Hypoxia Inducible Factors (HIF) functions are master regulators of oxygen homeostasis and have a key role in the physiological responses to hypoxia including angiogenesis and erythropoiesis. Under hypoxia, levels of HIF-α subunits increase, they hetereodimerise with HIF-1β sub unit and promote the initiation of transcription of target genes. Under normoxia, oxygen dependent HIF-α degradation is promoted by hydroxylation of either of two proline residues (Pro402 and Pro564). The interaction of prolylhydroxylated HIF-α with the Von Hippel-Lindau protein (pVHL) promotes hydrolytic degradation of HIFα through an E3 ubiquitin ligase proteasomal pathway. HIF prolyl hydroxylation is catalysed by three 2-oxoglutarate (2OG)-dependent oxygenases known as prolyl hydroxylase domain (PHD 1-3) proteins, through an Fe(II) mediated catalytic process using 2OG, and oxygen. The PHDs are part of the family of Fe(II) bound 2OG dependent oxygenases. There are approximately 70 human 2OG oxygenases many of which have biologically important roles. Small-molecule inhibitors have reached advanced clinical trials; however, many clinical candidates inhibit other structurally similar 2OG oxygenases (OGFOD1 and vCPH) potentially altering the therapeutic effect. This thesis describes the design and synthesis of potent and 2OG oxygenase selective inhibitors for the PHDs. The 1,3,8-triazaspiro[4.5]decane-2,4-dione and 4-hydroxy-2-(pyrazole)pyrimidine-5-amide series were chosen as initial 'hits' (reported in the patent literature). The main analogues of the series were characterised in vitro and in cells as potent and selective PHD inhibitors over structurally similar 2OG oxygenases (Chapter 2). Broad structure activity relationship (SAR) of both initial series demonstrated the sensitivity for PHD2 inhibition (Chapter 3). Combination of SAR work described in Chapters 2 and 3 lead to the development of the novel 4-hydroxy pyridine series. In-depth SAR resulted in optimised analogues including 1 (IC50 69 nM) and highly selective over structurally similar 2OG oxygenases including OGFOD1. The completed SAR work led to the development of two novel pharmacophores 2 and 3. Both pharmacophores displayed potent PHD inhibition and selectivity over OGFOD1. Analogues including 1 and 3 displayed on target cellular activity stabilising HIF-1α at 20 μM (Chapter 4). The 4-dimethylamine pyridine analogue displayed an increase in substrate hydroxylation on PHD2 in contrast to the DMSO control (Chapter 3). SAR and cellular characterisation indicated that the effect observed was not an assay artifact (Chapter 5). Fenofibrate was used as a starting point for the development of novel inhibitors of the oxygen consumption rate (OCR) via mitochondrial inhibition (Chapter 6). Analogues were synthesised in order to conduct broad SAR and on-target cellular activity was observed in a Seahorse XF assay (50% reduction in the OCR at 1 μM). A selection of amino and amide analogues warrant further investigation.

Prolyl Hydroxylase Domain 2 (PHD2) has been identified as a key oxygen sensor in humans along with Factor Inhibiting Hypoxia Inducible Factor (FIH). As such PHD2 and FIH play critical roles in myriad pathways of medical relevance by hydroxylation of their target substrate hypoxia inducible factor (HIF), a transcription factor responsible for the regulation of over 100+ genes. With such critical roles in human physiology the ability to selectively regulate these two enzymes could potentially lead the way for novel therapeutic treatments of a vast array of disease states from cancer to myocardial infarction. We report on three classes of iron chelators which show promise for independent regulation of the HIF hydroxylases. Compounds representing the pyrones/pyridinones, pyridines and catechols were tested and found to have differential impacts on PHD2 and FIH under the same experimental conditions. The mode of inhibition is the result of binding to the active site iron and is supported by UV-visible and electroparamagnetic resonance spectroscopy. PHD2 at the current time does not have a well resolved mechanistic understanding regarding its catalytic cycle and subsequent rate determining steps. We have employed pH, solvent isotope, and X-ray absorption studies in an effort to gain further understanding regarding PHD2's overall mechanism. Our data support that dissociation of an iron(II)-OH2 bond centered about the active site contributes to a portion of the overall rate determining steps in the catalytic reaction of PHD2 that activates oxygen and ends with the production of hydroxylated substrate.

No description available.

La polyglobulie (PG) a un arrière plan clinique et physiopathologique varié, et dont la connaissance a beaucoup évolué depuis 5 ans. Elle a pour caractéristique nécessaire et commune une augmentation du volume globulaire total qui peut résulter de mécanismes moléculaires différents. L'érythrocytose est une forme de la PG ou` seule la lignée érythroïde semble être augmentée. Dans ce contexte, nous nous sommes intéressés à la mutation JAK2 (Val617Phe) et aux gênes EPOR, VHL, PHD2 chez des patients présentant une érythrocytose d'origine inconnue (98 familles). Nous avons trouvé 3 nouvelles mutations délétionnelles et d'autres mutations faux-sens et silencieuses sur EPOR. Les cellules FDCP-1 et 32D transfectées, qui expriment les récepteurs tronqués ont montré une hypersensibilité à l'EPO à concentration basse. Les analyses EPO-dose-réponse pour une mutation faux-sens étaient semblables à celles du type sauvage. La mutation JAK2(Val617Phe) était présente avec une faible fréquence dans la série de patients étudiés. La recherche des mutations dans le gêne PHD2 nous a permis de trouver 3 nouvelles mutations frame-shift et non-sens, les seules connues à ce jour: Elles entraînent la perte d'une partie ou de l'ensemble du site catalytique de PHD2 dans l'hypothèse où elles seraient synthétisées. Ainsi, ces mutations et les deux autres mutations faux-sens décrites démontrent l'importance de PHD2 dans la régulation de la voie de HIF-a, et qu'une perturbation de cette régulation pourrait conduire à une PG. La majorité des patients reste sans défaut moléculaire identifié, et des recherches complémentaires sont nécessaires. Nous avons également étudié deux Hbs augmentant l'affinité pour l'oxygène: les Hbs Nantes et La Coruña. Enfin, nous avons cherché des mutations dans les 3 exons de DPGM chez 4 patients avec un taux de 2,3-DPG bas. Une seule mutation a été trouvée dans la région 5`, dans l'exon 1 non traduit, elle nécessite des études complémentaires. / Polycythemia has a varied clinical and physiopathological background, of which our knowledge has greatly evaluated since 5 years. It is characterised by augmentation of the red cell mass which can be resulted from different molecular mechanisms. Erythrocytosis is a form of polycythemia where only the erythrocytes are augmented. In this context, we looked for the mutation JAK2 (Val617Phe), and other mutations in the genes of EPOR, VHL, PHD2 in a series of patients with erythrocytosis of unknown origin (98 families). We found 3 new frame-shift mutations and other missense and silent mutations in the EPOR. Transfected FDCP-1 and 32D cell lines expressing a truncated EpoR showed increased sensitivity at low concentration of Epo. However, in the case of a missense EPOR mutation, the Epo-dose response assays were similar to that of the wild type. The mutation JAK2 (Val617Phe) was found at a low frequency in the studied patients. Looking for mutations in the PHD2 gene allowed us to find 3 new frame-shift and nonsense mutations, the only reported to date. The encoded PHD2, if synthesized, would lose its active site or a part of it. These mutations with the other two missense mutations reported provide the evidence of the importance of PHD2 in the regulation of HIF-a pathway, and that disturbing the oxygen-sensing pathway might be a cause of polycythemia. The molecular basis of polycythemia in the majority of patients is still to be identified, and complementary studies are necessary. We studied then two examples of Hbs with a high oxygen affinity: Hb Nantes et La Coruña. Finally, we looked for mutations in the 3 exons of DPGM in a series of 4 patients. One mutation in exon 1 in the 5`region was found, and needs further studies.

No description available.

La polyglobulie (PG) a un arrière plan clinique et physiopathologique varié, et dont la connaissance a beaucoup évolué depuis 5 ans. Elle a pour caractéristique nécessaire et commune une augmentation du volume globulaire total qui peut résulter de mécanismes moléculaires différents. L'érythrocytose est une forme de la PG ou` seule la lignée érythroïde semble être augmentée. Dans ce contexte, nous nous sommes intéressés à la mutation JAK2 (Val617Phe) et aux gènes EPOR, VHL, PHD2 chez des patients présentant une érythrocytose d'origine inconnue (98 familles). Nous avons trouvé 3 nouvelles mutations délétionnelles et d'autres mutations faux-sens et silencieuses sur EPOR. Les cellules FDCP-1 et 32D transfectées, qui expriment les récepteurs tronqués ont montré une hypersensibilité à l'Epo à concentration basse. Les analyses Epo-dose-réponse pour une mutation faux-sens étaient semblables à celles du type sauvage. La mutation JAK2(Val617Phe) était présente avec une faible fréquence dans la série de patients étudiés. La recherche des mutations dans le gène PHD2 nous a permis de trouver 3 nouvelles mutations frame-shift et non-sens, les seules connues à ce jour: Elles entraînent la perte d'une partie ou de l'ensemble du site catalytique de PHD2 dans l'hypothèse où elles seraient synthétisées. Ainsi, ces mutations et les deux autres mutations faux-sens décrites démontrent l'importance de PHD2 dans la régulation de la voie de HIF-α, et qu'une perturbation de cette régulation pourrait conduire à une PG. La majorité des patients reste sans défaut moléculaire identifié, et des recherches complémentaires sont nécessaires. Nous avons également étudié deux Hbs augmentant l'affinité pour l'oxygène: les Hbs Nantes et La Coruña. Enfin, nous avons cherché des mutations dans les 3 exons de DPGM chez 4 patients avec un taux de 2,3-DPG bas. Une seule mutation a été trouvée dans la région 5`, dans l'exon 1 non traduit, elle nécessite des études complémentaires.

Indiana University-Purdue University Indianapolis (IUPUI) / FGF23 is an osteocyte produced hormone necessary for maintaining systemic phosphate handling, and thus bone structure and function in both rare and common disorders such as chronic kidney disease (CKD). FGF23 is a critical factor in CKD, with elevated levels causing alterations in mineral metabolism and increased odds for mortality. However, the mechanisms directing the production of key modulators of skeletal homeostasis and biomineralization within osteocytes, and how this is altered in chronic kidney disease, remain unclear. The experimental focus of this dissertation was to dissect the molecular systems and role of oxygen sensing in the regulated production of FGF23. In CKD, up to 75% of patients have anemia and concomitant marked elevations in FGF23, increasing mortality odds. Anemia is a potent driver of FGF23 secretion, therefore, current and emerging therapies, including recombinant EPO and the hypoxia inducible factorprolyl hydroxylase inhibitors (HIF-PHI) FG-4592 and BAY 85-3934, were used to improve anemia in the adenine diet-induced mouse model of CKD. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by 65-85% after delivery of EPO or HIF-PHI, with no changes in serum phosphate. This was associated with improved systemic iron utilization and reductions in mRNA markers of renal fibrosis. In osteocyte-like cell cultures treated with HIF-PHI, integrative RNAseq and ATACseq analysis identified candidate genes upregulated in response to mimicked hypoxia, concomitant with elevated Fgf23 expression. These genes were found to be downregulated in CKD bone, therefore, knock-out cells were generated using CRISPR/Cas9 technology. These cells were found to be functionally similar to in vivo conditional knockout models that have enhanced bone mass and elevated FGF23. Taken together, these results further define novel factors involved in the regulation of FGF23 and identify new therapeutic targets. / 2023-05-26

No description available.

No description available.

Vaccinia virus (VACV) is the most intensively studied orthopoxvirus and acts as an excellent model to investigate host-pathogen interactions. VACV encodes about 200 proteins, many of which modulate the immune response. This study focusses on two of these: C16 and C4, that share 43.7 % amino acid identity. Given the sequence similarity, we explored whether C16 and C4 have any shared functions, whilst also searching for novel functions. To gain mechanistic insight, we sought to identify binding partners and determine the residues responsible. C16 has two reported functions. Firstly, it inhibits DNA-PK-mediated DNA sensing, and this study found that C4 can perform this function as well. Like C16, C4 associates with the Ku heterodimer to block its binding to DNA leading to reduced production of cytokines and chemokines. For both proteins, the function localised to the C termini and was abrogated by mutating three residues. Secondly, C16 induces a hypoxic response by binding to PHD2. This function was mapped to the N-terminal 156 residues and a full length C16 mutant (D70K,D82K) lost the ability to induce a hypoxic response. In contrast, C4 did not bind PHD2. C4 inhibits NF-κB signalling by an unknown mechanism. Reporter gene assays showed that C16 also suppresses NF-κB activity and, intriguingly, this was carried out by both the N and C termini. C16 acts at or downstream of p65 and the N terminus of C16 associated with p65 independently of PHD2-binding. Conversely, C4 acted upstream of p65, did not display an interaction with p65, and the function was restricted to its C-terminal region. Novel binding partners were identified by a screen utilising tandem mass tagging and mass spectrometry, and selected hits were validated. The C terminus of C16 associated with VACV protein K1, a known NF-κB inhibitor. Additionally, C16 bound to the transcriptional regulator ARID4B. C4 did not interact with these proteins, but the N-terminal region of C4 associated with filamins A and B. The functional consequences of these interactions remain to be determined. In vivo, C4 and C16 share some redundancy in that a double deletion virus exhibits an attenuated virulence phenotype that is not observed by single deletion viruses in the intradermal model of infection. However, non-redundant functions also contribute to virulence in that both single deletion viruses display attenuated virulence compared to a wild-type Western Reserve virus in the intranasal model of infection. Data presented also reveal that C4 inhibits the recruitment of immune cells to the site of infection, as was previously described for C16. Overall, this investigation highlights the complexity of host-pathogen interactions showing that VACV encodes two multifunctional proteins with both shared and unique functions. Moreover, their inhibition of DNA-PK emphasises the importance of this PRR as a DNA sensor in vivo.