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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22484号 / 農博第2388号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5264(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 加納 健司, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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An extensive overview of the current state of the research in the field of the development of acyclic nucleoside phosphonates (ANPs) was elaborated, which quotes from 196 publications in abstracted journals. A new microwave-assisted methodology for the preparation of dialkyl haloalkylphosphonates was developed. Through strict control of the reaction temperatures in microwave reactor, it was possible to lower the amount of the reactants all the way to the ideal ratio of 1:1. With the use of a continuous-flow microwave reactor, it was possible to prepare the key building blocks for the subsequent syntheses of ANPs in large quantities (100 g), which significantly accelerates research in this area. The new method was patented and published. While studying various ANP prodrugs, a new highly effective methodology for the preparation of the diamides of ANPs was developed. The method starts directly from ANP diesters, which react with trimethylsilylbromide to form the corresponding bis(trimethylsilyl)esters of ANPs, which are well soluble in organic solvents and react smoothly during the subsequent introduction of aminoacid esters. Moreover, the reaction with trimethylsilylbromide protects the reactive groups present in the rest of the molecule and thus prevents undesired side reactions. Furthermore, using...

In Chapter 1 a review of the literature concerning aspects of erythrocyte membrane transport and metabolism, and purine and pyrimidine metabolism is presented. The effects of pH, pO₂ and inorganic phosphate (Pi) on the uptake and metabolism of hypoxanthine by erythrocytes has been studied in Chapter 2. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low pO₂. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favouring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate (PRPP). Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3- bisphosphoglycerate (2,3-DPG). These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely PRPP synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue. In Chapter 3 the kinetics and mechanism of transport of orotate across the human erythrocyte membrane and the effect of pH and inorganic phosphate on its metabolism (in the erythrocyte) have been studied. It has been shown that orotate enters erythrocytes with non-saturable kinetics and with a capacity of 190 μmoles/1 packed cells/min at a concentration of 4-6 mmolar. The presence of competition for transport by a number of anions and the lack of competition by uridine is indicative of transport by a general anion transporter, with the ability for concentrative uptake in the absence of other external anions being compatible with transport via a ping-pong mechanism. Inhibition of transport by the specific band 3 inhibitors DIDS and CHCA confirm that transport is via the band 3 anion transporter. This explains the lack of significant uptake of orotate by most differentiated tissues which lack the intact band 3 protein. However, the demonstration of band 3 in rat hepatocytes (Cheng and Levy, 1980) provides a mechanism for the orotate transport which has been observed in liver (Handschumacher and Coleridge, 1979). Changes in pH and inorganic phosphate (Pi) concentrations have been shown to have marked effects on the relative quantities of metabolic products produced by the erythrocyte from orotate. There was an increase in orotate metabolised with increasing Pi, an effect augmented by lowering the pH, and most easily explained by the allosteric activation of PRPP synthetase by Pi. The increase in UTP levels with decreasing pH may be the consequence of both increased PRPP availability for the formation of uridine nucleotide from orotate, and decreased conversion of UMP to uridine by pyrimidine 5'-nucleotidase, which is known to be inhibited by phosphate. The accumulation of UDP sugars is optimal at a phosphate concentration of 10 mmolar, which is unexplained but would be compatible with an inhibitory effect of Pi on CTP synthetase. A PRPP wasting cycle at alkaline pH values is proposed to explain the apparent paradox where no PRPP was observed to accumulate in erythrocytes (Chapter 2) at pH values of 7.6 and above in the presence of 10 mmolar phosphate and no added hypoxanthine, yet the metabolism of orotate, which is a PRPP utilising reaction, at alkaline pH values was readily demonstrable here. This (apparent paradox) can be resolved if one assumes that even in the absence of added hypoxanthine and demonstrable intracellular IMP there are sufficient quantities of hypoxanthine and/or IMP to maintain a PRPP wasting cycle at alkaline pH values. The cycle is interrupted at acidic pH values as phosphate levels rise and inhibit 5'-nucleotidase, an effect augmented by the decreasing levels of 2,3-DPG which accompany decreasing pH. This wasting cycle has recently been confirmed by P. Berman (unpublished). The kinetics of orotate uptake by erythrocytes and its eventual release as uridine provides a role for the erythrocyte in the transport and distribution of pyrimidines to peripheral tissues. A model is proposed and involves the de novo production of orotate in the liver. In the next step erythrocytes take up the orotate secreted by the liver into the circulation, convert it into an intermediate buffer store of uridine nucleotides, whose distribution is a function of pH and phosphate concentration, and eventually release it as uridine, which is a readily available form of pyrimidine for utilisation by peripheral nucleated cells. The enhancement of uptake of labelled orotate into nucleic acids of cultured cells is demonstrated here. The degradative half of the cycle proposes that uracil and palanine are the predominant degradative forms of pyrimidines produced by peripheral cells, and their ultimate metabolic fate is complete catabolism in the liver to CO₂ and water. In the final chapter the possible role of the human erythrocyte in the prevention of reperfusion injury has been investigated. The development of a model of renal ischaemia in the rat is described. The ability of human erythrocytes, "primed" by preincubating in acid medium of high Pi concentration and low pO₂, to take up hypoxanthine in a concentrative manner when perfused through ischaemic rat kidney is demonstrated. Attempts to demonstrate improved survival and renal function in rats with "primed" human erythrocytes prior to reperfusion were, however, unsuccessful. It is further demonstrated that "unprimed" human erythrocytes, resident in ischaemic rat kidney for 3 hours, take up hypoxanthine and convert it to IMP. that erythrocytes could play a physiological prevention of reperfusion injury.

These studies have examined the effect of hypoxia inducible factor 1α (HIF-1α) on nucleotide metabolism in the ischemic heart using a genetic mouse model with heart-specific and regulated expression of a stable form of HIF-1α. We find that AMP deaminase (AMPD), the entry point of the purine nucleotide cycle (PNC), is induced by HIF-1α at the level of mRNA, protein, and activity. AMP that accumulates during ischemia can be metabolized to adenosine by 5'-nucleotidase or to IMP by AMPD. Consistent with the finding of AMPD induction, adenosine accumulation during ischemia was much attenuated in HIF-1α-expressing hearts. Further investigation of nucleotide salvage enzymes found that hypoxanthine phosphoribosyl transferase (HPRT) is also upregulated in HIF-1α-expressing hearts. Treatment of hearts with an inhibitor of the PNC, hadacidin, hastens the fall of the adenylate energy charge during ischemia and the accumulation of AMP. The results provide new insight into the role of the PNC in the heart, especially as it relates to ischemia, and indicate that HIF-1α regulates nucleotide metabolism as a compensatory response to hypoxia.

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Le travail s’est articulé autour de la synthèse et de l’évaluation biologique de nouveaux inhibiteurs de protéines kinases dont essentiellement de kinases dépendantes de cyclines (CDKs). La dérégulation de ces kinases est mise en cause dans l’apparition de nombreuses pathologies prolifératives telles que le cancer ou non prolifératives telles que les maladies neurodégénératives. Deux types d’inhibiteurs ont été préparés. La première famille de composés étudiée est la famille des purines, sur laquelle a été introduite des substructures de type aminodiols en position C-2. Ces aminodiols ont été préparés de manière stéréosélective via la réaction de dihydroxylation asymétrique décrite par Sharpless ou à partir de dérivés d’acides aminés. Parmi les inhibiteurs réalisés, plusieurs ont montré une inhibition envers les protéines CDK5, CDK9 et CK1 très supérieure (IC50 de l’ordre de 100 nM) à celle de la (R)-Roscovitine, molécule actuellement en phase clinique II dans plusieurs cancers. Ces inhibitions des cibles enzymatiques s’accompagnent d’un effet antiprolifératif sur plusieurs lignées tumorales. Enfin l’étude d’une des molécules a été complétée par des tests très favorables sur des enzymes de microsomes hépatiques (IC50 > 5 μM). Dans une seconde partie, des imidazo[1,2-b]pyridazines ont été préparées. Une synthèse originale conduisant aux imidazo[1,2-b]pyridazines 3,6,8-trisubstituées a été mise au point. Les produits présentent une inhibition forte envers les protéines kinases telles que CDK5 et CK1 (IC50 < 50 nM). Ils montrent également des propriétés antiprolifératives sur les cellules tumorales SH-SY5Y. Enfin, des imidazo[1,2-b]pyridazines 3,6-disubstituées se révèlent être des inhibiteurs sélectifs de la protéine CLK1 (IC50 < 100 nM). / This thesis focuses on the synthesis and biological evaluation of new kinase inhibitors. Kinase deregulation is associated with numerous diseases such as cancer or neurodegenerative diseases. In the first part of this work, 2,6,9-trisubstituted purines bearing at C-2 position aminodiols derivatives were prepared. Aminodiols were obtained either via Sharpless asymmetric dihydroxylation or by reduction of amino esters. The compounds appeared to be more potent against kinases than (R)-roscovitine which is presently undergoing phase II clinical tests. In particular, inhibition of CK1, CDK5 and CDK9 were observed with IC50 < 200 nM. The compounds prepared showed an antiproliferative effect against tumor cell-lines (SH-SY5Y). Eventually, one of the most promising compounds was assayed against a series of cyt P450 enzymes and did not showed any inhibition (IC50 > 5 μM). The second family of compounds prepared in this work is imidazo[1,2-b]pyridazines. A new route to 3,6,8-trisubstituted imidazo[1,2-b]pyridazines was first developed. These products were shown to be highly potent inhibitors of several kinases such as CDK5 and CK1 (IC50 < 50 nM). The kinase inhibitions were accompanied by antiproliferative activities against tumor cell-lines. Finally, a series of 3,6-disubtituted imidazo[1,2-b]pyridazines was also prepared and appeared to be selective inhibitors of CLK1 (IC50 < 100 nM).

Molecular association is part of many important processes in living cells. Computational methods for calculating binding free energies allows for a quantitative examination of biomolecular structures and hypotheses drawn from biochemical experiments. Here, binding free energy calculations for tRNAs and release factors binding to mRNA codons on the ribosome, sugars binding to lectins and purine analogs binding to the purine riboswitch are presented. The relative affinities between cognate and non-cognate tRNAs for different states involved in codon reading on the ribosome were determined. The calculations show that tRNA discrimination varies between different conformations of the 30S subunit, where the existence of both low and high selectivity states provides an efficient common mechanism for initial selection and proofreading. The simulations reveal a desolvation mechanism for the 30S conformational switch with which the accuracy of peptide bond formation can be amplified. When an mRNA stop codon (UAA, UAG or UGA) is located in the ribosomal A-site release factors bind to the ribosome and the synthesized protein is released. RF1 is specific for UAA and UAG whereas RF2 is specific for UAA and UGA. The free energy calculations and an analysis of the performed simulations show the mechanisms for how RF1 and RF2 are able to read the stop codons with different specificities. Also mitochondrial release factors were investigated. Vertebrate mitochondria have four stop codons, UAA, UAG, AGA and AGG and two release factors mtRF1 and mtRF1a. The calculations show how the specificities of both mtRF1 and mtRF1a agree with RF1 and that none of them are likely to read the non-standard stop codons AGA and AGG. The linear interaction energy method has also been examined for the RSL and PA-IIL lectins and for the purine riboswitch. The standard parameterization of the method works well for RSL, but fails for PA-IIL and the purine riboswitch due to compositions of the active sites in these systems. The development of new parameterizations to overcome these problems leads to a better understanding of both the method and the binding mechanisms in these systems.

A presente tese foi elaborada em três capítulos, correspondente às seções 2, 3 e 4. No primeiro objetivou-se avaliar a associação de quitosana com grão de soja integral nas dietas sobre o consumo, fermentação ruminal, digestibilidade e metabolismo de nutrientes e seus efeitos sobre a produção de leite, perfil de ácidos graxos do leite e a eficiência produtiva de vacas em lactação. Para tal, 24 vacas multíparas da raça Holandesa, oito dessas com cânula ruminal, foram distribuídas em quadrados latinos 4×4 replicados. Os tratamentos foram obtidos por esquema fatorial 2×2 pela combinação de quitosana e grão de soja integral nas dietas. De modo geral, a associação de quitosana com grão de soja aumentou a eficiência de fermentação ruminal, reduziu as populações bacterianas ligadas à biohidrogenação e aumentou o teor de ácidos graxos insaturados na gordura do leite, mas houve significativa redução do consumo e digestibilidade dos nutrientes, da síntese de proteína microbiana e da produção de leite. No entanto, a adição de quitosana em dietas sem suplementação lipídica pode ser uma boa estratégia nutricional para aumentar a retenção de N e a eficiência alimentar de vacas em lactação, e adicionalmente, aumentar o teor de ácidos graxos insaturados e de cis-9, trans-11 CLA no leite. No segundo capítulo, o objetivo geral foi avaliar os indicadores externos Cr2O3 e TiO2 e os internos MSi, FDNi e FDAi, estes analisados em diferentes sacos de incubação in situ, a partir da acurácia, precisão e robustez das estimativas de excreção fecal em ensaio de digestão com vacas leiteiras consumindo diferentes dietas. Foram utilizadas oito vacas canuladas no rúmen para a realização de coletas totais de fezes com 24, 48 e 72 h de duração. De modo geral, o Cr2O3 gerou estimativas acuradas e precisas, no entanto a recuperação fecal e a robustez foram afetadas pelo consumo de extrato etéreo das vacas. O indicador TiO2 apresentou incompleta recuperação fecal, baixa acurácia, precisão e robustez. Dentre as combinações avaliadas para os indicadores internos, a FDNi em sacos de poliéster teve completa recuperação fecal e gerou as estimativas mais acuradas, precisas e robustas. A falta de acurácia e precisão dos indicadores têm grande impacto final sobre as estimativas de digestibilidade. No terceiro capítulo, o objetivo foi avaliar o uso da creatinina como indicador do volume urinário para estimativas de excreção total de derivados de purinas como técnica de avaliação da produção microbiana ruminal em vacas leiteiras alimentadas com diferentes dietas. Também foram utilizadas as oito vacas canuladas no rúmen para a realização de coletas totais de urina com 24, 48 e 72 h de duração. De modo geral, a creatinina foi afetada pelas condições experimentais do estudo, incluindo efeito de dieta e variações conforme o período experimental e animais. Apesar da relação entre excreção observada e estimada de derivados de purinas, essa deve ser vista com cautela, em razão de que o volume urinário foi subestimado e que as estimativas não identificaram corretas diferenças entre as dietas, comprometendo as avaliações sobre a produção microbiana ruminal. / The present thesis was elaborated in three chapters, corresponding to sections 2, 3 and 4. In the first one, the aim was to evaluate the association of chitosan (C) and whole raw soybean (WRS) in diets, evaluating the intake, ruminal fermentation, digestibility and nutrient metabolism and its effects on milk production, milk fatty acids profile and the productive efficiency of lactating cows. Twenty four multiparous Holstein cows, eight of them fitted with ruminal cannula, were distributed in a replicated 4 × 4 Latin squares. The treatments were a combination of C and WRS diets arranged in a 2 × 2 factorial design. This association increased the efficiency of ruminal fermentation, reducing the bacterial population responsible for the biohydrogenation and increased the unsaturated fatty acids in milk. The C and WRS diets caused a significant reduction in DMI and nutrient digestibility, in microbial protein synthesis and milk yield. However, the addition of C in diets without a lipid supplementation may be a good nutritional strategy to increase the N retention and increase the food efficiency of lactating cows, and, indeed, to increase the unsaturated fatty acids content, mainly cis-9, trans-11 CLA. In the second chapter, the objective was to evaluate the external indicators Cr2O3 and TiO2 and the indigestible internal DM, NDF and ADF that were determined in in situ different incubation bags from the accuracy, precision and robustness of the fecal excretion estimates in a digestion assay with different dairy cow diets. Eight cows fitted with ruminal cannula were used to perform total fecal collection over 24, 48 and 72 hours. The Cr2O3 generated accurate estimates, however, the fecal recovery and robustness were affected by the ether extract intake. The TiO2 indicator showed incomplete fecal recovery, low accuracy, precision and robustness. Among the combinations for the internal indicators, the iNDF in polyester bags had a complete recovery and generated the most accurate and robust estimates. The lack of accuracy and precision of the indicators have a big impact on the digestibility estimates. In the third chapter, the aim was to evaluate the use of creatinine as an indicator of urinary volume. It would be possible to estimate the total excretion of purine derivatives to evaluate the ruminal microbial protein synthesis in different cow diets. Also, eight multiparous cows fitted with ruminal cannula were used to make total urine collection over 24, 48 and 72 hours. The creatinine was affected by the study experimental conditions, including dietary effect and variations according to period and animals. The relationship between observed and estimated excretion of purine derivatives should be viewed with caution, because the urinary volume was underestimated and the estimates did not identify the correct differences between diets, that could compromise the evaluations of ruminal microbial production.