DnaQ核酸水解外切酶家族之可能的抑制劑之鑑定

Identification of the potential inhibitors for the DnaQ-like exonucleases

黃冠偉(Kuan-Wei Huang)

指導教授 ： 袁小琀

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.00440

摘要

DnaQ核酸水解外切酶家族由超過萬種蛋白質成員組成，廣泛分布於原核與真核生物中。此家族成員具有一高度保留性3端至5端核酸水解外切酶結構區域，含有5個高度保留性胺基酸。此類核酸水解蛋白質在細胞中主要參與DNA或RNA的代謝，例如DNA的複製，DNA的修復、DNA的降解，或RNA成熟修飾過程。近來研究發現，部分病毒會利用DnaQ核酸水解外切酶家族成員，來幫助自身感染宿主細胞。故篩選抑制劑來抑制DnaQ核酸水解酶的活性，可能有助於抗病毒感染藥物的研發。
本論文使用DnaQ核酸水解外切酶家族成員之一的CRN-4與RNase T做為模型，用以篩選11種可能的核酸水解酶抑制劑。CRN-4核酸水解外切酶的活性實驗結果顯示，相對於其他9種可能的抑制劑而言，4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoicacid 與 5,5'-dithiobis(2-nitrobenzoate) 具有相對較好的抑制效果。而在 RNase T核酸水解外切酶活性實驗中，則是p-chloromercuriphenyl sulfonate 與Aurintricarboxylic acid 具有相對較好的抑制效果。此外根據CRN-4和2-(N-morpholino)ethanesulfonic acid的複合物之晶體結構，顯示出此化合物結合在CRN-4的活性中心位置，造成CRN-4活性中心發生構形變化，而導致CRN-4的核酸水解外切酶活性受到抑制。我們的研究結果顯示篩選的部分化合物，的確可以抑制DnaQ核酸水解外切酶家族成員的活性，然而這些化合物的抑制效果與專一性需要再加改善。

關鍵字

DnaQ核酸水解外切酶家族 ； 核酸水解酶抑制劑 ； CRN-4 ； RNase T ； 蛋白質結晶結構

並列摘要

The family of the DnaQ-like exonucleases contains more than ten thousand members widely distributed in prokaryotes and eukaryotes. These exonucleases all contain a highly conserved DEDDh domain with four acidic residues for metal ion binding and one general base residue in the active site. Members in this family play key roles in DNA or RNA metabolism, such as proofreading in DNA replication, DNA processing in DNA repair, DNA degradation in apoptosis and RNA processing in RNA maturation. Recent studies show that several exonucleases in this superfamily are important for viral infections. It is thus important to identify inhibitors for this family of nucleases that may be helpful for the development of anti-viral agents.
Here using CRN-4 and RNase T, members of DnaQ-like exonuclease, as the model system, we screened 11 inhibitor candidates. We found that two compounds, 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-
benzoicacid and 5,5'-dithiobis(2-nitrobenzoate), could weakly inhibit the exonuclease activity of CRN-4, whereas two compounds, p-chloromercuriphenyl sulfonate and aurintricarboxylic acid, could strongly inhibit the exonuclease activity of RNase T. Moreover, we co-crystallized CRN-4 with one of the weak inhibitors, 2-(N-morpholino)ethanesulfonic acid (MES). The crystal structure of CRN-4 in complex with MES shows that MES was bound in the active site and the general base His179 was flipped out of the active site. In summary, we identified potential inhibitors for the DnaQ-like exonucleases; however, the inhibition activity and specificity of these compounds need to be further improved.

並列關鍵字

DnaQ-like exonucleases ； nuclease inhibitor ； CRN-4 ； RNase T ； protein crystal structure

參考文獻

1. Bailey, S.L., Harvey, S., Perrino, F.W., and Hollis, T. (2012). Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease. DNA repair 11, 65-73.

2. Barber, G.N. (2011). Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses. Current opinion in immunology 23, 10-20.

3. Chowdhury, D., Beresford, P.J., Zhu, P.C., Zhang, D., Sung, J.S., Demple, B., Perrino, F.W., and Lieberman, J. (2006). The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death. Molecular cell 23, 133-142.

4. Deutscher, M.P., Marlor, C.W., and Zaniewski, R. (1984). Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA. Proc Natl Acad Sci U S A 81, 4290-4293.

5. Eckerle, L.D., Becker, M.M., Halpin, R.A., Li, K., Venter, E., Lu, X., Scherbakova, S., Graham, R.L., Baric, R.S., Stockwell, T.B., et al. (2010). Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing. Plos Pathog 6, e1000896.