis (pond snail). The interaction of CaM with a number of peptides representing the different regulatory motifs (IQ and NSCaTE) for both mammalian and snail isoforms was characterized in an attempt to better understand their role in CDI. Biophysical work with peptides as well as electrophysiology recordings with an N-terminal truncation mutant of Lymnaea CaV1 homolog were performed to expand our understanding of how the interplay between these channel elements might occur. In brief, the most striking feature of the interaction concerns the strong evidence for a CaM-mediated bridge between the N- and C-terminal elements of L-type channels. Further investigation of the CaM interaction with both IQ and NSCaTE peptides using Ca2+-deficient CaM mutants reveals a preference of both peptides for the Ca2+-C-lobe of CaM, and a much higher affinity of CaM for the IQ peptide, suggesting that the N-lobe of CaM is the main interaction responsible for the physiological effects of NSCaTE. These results are consistent with our electrophysiology findings that reveal a distinct buffer-sensitive CDI in wild type LCaV1 that can be abolished by the N-terminal truncation spanning the NSCaTE region. In addition to L-type channels, CaM has also been shown to have an indirect role in the regulation of low-voltage activated (LVA) or T-type channels (CaV3.x), via their phosphorylation by CaM-dependent protein kinase II (CaMKII). Using a primary sequence scanning algorithm, a CaM-binding site was predicted in a cytoplasmic region of these channels that was also previously shown to be important in channel gating. Biophysical experiments with synthetic peptides spanning this gating brake region from the three human and the single Lymnaea isoform strongly suggest that there is a novel, bona fide CaM interaction in this channel region, and also hint that this interaction may be a Ca2+-dependent switch of some sort. The results confirm a possible new role for CaM in the direct regulation of these channels, although the exact mechanism remains to be elucidated.

Synaptic plasticity is thought to be integral to learning and memory. The two most common forms of plasticity are long-term potentiation (LTP) and long-term depression (LTD), both of which can be supported either by presynaptic changes in transmitter release probability (Pr), or by postsynaptic changes in AMPA receptor number. It is generally thought that the induction of LTP and LTD at Schaffer-collateral synapses in the hippocampus depends on the activation of NMDA receptors (GluN). Recent studies, however, have demonstrated that both increases and decreases in Pr can be induced under blockade of postsynaptic GluN receptors, suggesting that the activation of postsynaptic GluN receptors by glutamate is only a strict requirement for postsynaptic plasticity. In this thesis, I therefore re-examined the role of glutamate in presynaptic plasticity. I used single synapse imaging along with electrophysiological and pharmacological techniques to independently manipulate and monitor the levels of glutamatergic signalling during synaptic activity. I discovered that glutamate is inhibitory and unnecessary for the induction of LTP at the presynaptic locus. My findings support a novel model of presynaptic plasticity in which the net activity-dependent changes in Pr at an active presynaptic terminal is jointly determined by two opposing processes that can be simultaneously active: 1) postsynaptic depolarization, which, via the activation of L-type voltage-gated Ca²⁺ channels, increases Pr by driving the synthesis and release of nitric oxide from neuronal dendrites and 2) glutamate release, which through the activation of presynaptic GluN receptors, decreases Pr. Computationally, this model suggests that plasticity functions to reduce prediction-errors that arise during synaptic activity, and, thereby offers a biologically plausible mechanism by which neuronal networks may optimize learning at the level of single synapses.

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.

Compromised calcium homeostasis in the central nervous system (CNS) is implicated as a major contributor in the pathology of neurodegeneration. Dysregulation of Ca2+ homeostasis initiates downstream Ca2+–dependent events that lead to apoptotic and/or necrotic cell death. Increases in the intracellular free calcium concentration ([Ca2+]i) may be the result of Ca2+ influx from the extracellular environment or Ca2+ release from intracellular Ca2+ stores such as the endoplasmic reticulum (ER). Influx from the extracellular environment is controlled predominantly by voltage gated calcium channels (VGCC), such as L–type calcium channels (LTCC) and ionotropic glutamate receptors, such as the N–methyl–D–aspartate (NMDA) receptors. Ca2+ release from the ER occurs through the inositol–1,4,5–triphosphate receptors (IP3Rs) or ryanodine receptors (RyRs) via IP3–induced or Ca2+–induced mechanisms. Mitigation of Ca2+ overload through these Ca2+ channels offers an opportunity for pharmacological interventions that may protect against neuronal death. In the present study the ability of a novel series of polycyclic compounds, both the pentacycloundecylamines and triquinylamines, to regulate calcium influx through LTCC was evaluated in PC12 cells using calcium imaging with Fura–2/AM in a fluorescence microplate reader. We were also able for the first time to determine IC50 values for these compounds as LTCC blockers. In addition, selected compounds were evaluated for their ability to offer protection in apoptosis–identifying assays such as the lactate dehydrogenase release assay (LDH–assay), trypan blue staining assay and immunohistochemistry utilizing the Annexin V–FITC stain for apoptosis. We were also able to obtain single crystal structures for the tricyclo[6.3.0.02,6]undecane–4,9–dien–3,11–dione (9) and tricyclo[6.3.0.02,6]undecane–3,11–dione (10) scaffolds as well as a derivative, N–(3–methoxybenzyl)–3,11–azatricyclo[6.3.0.02,6]undecane (14f). We also evaluated the possibility that the polycyclic compounds might be able to modulate Ca2+ flux through intracellular Ca2+ channels. Computational methods were utilized to accurately predicted IC50 values and develop a QSAR model with marginal error. The linear regression model delivered r2 = 0.83, which indicated a favorable correlation between the predicted and experimental IC50 values. This model could thus serve as valuable predictor for future structural design and optimization efforts. Data obtained from the crystallographic analysis confirmed the NMR–data based structural assignments done for these compounds in previous studies. Obtaining structural information gave valuable insight into the differences in size and geometric constrains, which are key features for the LTCC activity of these compounds. vii In conclusion, we found that all of the compounds evaluated were able to attenuate Ca2+ influx through the LTCC, with some compounds having IC50 values comparable with known LTCC blockers such as nimodipine. Representative compounds were evaluated for their ability to afford protection against apoptosis induced by 200 ?M H2O2. With the exception of compound 14c (the most potent LTCC blocker in the series, IC50 = 0.398 ?M), most compounds were able to afford protection at two or more concentrations evaluated. Compound 14c displayed inherent toxicity at the highest concentrations evaluated (100 ?M). We concluded that compounds representing both types of structures (pentacycloudecylamines and triquinylamines) have the ability to attenuate excessive Ca2+ influx through the LTCC. In general the aza–pentacycloundecylamines (8a–c) were the most potent LTCC blocker which also had the ability to offer protection in the cell viability assays. However, NGP1–01 (7a) had the most favorable pharmacological profile overall with good activity as an LTCC blocker (IC50 = 86 ?M) and the ability to significantly attenuate cell death in the cell viability assays, exhibiting no toxicity. In addition to their ability to modulate Ca2+ influx from the extracellular environment, these compounds also displayed the ability to modulate Ca2+ flux through intracellular Ca2+ channels. The mechanisms by which they act on intracellular Ca2+ channels still remains unclear, but from this preliminary study it would appear that these compounds are able to partially inhibiting Ca2+–ATPase activity whilst possibly simultaneously inhibiting the IP3R. In the absence of extracellular Ca2+ these compounds showed the ability in inhibit voltage–induced Ca2+ release (VICaR), possibly by modulating the gating charge of the voltage sensor being the dihydropyridine receptors. In future studies it might be worthwhile to do an expanded QSAR study and evaluate the aza–pentacycloundecylamines. To clarify the mechanisms by which the polycyclic compounds interact with intracellular Ca2+ channels we should examine the direct interaction with the individual Ca2+ channels independently. The polycyclic compounds evaluated in this study demonstrate potential as multifunctional drugs due to their ability to broadly regulate calcium homeostasis through multiple pathways of Ca2+ entry. This may prove to be more effective in diseases where perturbed Ca2+ homeostasis have devastating effects eventually leading to excitotoxicity and cell death. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Compromised calcium homeostasis in the central nervous system (CNS) is implicated as a major contributor in the pathology of neurodegeneration. Dysregulation of Ca2+ homeostasis initiates downstream Ca2+–dependent events that lead to apoptotic and/or necrotic cell death. Increases in the intracellular free calcium concentration ([Ca2+]i) may be the result of Ca2+ influx from the extracellular environment or Ca2+ release from intracellular Ca2+ stores such as the endoplasmic reticulum (ER). Influx from the extracellular environment is controlled predominantly by voltage gated calcium channels (VGCC), such as L–type calcium channels (LTCC) and ionotropic glutamate receptors, such as the N–methyl–D–aspartate (NMDA) receptors. Ca2+ release from the ER occurs through the inositol–1,4,5–triphosphate receptors (IP3Rs) or ryanodine receptors (RyRs) via IP3–induced or Ca2+–induced mechanisms. Mitigation of Ca2+ overload through these Ca2+ channels offers an opportunity for pharmacological interventions that may protect against neuronal death. In the present study the ability of a novel series of polycyclic compounds, both the pentacycloundecylamines and triquinylamines, to regulate calcium influx through LTCC was evaluated in PC12 cells using calcium imaging with Fura–2/AM in a fluorescence microplate reader. We were also able for the first time to determine IC50 values for these compounds as LTCC blockers. In addition, selected compounds were evaluated for their ability to offer protection in apoptosis–identifying assays such as the lactate dehydrogenase release assay (LDH–assay), trypan blue staining assay and immunohistochemistry utilizing the Annexin V–FITC stain for apoptosis. We were also able to obtain single crystal structures for the tricyclo[6.3.0.02,6]undecane–4,9–dien–3,11–dione (9) and tricyclo[6.3.0.02,6]undecane–3,11–dione (10) scaffolds as well as a derivative, N–(3–methoxybenzyl)–3,11–azatricyclo[6.3.0.02,6]undecane (14f). We also evaluated the possibility that the polycyclic compounds might be able to modulate Ca2+ flux through intracellular Ca2+ channels. Computational methods were utilized to accurately predicted IC50 values and develop a QSAR model with marginal error. The linear regression model delivered r2 = 0.83, which indicated a favorable correlation between the predicted and experimental IC50 values. This model could thus serve as valuable predictor for future structural design and optimization efforts. Data obtained from the crystallographic analysis confirmed the NMR–data based structural assignments done for these compounds in previous studies. Obtaining structural information gave valuable insight into the differences in size and geometric constrains, which are key features for the LTCC activity of these compounds. vii In conclusion, we found that all of the compounds evaluated were able to attenuate Ca2+ influx through the LTCC, with some compounds having IC50 values comparable with known LTCC blockers such as nimodipine. Representative compounds were evaluated for their ability to afford protection against apoptosis induced by 200 ?M H2O2. With the exception of compound 14c (the most potent LTCC blocker in the series, IC50 = 0.398 ?M), most compounds were able to afford protection at two or more concentrations evaluated. Compound 14c displayed inherent toxicity at the highest concentrations evaluated (100 ?M). We concluded that compounds representing both types of structures (pentacycloudecylamines and triquinylamines) have the ability to attenuate excessive Ca2+ influx through the LTCC. In general the aza–pentacycloundecylamines (8a–c) were the most potent LTCC blocker which also had the ability to offer protection in the cell viability assays. However, NGP1–01 (7a) had the most favorable pharmacological profile overall with good activity as an LTCC blocker (IC50 = 86 ?M) and the ability to significantly attenuate cell death in the cell viability assays, exhibiting no toxicity. In addition to their ability to modulate Ca2+ influx from the extracellular environment, these compounds also displayed the ability to modulate Ca2+ flux through intracellular Ca2+ channels. The mechanisms by which they act on intracellular Ca2+ channels still remains unclear, but from this preliminary study it would appear that these compounds are able to partially inhibiting Ca2+–ATPase activity whilst possibly simultaneously inhibiting the IP3R. In the absence of extracellular Ca2+ these compounds showed the ability in inhibit voltage–induced Ca2+ release (VICaR), possibly by modulating the gating charge of the voltage sensor being the dihydropyridine receptors. In future studies it might be worthwhile to do an expanded QSAR study and evaluate the aza–pentacycloundecylamines. To clarify the mechanisms by which the polycyclic compounds interact with intracellular Ca2+ channels we should examine the direct interaction with the individual Ca2+ channels independently. The polycyclic compounds evaluated in this study demonstrate potential as multifunctional drugs due to their ability to broadly regulate calcium homeostasis through multiple pathways of Ca2+ entry. This may prove to be more effective in diseases where perturbed Ca2+ homeostasis have devastating effects eventually leading to excitotoxicity and cell death. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.