A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

Kynurenine 3-monooxygenase (K3MO) lies on the kynurenine pathway, the major pathway for the catabolism of L-tryptophan. It converts kynurenine to 3-hydroxy kynurenine. Inhibition of K3MO is important in several neurological diseases and there is evidence that inhibition of K3MO could also be targeted for the prevention of multiple organ failure, secondary to acute pancreatitis. A structure activity relationship based upon the 1,2,4-oxadiazoles motif was carried out which revealed amide 207 as an inhibitor of P. fluorescens K3MO. Further structure activity relationships were developed based upon 207. This revealed 3,4-dichloro substitution in 235 and 245 as optimum for inhibition. Co-crystalisation of these inhibitors with P. fluorescens K3MO revealed their interactions with the enzyme. It also highlighted new, potential interactions between the inhibitors and K3MO. This led to the synthesis of 271 and 272, which were also potent inhibitors of K3MO. These amides were successfully co-crystalised with P. fluorescens K3MO. Further development of the amides followed, with amide 282 providing the most potent inhibitor of P. fluorescens K3MO to date (Kᵢ = 29.1 nM).

"A thesis submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy". / Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: p. 243-277. / Introduction -- A convenient synthesis of 30HKG -- Facile synthesis of the UV filter compounds 30HKyn and AHBG -- Synthesis, identification and quantification of novel human lens metabolites -- Modification of bovine lens protein with UV filters and related metabolites -- Effect of UV light on UV filter-treated lens proteins -- Conclusions and future directions. / The kynurenine-based UV filters are unstable under physiological conditions and undergo side chain deamination, resulting in α,β-unsaturated carbonyl compounds. These compounds can react with free or protein bound nucleophiles in the lens via Michael addition. The key sites of the UV filters kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) modification in human lenses include cysteine (Cys), and to a lesser extent, lysine (Lys) and histidine (His) residues. Recent in vivo studies have revealed that 3-hydroxykynurenine-O-β-D-glucoside (3OHKG) binds to Cys residues of lens crystallins in older normal human lenses. As a result of this binding, human lens proteins become progressively modified by UV filters in an age-dependent manner, contributing to changes that occur with the development of age-related nuclear (ARN) cataract. Upon exposure to UV light, free UV filters are poor photosensitisers, however the role of protein-bound species is less clear. It has been recently demonstrated that Kyn, when bound to lens proteins, becomes more susceptible to photo-oxidation by UV light. Therefore, the investigation of 3OHKG binding to lens proteins, and the effect of UV light on proteins modified with 3OHKG and 3OHKyn, were major aims of this study. As a result of the role of these compounds as UV filters and their possible involvement in ARN cataract formation, it is crucial to understand the nature, concentration and modes of action of the UV filters and their metabolites present in the human lenses. Therefore, an additional aim was to investigate human lenses for the presence of novel kynurenine-based human lens metabolites and examine their reactivity.--As 3OHKG is not commercially available, to conduct protein binding studies, an initial aim of this study was to synthesise 3OHKG (Chapter 2). Through the expansion and optimisation of a literature procedure, 3OHKG was successfully synthesised using commercially available and inexpensive reagents, and applying green chemistry principles, where toxic and corrosive reagents were replaced with benign reagents and solvent-free and microwave chemistry was used. A detailed investigation of different reaction conditions was also conducted, resulting in either the improvement of reaction yields or reaction time compared to the literature method. Applying the same synthetic strategy, and using key precursors from the synthesis of 3OHKG, the UV filters 3OHKyn and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG), were also successfully synthesised (Chapter 3). / Chapter 4 describes the investigation of both normal and cataractous human lenses in an attempt to identify novel human lens metabolites derived from deaminated Kyn and 3OHKyn (Chapter 4, Part A). Initially, 4-(2-aminophenyl)-4-oxobutanoic acid (AHA), glutathionyl-kynurenine (GSH-Kyn), kynurenine yellow (Kyn yellow), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid (AHB), glutathionyl-3-hydroxykynurenine (GSH-3OHKyn) and 3-hydroxykynurenine yellow (3OHKyn yellow) were synthesised and human lenses were examined for their presence. AHA and AHB were synthesised from similar precursors to those used in the synthesis of 3OHKG, while the GSH adducts and yellow compounds were synthesised from Kyn and 3OHKyn via base induced deamination. Following isolation and structural elucidation, AHA, AHB and GSH-Kyn were confirmed as novel human lens metabolites. They were quantified in low pmol/mg lens (dry mass) levels in normal and cataractous lenses of all ages, while GSH-3OHKyn, Kyn yellow and 3OHKyn yellow were not detected. In contrast to AHA, the lens metabolites AHB, GSH-Kyn and GSH-3OHKyn were found to be unstable at physiological pH. The spectral properties of these compounds suggest that they may act as UV filters. --Chapter 4 (Part B) also describes the identification and characterisation of a novel human lens UV filter, cysteinyl-3-hydroxykynurenine -O-β-D-glucoside (Cys-3OHKG). An authentic standard was synthesised via Michael addition of cysteine to deaminated 3OHKG. Cys-3OHKG was detected in low pmol/mg lens (dry mass) levels in normal lenses only after the 5th decade of life and was absent in cataractous lenses. Cys-3OHKG showed rapid decomposition at physiological pH. / Chapter 5 describes the identification and quantification of amino acids involved in covalent binding of 3OHKG to lens proteins. Model studies with bovine lens proteins and 3OHKG at pH 7.2 and 9.5 were undertaken. The amino acid adducts were identified via total synthesis and spectral analysis, and subsequently quantified upon acid hydrolysis of the modified lens proteins. Under both pH conditions, 3OHKG was found to react with lens proteins predominantly via Cys residues with low levels of binding also detected at Lys residues. Comparative studies with Kyn (pH 9.5) and 3OHKyn (pH 7.2 and 9.5) resulted in modified lens proteins at Cys residues, with only minor modification at Lys residues at pH 9.5. The extent of modification was found to be significantly higher at pH 9.5 in all cases. His adducts were not identified. 3OHKG-, Kyn- and 3OHKyn-modified lens proteins were found to be coloured and fluorescent, resembling those of aged and ARN cataractous lenses. In contrast, AHB and AHA, which can not form α,β-unsaturated carbonyl compounds, resulted in non-covalent modification of lens proteins. AHB may contribute to lens colouration and fluorescence as further reactions of this material yielded species that have similar characteristics to those identified from 3OHKyn modification. These species are postulated to arise via auto-oxidation of the o-aminophenol moiety present in both 3OHKyn and AHB.--In Chapter 6, the potential roles of 3OHKG and 3OHKyn, and the related species AHA and AHB, in generating reactive oxygen species and protein damage following illumination with UV light was examined. The UV filter compounds were examined in both their free and protein-bound forms. Kyn-modified proteins were used as a positive control. Exposure of these compounds to UV light (λ 305-385 nm) has been shown to generate H2O2 and protein-bound peroxides in a time-dependent manner, with shorter wavelengths generating more peroxides. The yields of peroxides were observed to be highly dependent on the nature of the UV filter compound and whether these species were free or protein bound, with much higher levels being detected with the bound species. Thus, protein-bound 3OHKyn yielded higher levels of peroxide than 3OHKG, with these levels, in turn, higher than for the free UV filter compounds. AHB-treated lens proteins resulted in formation of low but statistically significant levels of peroxides, while AHA-treated lens proteins resulted in insignificant peroxide formation. The consequences of these photochemical reactions have been examined by quantifying protein-bound tyrosine oxidation products (3,4-dihydroxyphenylalanine [DOPA], di-tyrosine [di-Tyr]) and protein cross-linking. 3OHKG-modified proteins gave elevated levels of di-Tyr, but not DOPA, whereas 3OHKyn-modified protein gave the inverse. DOPA formation was observed to be independent of illumination and most likely arose via o-aminophenol auto-oxidation. AHB- and AHA-treated lens proteins resulted in statistically insignificant di-Tyr formation, while a light independent increase in DOPA was observed for both samples. Both reducible (disulfide) and non-reducible cross-links were detected in modified proteins following illumination. These linkages were present at lower levels in modified, but non-illuminated proteins, and absent from unmodified protein samples. / This work has provided an optimised synthetic procedure for 3OHKG and other lens metabolites (Chapters 2 and 3). Four novel lens metabolites have been identified and quantified in normal and cataractous human lenses (Chapter 4). Subsequent experiments, described in Chapter 5, identified the major covalent binding sites of 3OHKG to lens proteins, while AHA and AHB showed non-covalent binding. Further work described in Chapter 6 showed that protein-bound 3OHKG, Kyn and 3OHKyn were better photosensitisers of oxidative damage than in their unbound state. Together, this research has provided strong evidence that post-translational modifications of lens proteins by kynurenine-based metabolites and their interaction with UV light appear, at least in part, responsible for the age-dependent colouration of human lenses and an elevated level of oxidative stress in older lenses. These processes may contribute to the progression of ARN cataract. / Mode of access: World Wide Web. / xxxix, 308 p. ill. (some col.)