細菌轉醣酶之研究:受質與抑制劑的設計、合成與活性評估

施皓維

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細菌轉醣酶之研究:受質與抑制劑的設計、合成與活性評估

Bacterial Transglycosylase Studies : Design, Synthesis and Bio-evaluation of Substrates and Inhibitors

施皓維 , Ph.D　　Advisor：翁啟惠　　Co-advisor ：鄭偉杰

英文

抗生素；細胞壁轉醣酶Lipid II ；結構活性分析；抑制劑； antibiotics ； bacterial transglycosylase ； structural activity study ； inhibitor ； Lipid II

Abstract │ Reference (34) │ Altmetrics

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Due to the increasing drug resistance, design and synthesis of potent antibiotics is urgent and critical to scientists. Bacterial TGase is considered as a potential target because of the lack of a eukaryotic counterpart, easy access and no resistance to current drugs. In order to develop new antibiotics against TGase, the detailed study in protein-substrate interaction and the establishment of TGase assay are required. However, preparation of the key component, TGase substrate-Lipid II, is difficult due to the low natural abundance and complex structure. Through our efforts, we established enzymatic and chemical synthetic approaches to prepare natural Lipid II and derived analogues. Also, we developed a new chemical synthetic route and successfully applied to Lipid II and Lipid IV preparation. With Lipid II and its derivatives in hand, a systematic investigation from structural study, assay development, protein characterization, inhibitor design and evaluation was initiated.
  First, we focused on the structural activity study of substrate, Lipid II, and prepared several Lipid II analogues to examine the role of the peptide moiety with regard to their contribution to substrate activity. As a result, we have discovered that the first two amino acids (D-Lac-L-Ala) within the peptide chain of Lipid II is essential for substrate binding-affinity and activity in our SPR analysis and kinetic studies. Besides, we found the fluorophore modified at the ε-NH2 of lysine side chain does not affect the substrate affinity.
Based on this result, we synthesized the fluorescent Lipid II with a NBD-tag at the lysine side chain as our TGase probe. The fluorescent probe not only reduces the usage amount of substrate and protein, but enhances the sensitivity of assay system. By using this NBD-Lipid II, the TGase activity assay has been established to characterize protein behaviors and to evaluate inhibitory activity against the TGase enzyme.
From the view of inhibitor design, we developed a combinatorial approach to prepare iminocyclitol-based libraries and screened out the first iminicyclitol-based inhibitor with IC50 value of 52 μM, which potentially has a similar binding feature with Moenomycin A in TGase after computer modeling study. We also designed and synthesized some substrate-based inhibitors guided from the structural activity study of Lipid II. The most potent substrate-based inhibitor, GalNAc-Lipid II, has a IC50 value of 5 μM and was delivered to crystallization with TGase for binding-site and binding-mode study

Reference ( 34 ) 〈TOP〉

4. (a) de Kruijff, B.; van Dam, V.; Breukink, E., Lipid II: A central component in bacterial cell wall synthesis and a target for antibiotics. Prostaglandins, Leukotrienes Essent. Fatty Acids 2008, 79, 117-121. (b) Ritter, T. K.; Wong, C. H., Carbohydrate-based antibiotics: A new approach to tackling the problem of resistance. Angew. Chem., Int. Ed. 2001, 40, 3509-3533. (c) Cheng, T. J. R.; Sung, M. T.; Liao, H. Y.; Chang, Y. F.; Chen, C. W.; Huang, C. Y.; Chou, L. Y.; Wu, Y. D.; Chen, Y.; Cheng, Y. S. E.; Wong, C. H.; Ma, C.; Cheng, W. C., Domain requirement of moenomycin binding to bifunctional transglycosylases and development of high-throughput discovery of antibiotics. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 431-436. (d) Shih, H. W.; Chen, K. T.; Chen, S. K.; Huang, C. Y.; Cheng, T. J. R.; Ma, C.; Wong, C. H.; Cheng, W. C., Combinatorial approach toward synthesis of small molecule libraries as bacterial transglycosylase inhibitors. Org. Biomol. Chem. 2010, 8, 2586-2593. (e) Cheng, T. J. R.; Wu, Y. T.; Yang, S. T.; Lo, K. H.; Chen, S. K.; Chen, Y. H.; Huang, W. I.; Yuan, C. H.; Guo, C. W.; Huang, L. Y.; Chen, K. T.; Shih, H. W.; Cheng, Y. S. E.; Cheng, W. C.; Wong, C. H., High-throughput identification of antibacterials against methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase. Bioorg. Med. Chem. 2010, 18, 8512-8529. (f) Stembera, K.; Vogel, S.; Buchynskyy, A.; Ayala, J. A.; Welzel, P., A surface plasmon resonance analysis of the interaction between the antibiotic moenomycin A and penicillin-binding protein 1b. Chembiochem 2002, 3, 559-565.
連結：
5. (a) Lovering, A. L.; de Castro, L. H.; Lim, D.; Strynadka, N. C. J., Structural insight into the transglycosylation step of bacterial cell-wall biosynthesis. Science 2007, 315, 1402-1405. (b) Yuan, Y. Q.; Barrett, D.; Zhang, Y.; Kahne, D.; Sliz, P.; Walker, S., Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 5348-5353. (c) Sung, M. T.; Lai, Y. T.; Huang, C. Y.; Chou, L. Y.; Shih, H. W.; Cheng, W. C.; Wong, C. H.; Ma, C., Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 8824-8829.
連結：
6. (a) Ye, X. Y.; Lo, M. C.; Brunner, L.; Walker, D.; Kahne, D.; Walker, S., Better substrates for bacterial transglycosylases. J. Am. Chem. Soc. 2001, 123, 3155-3156. (b) Fraipont, C.; Sapunaric, F.; Zervosen, A.; Auger, G.; Devreese, B.; Lioux, T.; Blanot, D.; Mengin-Lecreulx, D.; Herdewijn, P.; Van Beeumen, J.; Frere, J. M.; Nguyen-Disteche, M., Glycosyl transferase activity of the Escherichia coli penicillin-binding protein 1b: Specificity profile for the substrate. Biochemistry 2006, 45, 4007-4013. (c) Liu, C. Y.; Guo, C. W.; Chang, Y. F.; Wang, J. T.; Shih, H. W.; Hsu, Y. F.; Chen, C. W.; Chen, S. K.; Wang, Y. C.; Cheng, T. J. R.; Ma, C.; Wong, C. H.; Fang, J. M.; Cheng, W. C., Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl Moiety. Org. Lett. 2010, 12, 1608-1611. (d) Perlstein, D. L.; Wang, T. S. A.; Doud, E. H.; Kahne, D.; Walker, S., The Role of the Substrate Lipid in Processive Glycan Polymerization by the Peptidoglycan Glycosyltransferases. J. Am. Chem. Soc. 2010, 132, 48-49.
連結：
9. (a) Schwartz, B.; Markwalder, J. A.; Wang, Y., Lipid II: Total synthesis of the bacterial cell wall precursor and utilization as a substrate for glycosyltransfer and transpeptidation by penicillin binding protein (PBP) 1b of Eschericia coli. J. Am. Chem. Soc. 2001, 123, 11638-11643. (b) VanNieuwenhze, M. S.; Mauldin, S. C.; Zia-Ebrahimi, M.; Winger, B. E.; Hornback, W. J.; Saha, S. L.; Aikins, J. A.; Blaszczak, L. C., The first total synthesis of lipid II: The final monomeric intermediate in bacterial cell wall biosynthesis. J. Am. Chem. Soc. 2002, 124, 3656-3660. (c) Schwartz, B.; Markwalder, J. A.; Seitz, S. P.; Wang, Y.; Stein, R. L., A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP1b and development of a continuous fluorescence assay. Biochemistry 2002, 41, 12552-12561. (d) Chang, Y. F.; Liu, C. Y.; Guo, C. W.; Wang, Y. C.; Fang, J. M.; Cheng, W. C., Solid-phase organic synthesis of polyisoprenoid alcohols with traceless sulfone linker. J. Org. Chem. 2008, 73, 7197-7203.
連結：
13. (a) Debenham, J. S.; Rodebaugh, R.; FraserReid, B., TCP- and phthalimide-protected n-pentenyl glucosaminide precursors for the synthesis of nodulation factors as illustrated by the total synthesis of NodRf-III (C18:1, MeFuc). J. Org. Chem. 1997, 62, 4591-4600. (b) Pollastri, M. P., Microwave-mediated synthesis: A green chemistry technology. Abstracts of Papers of the American Chemical Society 2005, 229, U575-U575.
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