Biomedical Application of Activity Probe for Neuraminidase and Steroid Sulfatase
Date Issued
2007
Date
2007
Author(s)
Lu, Chun-Ping
DOI
en-US
Abstract
Depending on their enzymatic activities, the small molecular activity probe can be used to tag, enrich or isolate distinct sets of proteins. It only selectively reacts with enzymes that have a catalytic activity and covalent linkage with targeted enzyme. Activity probe contains four major components: a recognition head, a trapping device, a linker and a reporter group. The designed probe 6 was employed by this study to test its labeling effects on influenza virus neuraminidase (NA), whereas for the application of steroid sulfatase (STS), probes 11 and 12 were adopted. Compounds 17, 18 and 19, whose structures were designed on the basis of that of probe 12, were used for inhibiting STS activity.
Part I
For the detection and inhibition of influenza virus NA, the structure of probe 6 was designed to contains four fragments, a sialic acid for recognition, a latent quinone methide for trapping device, a linker and a biotin as reporter for detection and separation. Once a sialic acid is released by the hydrolysis of NA, the enzyme can be covalently modified by the resulting highly reactive quinone methide. In our study, the biotin labeled Arthrobacter ureafaciens NA were observed by Western blotting. For virus capturing experiment, the probe was be used to attach the microplates through avidin-biotin interactions and the captured virus were successfully demonstrated by the ELISA assay. To identify the captured virus, RT-PCR method combined with specific primer sets was future implemented. Overall, the novel approach adopted in this study offers opportunities for the rapid screening of antibodies against influenza virus and development of sensitive, rapid diagnostic methods.
Part II
Steroid sulfatase (STS) facilitates the estrogen uptake of breast cancer cells by mediating the desulfation of estrone sulfate. Human steroid sulfatase has become a target for drug development due to its increased expression and activity in breast carcinoma. In this study, two activity probes (probes 11 and 12) and three inhibitors (compounds 17, 18, and 19) were used for STS labeling and inhibition. Regarding STS labeling, probe 11 was shown to specifically react with STS. The dot signal intensity of probe 11 was illustrated by dot blot analysis to be correlated with enzymatic activity. Compared with probe 11, probe 12 demonstrated that higher specificity and less cross-reactivities. For STS inactivation study, compound 19, trivalent tyrosine sulfate ester derivative, revealed better inhibition ability than compounds 17 and 18. For compound 19, moderate cell permeability and no cytotoxicity for CHO cells were demonstrated in live cell assay. As a result, for the development of breast cancer treatment, probe 12 and compound 19 may be two effective chemical devices in STS identification, STS inhibition, and functional state determination in complex proteome.
Subjects
流行性感冒病毒
活性探針
唾液酸酶
唾液酸
生物素
固醇硫酸酶
抑制劑
influenza virus
activity probe
quinone methide
neuraminidase
sialic acid
biotin
steroid sulfatase
inhibitor
SDGs
Type
other
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