Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Pantothenate kinase (PanK) enzymes catalyze the first reaction in the five step biosynthesis of the essential cofactor coenzyme A. Enzymes representing each of the three identified PanK types have been studied and characterized and these PanK types exhibits a unique diversity between different organisms, therefore highlighting them as potential drug targets. In this study the type III PanK of specifically pathogenic bacteria were characterized with the goal of developing type-specific inhibitors. Several questions about the activity of the Mycobacterium tuberculosis enzyme was answered, which addresses the contradicting results achieved in related PanK studies performed to date. Furthermore the first inhibitors, that are competitive to the pantothenate binding site, were designed, synthesized and tested against the Pseudomonas aeruginosa enzyme. This resulted in the discovery of the most potent inhibitors of the type III PanKs to date. / AFRIKAANSE OPSOMMING: Pantoteensuurkinase-ensiem (PanK) kataliseer die eerste stap in die vyf stap biosintese van die lewens belangrike en essensiële kofaktor, koënsiem A (KoA). Die meerderheid patogeniese bakterieë, waaronder die organisme wat tuberkulose veroorsaak, besit ‘n unieke vorm van die PanK-ensiem. Gevolglik word hierdie ensieme as belangrike teikens vir die ontwikkeling van antibakteriële middels beskou. In hierdie studie is die aktiwiteit van die Mycobacterium tuberculosis ensiem gekarakteriseer wat verskeie teenstrydige bevindings oor hierdie ensiem beantwoord het. Verder is nuwe inhibitore vir die Pseudomonas aeruginosa ensiem ontwerp, gesintetiseer en getoets. Die beste inhibitore van hierdie tipe ensiem tot op hede is sodoende geïdentifiseer.

Pantothenate kinase (PanK) is an ubiquitous and essential enzyme that catalyzes the first step in the universal Coenzyme (CoA) biosynthesis pathway. In this step, pantothenate (Vitamin B5) is converted to 4′-phosphopantothenate, which subsequently forms CoA in four enzymatic steps. In bacteria, three types of PanK’s have been identified which exhibit wide variations in their distribution, mechanisms of regulation and affinity for substrates. Type I PanK is a key regulatory enzyme in the CoA biosynthesis pathway and its activity is feedback regulated by CoA and its thioesters. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on type I PanK from Mycobacterium tuberculosis (MtPanK) was initiated and the structure of this enzyme in complex with a CoA derivative has been reported earlier. To further elucidate the structural basis of the enzyme action of MtPanK, several crystal structures of the enzyme in complex with different ligands have been determined in the present study. In conjunction to this, solution studies on the enzyme were also carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion method was used for crystallization in all cases. The X-ray intensity data were collected using a MAR Research imaging plate system mounted on a Rigaku RU200 and Bruker-AXS Microstar Ultra II rotating anode X-ray generator. The data were processed using the HKL and MOSFLM and SCALA from the CCP4 suite. The structures were solved by the molecular replacement method using the program AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT and the refined structures were validated using PROCHECK and MOLPROBITY. Secondary structure was assigned using DSSP, structural superpositions were made using ALIGN and buried surface area was calculated using NACCESS. Solution studies on CoA binding and catalytic activity were carried out using Isothermal titration calorimetry (ITC). To start with, the crystal structures of the complexes of MtPanK were determined with (a) citrate, (b) the non-hydrolysable ATP analog AMPPCP and pantothenate (initiation complex), (c) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (end complex), (d) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (intermediate complex), (e) CoA, (f) ADP prepared by soaking and co-crystallization, which turned out to have identical structures and (g) ADP and pantothenate. Unlike in the case of the homologous E.coli enzyme (EcPanK), AMPPCP and ADP occupied different, though overlapping, locations in the respective complexes; the same was true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK was found to be substantially preformed while that of EcPanK exhibited considerable plasticity. The difference in the behavior of the E.coli and M.tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action and the changes in the locations of ligands exhibited by M.tuberculosis pantothenate kinase are remarkable and novel. The movement of ligands exhibited by MtPanK during enzyme action appeared to indicate that the binding site of the enzyme was less specific for a particular type of ligand than EcPanK. Kinetic measurements of enzyme activity showed that MtPanK had dual substrate specificity for ATP and GTP, unlike the enzyme from E.coli which showed a much higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes was provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate (2) GDP and phosphopantothenate (3) GDP (4) GDP and pantothenate (5) AMPPCP and (6) GMPPCP and the structures of the complexes of the two enzymes involving CoA and different adenyl nucleotides. The explanation was substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. Dual specificity of the type exhibited by this enzyme is rare and so are the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands and specificity. The crystal structures of MtPanK in binary complexes with nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP) provided insights about the natural location and conformation of nucleotides. In the absence of pantothenate, the NDP and the NTP bound with an extended conformation at the same site. In the presence of pantothenate, as seen in the initiation complexes, the NTP had a closed conformation and an altered location. However, the effect of the nucleotide on the conformation and the location of pantothenate were yet to be elucidated as the natural location of the ligand in MtPanK was not known. This lacuna was sought to be filled through X-ray analysis of the binary complexes of MtPanK with pantothenate and two of its derivatives, namely, pantothenol and N-nonyl pantothenamide (N9-Pan). These structures demonstrated that pantothenate, with a somewhat open conformation occupied a location similar to that occupied by phosphopantothenate in the “end” complexes, which was distinctly different from the location of pantothenate in “closed” conformation in the ternary “initiation” complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appeared to play a critical role in the movement of ligand that took place during enzyme action. The structure analysis of the binary complexes with the vitamin and its derivatives completed the description of the locations and conformations of nucleoside di and triphosphates and pantothenate in different binary and ternary complexes. These complexes provide snapshots of the course of action of MtPanK.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has long been the scourge of humanity, claiming millions of lives. It is the most devastating infectious disease of the world in terms of mortality as well as morbidity (WHO, 2009). The lack of a uniformly effective vaccine against TB, the development of resistance in the Mycobacterium tuberculosis against the present antitubercular drugs and its synergy with AIDS has made the situation very alarming. This therefore necessitates a search for new antitubercular drugs as well as the identification of new and unexplored drug targets (Broun et aI., 1992). Coenzyme A is an essential cofactor for all organisms and is synthesized in organisms from pantothenate by a universally conserved pathway (Spry et al., 2008; Sassetti and Rubin, 2003). The first enzyme of the pathway, pantothenate kinase catalyzes the most important step of the biosynthetic process, being the first committed step of CoA biosynthesis and the one at which all the regulation takes place (Gerdes et aI., 2002) This thesis describes the successful cloning of PanK from Mycobacterium tuberculosis, its expression in E. coli, single step affinity purification, and complete biochemical and biophysical characterization. In this work, pantothenol, a widely believed inhibitor of pantothenate kinase, has been shown to act as a substrate for the mycobacterial pantothenate kinase. Further it was shown that the product, 4'phosphopantothenol, thus formed, inhibited the next step of the CoA biosynthesis pathway in vitro. The study was extended to find outthe fate of pantothenol inside the cell and it was demonstrated that the CoA biosynthetic enzymes metabolized the latter into the pantothenol derivative of CoA which then gets incorporated into acyl carrier protein. Lastly, it was decisively shown that pantothenate kinase is not only regulated by feedback inhibition by CoA but, also regulated through feed forward stimulation by Fructose 1, 6 biphosphate (FBP), a glycolytic intermediate. The binding site of FBP was determined by docking and mutational studies of MtPanK. Chapter 1 presents a brief survey of the literature related to Coenzyme A biosynthesis pathway and describes the objective of the thesis. It also presents a history of TB and briefly reviews literature describing TB as well as the life cycle, biology, survival strategy, mode of infection and the metabolic pathways operational in the TB parasite, Mycobacterium tuberculosis. The chapter details the enzymes involved in CoA biosynthesis pathway from various organims. Chapter 2 In this chapter, cloning of the ORF (Rv1092c), annotated as pantothenate kinase in the Tuberculist database (http://genolist.pasteur.frfTubercuList), its expression in E. coli and purification using affinity chromatography has been described. Protein identity was confirmed by MALDI-TOF and by its ability to complement the pantothenate kinase temperature sensitive mutant, DV70. This chapter also illustrates the oligomeric status of MtPanK in solution and describes the biochemical characterization of MtPanK by means of two different methods, spectrophotometrically by a coupled assay and calorimetrically by using Isothermal Titration Calorimetry. Feedback inhibition of MtPanK by CoA is also discussed in this chapter. Chapter 3 describes the biophysical characterization of MtPanK. It discusses the enthalpy (~H) and free energy change (~G) accompanying the binding of a non-hydrolysable analogue of ATP; CoA; acetyl CoA and malonyl-CoA to MtPanK. The chapter details the energetics observed upon ATP binding to pantothenate-saturated MtPanK further elucidating the order of the reaction. This chapter also describes the various strategies which were designed and tested to remove CoA from the enzyme as the latter is always purified from the cell in conjunction with CoA. Validation of these strategies for complete CoA removal (by studying the n value from ITC studies) is further described. Chapter 4 discusses the interaction of the well-studied inhibitor of pantothenate kinases from other sources (e.g. the malarial parasite), pantothenol, with the mycobacterial enzyme. In order to investigate the interaction of this compound with MtPanK, its effect on the kinetic reaction carried out by the enzyme was studied by several methods. Surprisingly, a new band corresponding to 4'phosphopantothenol appeared when the reaction mix of MtPanK with pantothenol and ATP was separated on TLC. The identity of the new spot was confirmed by mass spectrometry analyses of the MtPanK reaction mixture.. These findings established the fact that pantothenol is a substrate of pantothenate kinase. To delve deeper into the mechanism of interaction of this compound with the enzymes of the coenzyme A biosynthesis pathway, the ability of pantothenol to serve as a substrate for the next step of the pathway, MtCoaBC was studied. Using various approaches it was established that pantothenol is actually a substrate for the MtPanK and the inhibition observed earlier (Saliba et aI., 2005) is actually due to the inability of CoaBC to utilize 4' -phosphopantothenol as substrate. Chapter 5 takes the story from Chapter 4 further detailing the effects of pantothenol on cultures of E. coli and M. smegmatis. I observed that pantothenol does not inhibit the culture of E. coli or M. smegmatis. So, further studies were carried out to know the fate of pantothenol once it is converted into 4'phosphopantothenoi. Since, the next enzyme of the pathway does not utilize 4'phosphopantothenol, I checked the further downstream enzyme of the pathway, CoaD, and found that it converts 4'-phosphopantothenol to thepantothenol derivative of dephospho-CoA. The next enzyme of the pathway, CoaE, took up this pantothenol derivative of dephospho-CoA as a substrate and converted it to the pantothenol derivative of CoA which was then transferred to apo-ACP by holo-ACP synthase. The holo-ACP thus synthesized enters into the dedicated pathway of fatty acid synthesis. Extensive investigations have been carried out on the regulation of pantothenate kinases, by the product of the pathway, Coenzyme A and its thioesters, xx establishing the latter as the feedback regulators of these enzymes. In order to determine if the cell employs mechanisms to sense available carbon sources and consequently modulate its coenzyme A levels by regulating activity of the enzymes involvedin CoA biosynthesis, glycolytic intermediates were tested against MtPanK for their possible role in the regulation of MtPanK activity. Chapter 6 details my identification of a novel regulator of MtPanK activity, fructose-I, 6-bisphosphate (FBP), a glycolytic intermediate, which enhances the MtPanK catalyzed phosphorylation of pantothenate by three fold. Further, the possible mechanisms through which FBP mediates MtPanK activation are also discussed. This chapter also describes the experiments carried out to identify the binding site of FBP on MtPariK.Interestingly, docking of FBP on MtPanK revealed that FBP binds close to the ATP binding site on the enzyme with one of its phosphates overlapping with the 3'~phosphate of CoA thereby validating its competitive binding relative to CoA on MtPanK. Based on these observations I propose that the binding of FBP to MtPanK lowers the activation energy of pantothenate phosphorylation by PanK. Chapter 7 presents a summary of the findings of this work. Coenzyme A biosynthesis pathway harbors immense potential in the development of drug against many communicable diseases, thanks to its essentiality for the pathogens and the differences between the pathogen and host CoA biosynthetic enzymes. The work done in this thesis extensively characterizes the first committed enzyme of the CoA biosynthetic pathway, pantothenate kinase, from Mycobacterium tuberculosis (MtPanK). The thesis also deals with the fate of a known inhibitor of PanK and proves it as a substrate for MtPanK. Finally this thesis describes a new link between glycolysis and CoA biosynthesis. Biotin, like coenzyme A, is another essential cofactor required by several enzymes in critical metabolic pathways. De novo synthesis of this critical metabolite has been reported only in plants and microorganisms. Therefore targeting the synthesis of biotin in the tubercular pathogen is another effective means of handicapping the tubercle pathogen. During the course of my studies, I also investigated the mycobacterial biotin biosynthesis pathway, studying the first enzyme of the pathway, 7-keto-8-aminopelargonic acid (KAPA) synthase (bioF) in extensive detail. Appendix 1 elucidates the kinetic properties of 7-keto-8aminopelargonic acid synthase (bioF) from Mycobacterium tuberculosis and proves beyond doubt that D-alanine which has previously been reported to act as a competitive inhibitor for the B. sphaericus enzyme (Ploux et al., 1999), is actually a substrate for the mycobacterial bioF.

N-substituted pantothenamides are analogues of pantothenate, the precursor of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive target for drug discovery due to low sequence homology between bacterial and human PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is located within a highly aromatic pocket, suggesting that an aromatic substituent may enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved. The structure reveals that the pyridine ring adopts alternative conformations in the aromatic pocket, providing the structural basis for further improvement of pantothenamide-binding to KpPanK.

N-substituted pantothenamides are analogues of pantothenate, the precursor of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive target for drug discovery due to low sequence homology between bacterial and human PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is located within a highly aromatic pocket, suggesting that an aromatic substituent may enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved. The structure reveals that the pyridine ring adopts alternative conformations in the aromatic pocket, providing the structural basis for further improvement of pantothenamide-binding to KpPanK.

This thesis consists of two parts. The first part is concerned with further structural and related studies of jacalin, one of the two lectins found in jack fruit seeds. The second part deals with the search of mycobacterial and archeal genomes for lectins. The β-prism I fold was identified as a lectin fold through the X-ray analysis of jacalin way back in 1996. Subsequent structural studies on jacalin are described in the first chapter in context of the overall efforts on lectins with particular reference to those on lectins with β-prism I fold. The structure of jacalin has been thoroughly characterized through the analysis of several crystals. The extended binding site of the lectin, made up of the primary binding site and secondary sites A and B, has also been characterized through studies on different jacalin-sugar complexes. However, nuances of jacalin-carbohydrate interactions remain underexplored with respect to two specific issues. The first issue is concerned with the structural basis for the lower affinity of jacalin for β-substituted sugars. The second has to do with the influence of the anomeric nature of the glycosidic linkage on the location of the reducing and non-reducing sugars in disaccharides when interacting with jacalin. Part of the work described in the thesis addresses these two issues. It was surmised that the lower affinity of β-galactosides to jacalin as compared to α-galactosides, is caused by steric interactions of the substituents in the former with the protein. This issue is explored both energetically and structurally in Chapter 2 using appropriately derivatized monosaccharide complexes of jacalin. It turns out that the earlier surmise is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in β-galactosides. The difference in energy, and therefore affinity, is caused by the distortion of the sugar ring in β-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands to jacalin. The crystal structures of jacalin complexed with α-linked oligosaccharides Gal α-(1,4) Gal and Gal α-(1,3) Gal β-(1,4) Gal, as described in Chapter 3, have been determined with the primary objective of exploring the effect of linkage on the location of reducing and non-reducing sugars in the extended binding site of the lectin, an issue which has not been studied thoroughly. Contrary to the earlier surmise based on simple steric considerations, the two structures demonstrate that α-linked sugars can bind to jacalin with non-reducing sugar at the primary binding site. This is made possible substantially on account of the hitherto underestimated plasticity of a non-polar region of the extended binding site. Modelling studies involving conformational search and energy minimization, along with available crystallographic and thermodynamic data, indicate a strong preference for complexation with Gal β-(1,3) Gal with the reducing Gal at the primary site, followed by that with Gal α-(1,3) Gal, with the reducing or non-reducing Gal located at the primary binding site. This observation is in consonance with the facility of jacalin to bind mucin type O-glycans containing T-antigen core. Crystal structures of jacalin in complex with GlcNAc β-(1,3) Gal-β-OMe and Gal β-(1,3) Gal-β-OMe have also been described in Chapter 4. The binding of the ligands to jacalin is similar to that of analogous α-substituted disaccharides. However, the β-substituted β-(1,3) linked disaccharides get distorted at the anomeric centre and the glycosidic linkage. The distortion results in higher internal energies of the ligands leading to lower affinity to the lectin. This confirms the possibility of using ligand distortion as a strategy for modulating binding affinity. Unlike in the case of β-substituted monosaccharides bound to jacalin, where a larger distortion at the anomeric centre was observed, smaller distortions are distributed among two centres in the structures of the two β-substituted β-(1,3) linked disaccharides presented here. These disaccharides, like the unsubstituted and α-substituted counterparts, bind jacalin with the reducing Gal at the primary binding site, indicating that the lower binding affinity of β-substituted disaccharides is not enough to overcome the intrinsic propensity of Gal β-(1,3) Gal based disaccharides to bind jacalin with the reducing sugar at the primary site. Although originally isolated from plants, lectins were also found subsequently in all forms of life, including bacteria. Studies on microbial lectins have not been as extensive as on those from plants and animals, although there have been some outstanding individual investigations on bacterial toxins like ADP-ribosylating toxins and neurotoxins. In addition to bacterial toxins, adhesins, β-trefoil lectins and cyanobacterial lectins form other important subgroups which have been explored using crystallography. Features pertaining to their three dimensional folds, carbohydrate specificity and biological properties are described in Chapter 5, to set the stage for the work discussed in the second part of the thesis. Studies on mycobacterial lectins were unexplored until work was initiated in the area in this laboratory some years ago. One of the lectins, identified on the basis of a bioinformatics search of M. tuberculosis H37Rv genome was cloned, expressed and crystallized. Also cloned, expressed and crystallized is another lectin from M. smegmatis. Biophysical and modelling studies were carried out on the full length protein containing this lectin. However, systematic efforts on mycobacterial lectins were conspicuous by their absence. The first chapter (Chapter 6) in the second part of the thesis is concerned with a genomic search for lectins in mycobacterial genomes. It was also realized that hardly anything is known about archeal lectins. Therefore, as discussed in the final chapter, a genomic search for archeal lectins was undertaken. Sixty-four sequences containing lectin domains with homologs of known three-dimensional structure were identified through a search of mycobacterial genomes and are described in detail in Chapter 6. They appear to belong to the β-prism II, the C-type, the Microcystis virdis (MV), and the β-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC, and the β-grasp domains, respectively while mycobacterial β-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three-dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and non-pathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one of the four lectin domains can be gleaned through the examination of homologous proteins, although the structure of none of them is available. Their biological role is yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins. As mentioned in Chapter 7, forty six lectin domains, which have homologues among well established eukaryotic and bacterial lectins of known three dimensional structure, have been identified through a search of 165 archeal genomes using a multi-pronged approach involving domain recognition, sequence search and analysis of binding sites. Twenty one of them have the 7-bladed β-propeller lectin fold while 16 have the β-trefoil fold and 7 the legume lectin fold. The remainder assumes the C-type lectin, the β-prism I and the tachylectin folds. Acceptable models for almost all of them could be generated using the appropriate lectins of known three dimensional structure as templates, with binding sites at one or more expected locations. The work represents the first comprehensive bioinformatics study of archeal lectins. The presence of lectins with the same fold in all domains of life indicates their ancient origin well before the divergence of the three branches. Further work is necessary to identify archeal lectins which have no homologues among eukaryotic and bacterial species.

Neurodegeneration with Brain Iron Accumulation (NBIA) disorders, such as Phospholipase A2G6-Associated Neurodegeneration (PLAN) and Pantothenate Kinase-Associated Neurodegeneration (PKAN), are a group of rare early-onset, genetic disorders characterized by neurodegeneration and iron accumulation inside of the basal ganglia (BG), which is accompanied by progressive motor symptoms. In order to address the limitations in available models of NBIA, a B6.C3-Pla2g6m1J/CxRwb mouse model of PLAN was characterized. This model demonstrated key hallmarks of the disease presentation in NBIA, including a severe and early-onset motor deficit, neurodegeneration inside of the substantia nigra (SN) including a loss of dopaminergic function and the formation of abnormal spheroid inclusions as well as iron accumulation. The capture of these hallmarks of NBIA makes this an ideal animal research model for NBIA. Additionally, exploration of candidate systemic biomarkers of NBIA was performed in a case study of a patient with PLAN and in a cohort of 30 patients with PKAN. These investigations demonstrated reductions in transfer and slight, but not significant elevations in soluble transferrin receptor. No significant difference was seen in serum iron parameters. A systemic disease burden including chronic oxidative stress; elevated malondialdehyde, and inflammation; elevated C-reactive protein (CRP), IL-6 and TNFα was noted in both investigations. A number of candidate protein biomarkers including: fibrinogen, transthyretin, zinc alpha-2 glycoprotein and retinol binding protein were also identified. These markers correlated with measures of the severity of iron loading in the globus pallidus (GP); based on R2* magnetic resonance imaging (MRI) and the severity of motor symptoms (Barry-Albright Dystonia Rating Scale) making them potential candidates markers of dysfunction in NBIA. In the patient with PLAN, 37 weeks of therapy with the iron chelator deferiprone (DFP) as well as 20 months of therapy with the antioxidants alpha lipoic acid (ALA) and n-acetylcysteine (NAC) were efficacious in reducing the systemic oxidative and inflammatory disease burden, but it did not significantly alter the progression of the disease. In the antioxidant therapy, this efficacy was primarily due to ALA. When the cohort of patients with PKAN were treated with DFP for 18 months it was highly efficacious in lowering brain iron accumulation in the GP. No significant reduction in the speed of disease progression was seen in DFP treated patients compared to placebo based on initial analysis. Similar to the PLAN patient, DFP also mitigated the systemic disease burden in PKAN patients. In both cases DFP was well tolerated and had minimal impact on serum iron levels, TIBC and transferrin saturation. Collectively these investigations provide valuable insights into disease progression in NBIA. They also provide tools to aid further investigations in NBIA. These are provided in the form of a well-characterized B6.C3-Pla2g6m1J/CxRwb model of PLAN, which robustly captures the disease presentation seen in patients, as well as a panel of systemic blood-based markers of disease burden in NBIA and candidate markers of dysfunction in NBIA. These markers were used to assess two novel therapies in NBIA chelation with DFP and antioxidant therapy with ALA and NAC. / Graduate / 2019-04-19