2016 Fiscal Year Annual Research Report
糖加水分解酵素蛍光プローブ群の開発による新規迅速がんイメージング手法の確立
| Project/Area Number |
16F16109
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| Research Institution | The University of Tokyo |
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Principal Investigator |
浦野 泰照 東京大学, 大学院薬学系研究科(薬学部), 教授 (20292956)
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| Co-Investigator(Kenkyū-buntansha) |
RIVAS CHARLOTTE 東京大学, 大学院薬学系研究科(薬学部), 外国人特別研究員
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| Project Period (FY) |
2016-10-07 – 2019-03-31
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| Keywords | Sialidase / Fluorescence activation / Sialic acid substrate / Cancer imaging |
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| Outline of Annual Research Achievements |
Two novel fluorescent sialidase substrates were assessed for their ability to report on the presence of sialidase via a switch on in fluorescence. Both probes were based on the conjugation of sialic acid to a HMRef scaffold and they differed by the presence of a methyl group on the sialic acid carboxylic function (1 = HMRef-Neu5Ac, 2 = HMRef-Neu5AcMe). The stability of the probes was assessed by spectrometric means and it was observed that probe 2 possessed greater stability owing to the presence of the methyl group preventing internal catalysis via the acid hydrolysis pathway. 1 was found to be unstable at pHs >= 4.5 but only over extended time periods e.g. 1.5 hr. In vitro tests showed that only probe 1 was a suitable substrate for sialidase (of bacterial origin), probe 2 was not recognised by the enzyme and therefore was not activated to give a fluorescence response. Probe 1 is being further assessed by in vitro experiments.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Probe 1 is being assessed in cell lysate studies using lysates from colon cancer and prostate cancer cell lines. The experiments are also being carried out on the commercially available fluorescent sialidase substate 4-MUNANA as a control. Both probes however show no activation in the lysate solutions. The sialidase enzyme overexpressed in certain cancer cells (NEU3) has been reported to have a stricter substrate specificity in comparison to bacterial or viral sialidases, preferring gangliosides over glycoproteins. The probe is therefore going to be assessed against a range of bacteria, specifically bacteria that have been shown to cause tooth decay; porphyromonas gingivalis which has been reported to have sialidase expression and Lactobacillus acidophiolus and Lactobacillus crispatus in which sialidase expression is as of yet unknown.
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| Strategy for Future Research Activity |
The stability of probe 1 is to be improved. Its instability is a factor of both the acidity of the phenolic residue of the HMRef moiety and the presence of a carboxylic acid function next to the glycosidic link resulting in internal catalysis. This situation could be ameliorated by incorporating a self-immolative spacer into the probe. In this design, one end of the spacer is connected to sialic acid and the other is attached to the HMRef phenol through a hydrolytically stable ether bond. The presence of sialidase cleaves the bond between sialic acid and the spacer, thus revealing a phenol that initiates release of the pendant phenol via a presumed quinone methide intermediate. These spacer groups will allow for the release of the HMRef phenol, i.e. activation of fluorescence, without complications from nonspecific background release reactions and with tunable rates of release.
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