臺灣博碩士論文加值系統

中文摘要
目前，fluoroquinolone和ampicillin抗生素在臨床上已有許多菌株產生抗藥性。因此，開發新藥為當務之急。本實驗室已開發出一種化合物，由ampicillin和6-fluoroquinolonic acid (FP-4)經由coupling reaction所合成之新抗生素, FQ-1。先前研究結果顯示FQ-1是一種廣效性抗生素，尤其對臨床分離之多重抗藥性綠膿桿菌(Pseudomonas aeruginosa)仍具有抗菌活性。本論文的主要目的在研究FQ-1的抗菌機制及其抗病毒活性。
FQ-1抗菌機制研究結果，發現FQ-1不會抑制細胞壁合成，但會抑制細菌分裂時膈膜(septum)的形成，而造成枯草桿菌(Bacillus subtilis)產生cell filamentation的現象。另外, FQ-1也會抑制枯草桿菌的spore formation。FQ-1抗菌機制可能是與抑制penicillin- binding protein 3 (PBP3)功能和抑制細菌細胞的分裂。另一方面，新合成的抗生素FQ-1因具有兩個多環狀平板分子結構可表現嵌入DNA的活性，這與一些會嵌入(intercalate) DNA的藥物結構相似(如actinomycin D)。研究結果顯示，FQ-1嵌入DNA上須有連續兩個GC以上的序列，且FQ-1的β-lactam ring結構若是被penicillinase水解後，FQ-1就失去嵌入DNA及抗菌活性，顯示FQ-1的β-lactam ring結構在嵌入DNA及抗菌活性上是必須的。
FQ-1抗病毒活性的研究結果發現，FQ-1會抑制double-stranded DNA的vaccinia virus複製而不會抑制single-stranded linear RNA病毒JEV RP-9的複製且其抗病毒活性與rifampin相近，而FQ-1對BHK 21細胞(baby hamster kidney cell)的毒性則比rifampin低7倍。由治療指數(therapeutic index)分析結果顯示，FQ-1在抗vaccinia virus用藥上其治療指數高於rifampin約7倍，顯示其開發為臨床用藥的潛力優於rifampin。

Abstract
Recently, more and more drug resistant bacteria emerge as a result of drug abuse by human beings. In order to develop a new antibiotic, compound N-(6-fluoroquinoloyl)-ampicillin, FQ-1 was synthesized by coupling the carboxyl group of 6-fluoroquinolone (FP-4) with the α-amino group of ampicillin in our laboratories. FQ-1 exhibits a broad-spectrum antibacterial activities. In particular, it is active against some clinical isolates of Pseudomonas aeruginosa, which are highly resistant to norfloxacin and/or gentamincin. Protoplast formation study revealed that FQ-1 did not inhibit biosynthesis of the cell wall but did induce cell filamentation of Bacillus subtilis at the level close to its MIC. This implied that FQ-1 might inhibit penicillin binding protein 3 (PBP3) which was required for septum formation. Interestingly, study on the mechanism of antimicrobial action of FQ-1 revealed that FQ-1 could intercalate into double-stranded DNA but not single-stranded DNA. Moreover, two tandem GC sequence was the minimal requirement for FQ-1 intercalation. However, FQ-1 lost its anti- bacterial activity and DNA intercalation activity after it was treated with penicillinase, indicating that the β-lactam ring structure in ampicillin moiety of FQ-1 might be hydrolyzed by penicillinase and the hydrolyzed structure do not own intercalation activity. In the aspect of antiviral activity, our results demonstrated that FQ-1 did inhibit vaccinia virus and replication but didn’t inhibit Japanese Encephalitis Virus (JEV) replication, a single- stranded RNA virus. As compared to rifampin, FQ-1 showed a much lower cytotoxicity to BHK 21 cells than did rifampin. Thus, the therapeutic index of FQ-1 was 7-fold higher than that of rifampin. This suggests that FQ-1 is more potential to be developed as an antiviral drug for clinical use than rifampin.

目 錄
目錄…………………………………………………………………………Ⅰ
表目錄……………………………………………………………………………Ⅳ
圖目錄………………………………………………………………………Ⅴ
中文摘要……………………………………………………………………Ⅶ
英文摘要…………………………………………………………………………Ⅷ
第一章 緒論
壹、新合成抗生素FQ-1藥物簡介………………………………………1
貳、β-lactam抗生素與Quinolone藥物簡介─藥物基本結構及其抗菌 機制……………………….…………………………………………………2
參、Vaccinia virus及抗Vaccinia virus藥物簡介……………………5
肆、研究目標…………………………………………………………………7
第二章 材料與方法
★實驗材料
一、細胞株、病毒株及菌株……………………………………………8
二、實驗所採用之藥品、試劑、培養基及套組等……………………8
三、主要儀器設備……………………………………………………………….10
四、實驗用培養基成份及緩衝液之配製………………………………10
★實驗方法
一、菌種的準備及儲存……………………………………………………12
二、細胞培養及病毒株之增殖……………………………………………12
三、FQ-1的抗菌活性測定…………………………………………………13
四、紙錠瓊脂擴散法測定FQ-1、norfloxacin、ampicillin之抗菌活性….…………………………………………………………………………14
五、細胞壁形成的抑制效果………………………………………………14
六、芽胞形成的抑制效果………………………………………………14
七、FQ-1嵌入DNA之作用…………………………………………………15
八、FQ-1的β-lactam ring結構與嵌入DNA的相關性………………15
九、電灑離子質譜分析 (Electrospray Ionization Mass Spectroscopy, ESI-MS)……………………………………………………………………15
十、FQ-1對DNA合成的影響…………………………………………16
十一、病毒斑形成的抑制效果……………………………………………16
十二、病毒複製的抑制效果………………………………………………17
十三、BHK 21細胞毒殺性試驗…………………………………………17
十四、藥物的治療指數評估…………………………………………17
十五、回歸曲線…………………………………………………………………18
第三章 結果
一、FQ-1的β-lactam ring結構與抗菌活性的相關性………………19
二、紙錠瓊脂擴散法測定FQ-1、norfloxacin、ampicillin之抗菌活性19三、FQ-1的抗菌機制………………………………………………………19
四、抑制芽胞形成之作用…..…………………………………………..20
五、FQ-1嵌入DNA之作用………………………………………………….20
六、電灑離子質譜分析……………………………………………………21
七、FQ-1的β-lactam ring結構與嵌入DNA活性的相關性…………21
八、FQ-1抑制DNA合成作用………………………………………………22
九、抑制病毒斑形成之作用………………………………………………22
十、抑制病毒複製之作用…………………………………………………22
十一、BHK 21細胞毒殺性試驗……………………………………………23
十二、藥物的治療指數評估………………………………………………23
第四章 討論
一、FQ-1抗微生物機制之探討……………………………………………24
二、FQ-1抗病毒活性之探討………………………………………………27
三、開發為臨床治療Vaccinia virus感染用藥之評估………………28
參考文獻…………………………………………………………………….51

參考文獻 (References)
1. Patrick R. Murray., Ken S. Rosenthal., George S. Kobayashi., Michael A.
Pfaller. Medical microbiology 4th . Mosby. p.39.
2. Lin WP., Yanga YW., Tsoub TL., Jib DD., Tangb ST., Shiauc CY., Chend CH. and Liu YT. In vitro and in vivo antipseudomonal activity and acute toxicity of the newly synthesized Fluoroquinolonyl ampicillin derivatives. Submitted to The Journal of Laboratory and Clinical Medicine.
3. Naoki , K., Haru, K., Kaori, T. —B., Kunitomo, W., and Kazue, U. J. 1997. Comparative in-vitro and in-vivo activity of AM-1155 against anaerobic bacterial . Antimicrobial Chemotherapy 40:631-637.
4. Eiji, W. and Susumu, M. 1994. In vitro antibacterial activity of AM-1155, a novel 6-fluoro-8-methoxy quinolone. Antimicrobial Agents and Chemotherapy. Mar. 38(3): 594-601.
5. Hiroshi, K., Akira, I., Satoshi, M., Segio, S. and Tsutomu, I. 1980. Structure- activity relationships of antibacterial 6,7-and 7,8-disubstituted 1-alkyl-1,4- dihydro-4-oxoquinolone-3-carboxylic acids. J. Med. Chem. 23:1358-1363.
6. Drlica, K. 1999. Mechanisms of fluoroquinolone action. Curr. Opin. Microbiol. 2:504—508.
7. Fiaccadori F. . Fluoroquinolones: a new era in antibiotic therapy ? Annali Italiani di Medicina Interna. 7(1):46-50, 1992 Jan-Mar.
8. E, Wakabayashi. and S, Mitsuhashi. In vitro antibacterial activity of AM-1155, a novel 6-fluoro-8-methoxy quinolone. 1994. Antimicrobial agents and chemotherapy 38:594-561.
9. 黃啟嘉, 醫用微生物學18th , 合記圖書出版社, 上冊p.30, 37-38, 220-221, 231-232 ; 下冊232-239.
10. 蔡文城, 微生物學3th ,藝軒圖書出版社, p.27, 143,174, 180-181.
11. George N. R. Forty years of β-lactam research. Journal of Antimicrobial Chemotherapy (1998) 41, 589—603.
12. Fleming, A. (1929). On the antibacterial action of cultures of Penicillium, with special reference to their use in the isolation of B.influenzae. British Journal of Experimental Pathology 10, 226—36.
13. J. A. Kelly, O. Dideberg, P. Charlier, J. P. Wery, M. Libert, P. C. Moews, J. R. Knox, C. Duez, CL. Fraipont, B. Joris, J. Dusart, J. M. Frere, and J. M. Ghuysen. On the origin of bacterial resistance to penicillin : comparison of a β-lactamase and a penicillin target. Science. 231(4744):1429-31, 1986 Mar 21.
14. Irina M. and Shahriar M. Minireview :Kinship and diversification of bacterial penicillin-binding proteins and β-lactamases. Antimicrobial Agents and Chemotherapy, Jan. 1998, p. 1—17 Vol. 42, No. 1.
15. David L., Meghan E. and Peter S. Roles of low-molecular-weight penicillin-binding proteins in Bacillus subtilis spore peptidoglycan synthesis and spore properties. J. of Bacteriology, Jan. 1999, p. 126—132 Vol. 181, No. 1.
16. Michael J. and Thomas J. Direct Quantitation of the numbers of individual penicillin-binding proteins per cell in Staphylococcus aureus. J. of Bacteriology, Jan. 2002, p. 588—591 Vol. 184, No. 2.
17. Ghuysen, J. M. 1991. Serine β-lactamases and penicillin-binding proteins. Annu. Rev. Microbiol. 45:37—67.
18. Mariana G., Se´Rgio R., Hermi´Nia De L, and Alexander T. Complementation of the essential peptidoglycan transpeptidase function of penicillin-binding protein 2 (PBP2) by the drug resistance protein PBP2A in Staphylococcus aureus. J. of Bacteriololgy, Nov. 2001, p. 6525—6531 Vol. 183, No. 22.
19. Hopper, D. C., Wolfson, J. S. 1991. Fluoroquinolone antimicrobial agents. The New England of Medicine. Feeb. 384-394.
20. Hopper, D. C. 1999. Mode of action of flurorquinolones. Drugs. 58 suppl. 2:6-10.
21. Drlica, K. and X. Zhao. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61:377—392.
22. Sugino, A., C. L. Peebles, K. N. Kruezer, and N. R. Cozzarelli. 1977. Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc. Natl. Acad. Sci. USA 74:4767—4771.
23. Palu G., Valisena S., Ciarrocchi G., Gatto B. and Palumbo M. Quinolone binding to DNA is mediated by magnesium ions. Proceedings of the National Academy of Sciences of the United States of America. 89(20):9671-5, 1992 Oct 15.
24. Albrecht, H. A., G. Beskid, K. K. Chan, J. G. Christenson, R. Cleeland, K. H. Deitcher, N. H. Georgopapadakou, D. D. Keith, D. L. Pruess, and J. Sepinwall. 1990. Cephalosporin 3’-quinolone esters with a dual mode of action. J. Med. Chem. 33:77—86.
25. Georgopapadakou, N. H., and A. Bertasso. 1993. Mechanisms of action of cephalosporin 3’-quinolone esters, carbamates, and tertiary amines in Escherichia coli. Antimicrob. Agents Chemother. 37:559—565. laboratory manual, p. 368—369. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
26. Geroge, M. E. 1999. Activity of newer fluoroquinolones in vitro against Gram -positive bacteria. Drugs. 58 suppl. 2:23-28.
27. Volmer, D. A., Mansoori, B., and Locke, S. J. 1997. Study of 4-quinolone antibiotics in biological samples by short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Anal. Chem. 69:4143-4155.
28. H. Kashiwase, T. Katsube, K. Iida, T. komai, T. Nishigaki, T. Kimura, and M. Yamashita. Investigation into the mode of action of R-91650, an arylpiperazinyl fluoroquinolone, on feline immunodeficiency virus replication inhibitory activity. Arch Virol. (2000) 145：859-869.
29. Scott J. Goebel, Gerard P. Johnson, Marion E. Perkus, Stephen W. Davis, Joseph P. Winslow, and Enzo Paoletti. 1990. The complete DNA sequence of Vaccinia virus. Virology 179, 247-266.
30. B. N. Fields., D. M. Knipe., P. M. Howley. Fields Virology, 3th . Chapter 83：Poxvirdae：The viruses and their replication. p.2367-2659.
31. Masaharu T. Poxviruses and tha origin of the eukaryotic nucleus. J. Mol. Evol. (2001) 52：419-25.
32. Erik De Clercq. Vaccinia virus inhibitors as a paradigm for the chemotherapy of poxvirus infections. Clinical Microbiololgy Reviews, Apr. 2001, p.382-397.
33 Clarissa R., A. Damaso, and S. J. Keller. Cyclosporin A inhibit vaccinia virus replication in vitro. Arch Virol. (1994) 134：303-319.
34. Sekiguchi J., and Shyman S. Novobiocin inhibits vaccinia virus replication by blocking virus assembly. Virology 1997 Aug 18：235(1)：129-37.
35. Mansun L., and G. L. Smith. Antibody neutralization of the extracellular enveloped form of Vaccinia virus. Virology 280, 132-142. (2001).
36. Bair CH., Chung CS., Irina A. Vasilevskaya, and Chang W. Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to Vaccinia virus killing. J. of Virology, July 1996, p.4655-4666.
37. Anna RE., and Bernard M. Restriction of Vaccinia virus replication in CHO cells occurs at the stage of viral intermediate protein synthesis. Virology 206, 984-993 (1995).
38. David U., Douglas F., and Dennis E. H. Brefeldin A inhibits Vaccinia virus envelopment but does not prevent normal processing and localization of the putative envelopment receptor P37. Journal of General Virology (1995), 76, p.103-111.
39. Erik De Clercq, Ria Bernaerts, Y. Fulmer S., and John A. Montgomery. Broad-spectrum antiviral activity of Carbodine, the Carbocyclic analogue of Cytidine. Biochemical Pharmacology. Vol. 39. No. 2. pp.319-325. 1990.
40. Albert Balows. Current techniques — for antibiotic — susceptibility testing. Chapter Ⅱ and Ⅲ. p.6 — 25. Springfield, Illinois, U.S.A.
41. David C. G., David R. G., Karen J. LW., and Richard D. Smith. Observation of duplex DNA-drug noncovalent complexes by electrospray ionization mass spectrometry. J. Am. Chem. Soc. 1994, Vol.116, No.13, 6027-6028.
42. Katty X. W., Toshimichi Shibue, and Michael L. G. Non-covalent complexes between DNA-binding drugs and double-stranded oligodeoxynucleotides : a study by ESI ion-trap mass spectrometry. J. Am. Chem. Soc. 2000,Vol. 122, No.2, 300-307.
43. Michelle L. R., Jennifer S. B., Sean M. K., and Devinder K. Evaluation of complexation of metal-mediated DNA-binding drugs to oligonucleotides via electrospray ionization mass spectrometry. Nucleic acids research, 2001, Vol.29, No.21, e103.
44. Amit K., Jennifer L. B. and Margaret M. S. Observation of Daunomycin and Nogalamycin complexes with duplex DNA using electrospray ionization mass spectrometry. Rapid commun. mass spectrum. 1999, Vol.13, 2489-2497.
45. Timothy D. Veenstra. Electrospray ionization mass spectrometry : a promising new technique in the study of protein / DNA noncovalent complexes. Biochemical and biophysical research communications. 1999, Vol.257, 1-5.
46. Frederic Rosu, Valĕrie Gabelica, Claude Houssier and Edwin De Pauw. Determination of affinity, stoichiometry and sequence selectivity of minor groove binder complexes with double — stranded oligodeoxynucleotides by electrospray ionization mass spectrometry. Nucleic acids reseatch, 2002, Vol.30, No.16, e82.
47. S. B. Primrose, R. M. Twyman and R. W. old. Principles of gene manipulation 6th . Chapter 15：Nucleic acid probes and their applications. p.331-3.
48. Thomas M., David L. P, Christine B. P., Arithur R. H., and Peter S. Analysis of outgrowth of Bacillus subtilis spores lacking penicillin-binding protein 2a. J. of Bacteriology, Dec. 1998, p. 6493—6502 Vol. 180, No. 24.
49. Liu YT., Yeh SJ., Liao CL., Chou CF., and Shou L. Mechanism of action of chounghwamycin A. Proceedings of the National Science Council, Republic of China - Part B, Life Sciences. 13(4):262-6, 1989 Oct.
50. Hou1MH., Howard R., Gao YG., Andrew H. and Wang J. Crystal structure of actinomycin D bound to the CTG triplet repeat sequences linked to neurological diseases. 4910-4917. Nucleic Acids Research, 2002, Vol. 30 No. 22.
51. Albrecht, H. A., G. Beskid, J. G. Christenson, K. H. Deitcher, N. H. Georgopapadakou, D. D. Keith, F. M. Konzelmann, D. L. Pruess, and C. C. Wei. 1994. Dual-action cephalosporins incorporating a 39-tertiary-amine- linked quinolone. J. Med. Chem. 37:400—407.
52. Ehrhardt, A. F., and C. C. Sanders. 1992. Structure/activity studies of quinolonyl β-lactam antimicrobials (QLA) using a genetically defined panel, abstr. 771, p. 239. In Program and Abstracts of the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C.
53. Lotte B. Pedersen, Esther R. Angert, and Peter Setlow. Septal localization of penicillin-binding protein 1 in Bacillus subtilis. J. of Bacteriology, May 1999, p. 3201—3211 Vol. 181, No. 10.
54. Andrea Feucht, Richard A. Daniel and Jeffery Errington*. Characterization of a morphological checkpoint coupling cell-specific transcription to septation in Bacillus subtilis. Molecular Microbiology (1999) 33(5), 1015-1026.
55. Lotte B. Pedersen, Thomas Murray, David L. Popham, and Peter Setlow. Characterization of dacC, which encodes a new low-molecular-weight penicillin-binding protein in Bacillus subtilis. J. of Bacteriology, Sept. 1998, p. 4967—4973 Vol. 180, No. 18.
56. Thomas Murray, David L. Popham, and Peter Setlow. Identification and characterization of pbpA encoding Bacillus subtilis penicillin-binding protein 2A. J. of Bacteriology, May 1997, p. 3021—3029 Vol. 179, No. 9.
57. David A. Cano, Chakob Mouslim, Juan A. Ayala, Francisco Garci´A-Del Portillo,and Josef Casadesu´ Sl. Cell division inhibition in Salmonella typhimurium histidine-constitutive strains: an ftsI-like defect in the presence of wild-type penicillin-binding protein 3 Levels. J. Bacteriology, Oct. 1998, p. 5231—5234 Vol. 180, No. 19.
58. Petra Anne Levin and Richard Losick. Characterization of a cell division gene from Bacillus subtilis that is required for vegetative and sporulation septum formation. J. of bacteriology, Vol. 176, No. 5, 1994, p. 1451-1459.
59. Derrell C. M., Adam D., and David L. P. Two class A high-molecular-weight penicillin-binding proteins of Bacillus subtilis play redundant roles in sporulation. J. of Bacteriology, Oct. 2001, p. 6046—6053 Vol. 183, No. 20.
60. David A. Cano, Chakib M., Juan A. A., Francisco Garci´A-Del P., and Josep Casadesu´ S. Cell division inhibition in Salmonella typhimurium histidine-constitutive strains: an ftsI-like defect in the presence of wild-type penicillin-binding protein 3 levels. J. of Bacteriology, Oct. 1998, p. 5231—5234 Vol. 180, No. 19.
61. Lee EJ., Yeo JA., Cho CB., Lee GJ., Han SW., and Kim SK. Amine group of guanine enhances the binding of norfloxacin antibiotics to DNA. Eur. J. Biochem. 267, p.6018-6024 (2000)
62. Bailly, C., Colson, P., and Houssier, C. 1998. The orientation of norfloxacin bound to double-stranded DNA. Biochemical and Biophysical Research Communications. 243:844-848.