Biosynthetic studies of thiosugar-containing natural products, BE-7585A and Lincomycin A

Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been well understood. On the other hand, a wide variety of sugar derivatives commonly found in natural products are often vital components for the efficacy and specificity of their parent molecules. Elucidation of such unusual sugar biosyntheses is important both for understanding their intriguing chemical mechanisms and creating unnatural compounds by altering their biosynthetic machineries, which could potentially exhibit enhanced or novel biological activities. This dissertation describes biosynthetic studies of two thiosugar-containing natural products, BE-7585A and lincomycin A, produced by Amycolatopsis orientalis and Streptomyces lincolnensis, respectively. While the former possess a C-2-thiosugar-containing disaccharide moiety, the latter contains a C-1-thio substituent on a characteristic eight-carbon backbone sugar. The focus of this research is to characterize the biological pathways and mechanisms responsible for the sulfur incorporation and the unique sugar scaffolds. BE-7585A, an angucycline-type natural product, contains the rare C-2-thiosugar moiety. PCR-based screening of a cosmid library constructed from the genomic DNA of A. orientalis led to the identification of the BE-7585A biosynthetic gene cluster. A gene, bexX, was found to be a candidate for a thiosugar synthase with moderate sequence similarity to a thiazole synthase. The gene, bexX, and a glycosyltransferase homologue, bexG2, were heterologously expressed in Escherichia coli. A variety of biochemical experiments provided a wealth of evidence supporting the proposed biosynthetic pathway for the C-2-thiodisaccharide moiety. Finally, whole genome sequencing and a genome mining approach led to the identification of a sulfur carrier protein to accomplish the in vitro enzymatic synthesis of the C-2-thiosugar for the first time. Lincomycin A is a lincosamide antimicrobial natural product with a C-1 methylthio substituent. Although the lincomycin A biosynthetic gene cluster has been reported, biochemical verification of the biosynthetic pathway has remained elusive. In this dissertation, the complete methlthiolincosamide biosynthetic pathway including the potential C-1 sulfur incorporation mechanism was proposed. Furthermore, two early intermediates of the pathway were characterized for the first time by demonstrating the LmbR (transaldolase) and LmbN (isomerase) reactions in vitro.Chemistr

Genomic Approaches Enable Evaluation of the Safety and Quality of Influenza Vaccines and Adjuvants

Vaccination is an effective means for prevention of the progression and spread of influenza virus infection. Nonetheless, there is a risk of adverse reactions, such as pain and fever, during the vaccination. In addition, because people from a wide age range, that is, from children to the elderly, are inoculated with vaccines, safety confirmation of these vaccines is important. Safety assessments of a vaccine, in the form of quality controls, have been carried out on animals. For example, the abnormal toxicity test is based on body weight changes as a toxicity index, and the leukopenic toxicity test can evaluate hematological toxicity. Meanwhile, since the 2000s, safety evaluation of drugs and chemicals by the genomic approach has been conducted frequently. The benefits with respect to safety evaluation are high sensitivity and abundant information about toxicity profiles. In this chapter, we describe the genes that are helpful as safety assessment markers and their usefulness for safety testing and vaccine development. In addition, this information may provide toxicity profiles, help understand the reactogenicity of nasal vaccines or adjuvants, and explain the prospects of genomic analyses in the development of novel vaccines and adjuvants

反応性代謝物および免疫・炎症関連因子を考慮したフェニトイン誘導性肝障害発症機序に関する研究

13301甲第4191号博士（創薬科学）金沢大学博士論文本文Full 以下に掲載：1.Toxicological Sciences 136(1) pp.250-263 2013. Oxford journal. 共著者：Sasaki E, Matsuo K, Iida A, Tsuneyama K, Fukami T, Nakajima M, Yokoi T 2.Toxicology Letters 232(1) pp.79-88 2015. Elsevier. 共著者：Sasaki E, Iwamura A, Tsuneyama K, Fukami T, Nakajima M, Kume T, Yokoi

A Novel Mouse Model for Phenytoin-Induced Liver Injury: Involvement of Immune-Related Factors and P450-Mediated Metabolism

Drug-induced liver injury is an important issue for drug development and clinical drug therapy; however, in most cases, it is difficult to predict or prevent these reactions due to a lack of suitable animal models and the unknown mechanisms of action. Phenytoin (DPH) is an anticonvulsant drug that is widely used for the treatment of epilepsy. Some patients who are administered DPH will suffer symptoms of drug-induced liver injury characterized by hepatic necrosis. DPH-induced liver injury occurs in 1 in 1000 or 1 in 10 000 patients. Clinically, 75% of patients who develop liver injury develop a fever and 63% develop a rash. In this study, we established a mouse model for DPH-induced liver injury and analyzed the mechanisms for hepatotoxicity in the presence of immune-related or inflammation-related factors and metabolic activation. Female C57BL/6 mice were administered DPH for 5 days in combination with l-buthionine-S,R-sulfoximine. Then, the plasma alanine aminotransferase (ALT) levels were increased, hepatic lesions were observed during the histological evaluations, the hepatic glutathione levels were significantly reduced, and the oxidative stress marker levels were significantly increased. The inhibition of cytochrome P450-dependent oxidative metabolism significantly suppressed the elevated plasma ALT levels and depleted hepatic glutathione. Among the innate immune factors, the hepatic mRNA levels of NACHT, LRR, pyrin domain-containing protein 3, interleukin-1β, and damage-associated molecular patterns were significantly increased. Prostaglandin E 1 treatment ameliorated the hepatic injury caused by DPH. In conclusion, cytochrome P450-dependent metabolic activation followed by the stimulation of the innate immune responses is involved in DPHinduced liver injury

Recent Advances in Detection, Isolation, and Imaging Techniques for Sulfane Sulfur-Containing Biomolecules

Hydrogen sulfide and its oxidation products are involved in many biological processes, and sulfane sulfur compounds, which contain sulfur atoms bonded to other sulfur atom(s), as found in hydropersulfides (R-S-SH), polysulfides (R-S-Sn-S-R), hydrogen polysulfides (H2Sn), etc., have attracted increasing interest. To characterize their physiological and pathophysiological roles, selective detection techniques are required. Classically, sulfane sulfur compounds can be detected by cyanolysis, involving nucleophilic attack by cyanide ion to cleave the sulfur–sulfur bonds. The generated thiocyanate reacts with ferric ion, and the resulting ferric thiocyanate complex can be easily detected by absorption spectroscopy. Recent exploration of the properties of sulfane sulfur compounds as both nucleophiles and electrophiles has led to the development of various chemical techniques for detection, isolation, and bioimaging of sulfane sulfur compounds in biological samples. These include tag-switch techniques, LC-MS/MS, Raman spectroscopy, and fluorescent probes. Herein, we present an overview of the techniques available for specific detection of sulfane sulfur species in biological contexts

Self-Assembly of Proteinaceous Multishell Structures Mediated by a Supercharged Protein

Engineered variants of the capsid-forming enzyme lumazine synthase can exploit electrostatic interactions to encapsulate complementarily charged guest macromolecules. Here we investigate the effect of ionic strength and cargo molecules on assembly of AaLS-13, a negatively supercharged lumazine synthase protein cage, and we show that multishell structures are produced upon mixing the capsid core with free capsomers and a positively supercharged variant of the green fluorescent protein GFP­(+36). The assembly process is mediated by favorable electrostatic interactions between the negatively charged capsid shells/capsomers and GFP­(+36) molecules, and it is therefore strongly dependent on ionic strength. The mechanism of formation of these assemblages is likely similar to the assembly of multishell structures of some virus-like particles, where outer shells organize as nonicosahedral structures with larger radii of curvature than the templating inner shell. In contrast to the viral multishell structures, the positively charged mediator was found to be essential for the assembly of multilayered structures of different shapes and sizes constituted of AaLS-13 capsomers. This mediator-bridging approach may be widely applicable to create protein-based hierarchical nanostructures for various nanotechnology applications such as drug delivery and bioimaging