發展抗金黃色葡萄球菌的新穎小分子藥物

Development of Novel Small-Molecule Agents against Staphylococcus aureus

陳逸洳(Yi-Ru Chen)

指導教授 ： 邱浩傑

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10223

摘要

Chapter I
發展針對金黃色葡萄球菌的新穎小分子化合物

耐甲氧苯青霉素之金黃色葡萄球菌(Methicillin-resistant Staphylococcus aureus , MRSA)具有能對抗多種抗生素的能力，在用藥方面增加困難度，在現今已成為了臨床治療上的很大威脅，因此開發新的抗生素是很重要的議題。我們自陽明大學的蕭崇瑋老師取得了157個化合物，分別對MRSA以及鮑氏不動桿菌(A. baumannii) 進行抗生素感受性試驗，篩選出14個具有潛力的化合物，再接著對細胞進行毒性測試，最後篩選出選擇性最高的前兩名，分別為SC78、SC80。
為了明白化合物的效果與應用性，我們將SC78、SC80與其他細菌進行測試，發現對45株自醫院分離出來的MRSA菌株與其他非金黃色葡萄球菌的革蘭氏陽性細菌有抑制效果，但在革蘭氏陰性細菌則不具有抑制能力。再來，我們將SC78、SC80與其他臨床上常用的抗生素比較效果，在MRSA與抗萬古霉素腸球菌(VR-E)這兩株多重抗藥細菌身上，SC78的效果明顯比其他抗生素為佳。
除此之外，為了瞭解SC78與SC80的作用特性，我們也進行殺菌效力評估試驗(time-kill assay)，發現4倍、8倍MIC濃度的SC78與SC80可以在24小時之內殺死99.9%的細菌，但是2倍MIC濃度的SC78與SC80則無此現象，顯示在高濃度的情況下為殺死細菌的效果，而在低濃度則為抑制細菌生長的效果。
另一方面，為了評估SC78是否有在臨床應用的潛力，我們將SC78給予受到MRSA感染的小鼠進行動物實驗，雖目前以此條件尚無法看到小鼠有治癒的現象，但即使如此，SC78這個先導藥物在抗生素的開發上仍具有相當的潛力。

Chapter II
以全基因體的策略去尋找由Celecoxib衍生的新穎抗金黃色葡萄球菌之化合物的作用機制

耐甲氧苯青霉素之金黃色葡萄球菌(Methicillin-resistant Staphylococcus aureus , MRSA)具有能對抗多種抗生素的能力，使得在用藥方面增加困難度，於現今已成為臨床治療上很大的威脅，因此開發新的抗生素是相當重要的議題。在先前的研究中，本實驗室以非類固醇類的抗發炎藥物希樂葆(Celecoxib)為基礎，合成並篩選出兩個可以有效抑制MRSA生長的化合物，暫命名為化合物-36(Cpd-36)與化合物-46(Cpd-46)。首先為了測試化合物的效果與應用性，本實驗使用革蘭氏陽性細菌例如葡萄球菌、腸球菌以及MRSA的標準菌株與35株台大醫院臨床分離的菌株；革蘭氏陰性細菌如大腸桿菌、沙門氏菌及孢氏不動桿菌和Cpd-36與Cpd-46進行抗生素感受性試驗，實驗結果發現革蘭氏陽性細菌對本實驗的藥物皆具有感受性，但在革蘭氏陰性菌上則沒有觀察到抑制效果。
為了提供資訊以利於合成更有效的化合物，我們想要瞭解化合物的作用機制，因此我們將化合物與金黃色葡萄球菌長期培養，挑選出對化合物具有抵抗能力的抗藥菌株，再抽取其全基因組去氧核醣核酸(genomic DNA)，利用全基因定序(next generation sequencing，NGS)去尋找並分析金黃色葡萄球菌的抗藥株與野型株在哪些基因上具有變異。
根據全基因定序的結果顯示，存在數個可能具有突變位點的基因，接著再使用傳統定序方式確認，最後找出一個最可能的yidC2基因，並發現突變株的YidC2蛋白其胺基酸確實有產生變化，因此我們推測此蛋白的改變和抗藥性的上升有關。同時我們將具有抗藥性的yidC2基因利用質體送入野型金黃色葡萄球菌當中，發現帶有抗藥基因的金黃色葡萄球菌，對於化合物的感受性會下降，此基因確實為化合物影響細菌的重要標的。
先前有研究指出，若細菌的YidC2蛋白失去功能，會影響其將氫離子排出菌體外的能力，因此對酸性環境會較敏感。本研究將化合物與金黃色葡萄球菌先行培養，再將細菌移入酸性環境中，發現細菌生長情形會下降，故證實此藥物會與YidC2蛋白作用。
另外，為了評估Cpd-36與Cpd-46是否有在臨床上實際應用的潛力，我們使用小鼠以腹腔注射感染MRSA後，再口服給予Cpd-46，觀察藥物是否可以降低因MRSA所造成的感染，或減緩感染情況並降低致死率，實驗結果顯示，Cpd-46應可以被腸胃道吸收，進而達成殺菌效果，推測Cpd-46應該具有應用在臨床上的潛力，因此值得去發展以Cpd-46為基礎的其他化合物，提高藥物的作用能力，進而發展更有效的抗耐甲氧苯青霉素之金黃色葡萄球菌的藥物。

關鍵字

金黃色葡萄球菌 ； 抗甲氧苯青霉素之金黃色葡萄球菌 ； 抗藥性 ； 抗生素之新藥開發

並列摘要

Chapter I
Development of Novel Small-Molecule Anti-Staphylococcal Agents

Methicillin-resistant Staphylococcus aureus (MRSA) possesses multi-drug resistance and increase difficulty in treatment, thus it has become a major threat in clinical treatment nowadays. We have tested a small compound library with antibiotic susceptibility testing against MRSA and A. baumannii. The results indicated that fourteen compounds show inhibitory efficacy against MRSA and A. baumannii. Further, we applied these fourteen compounds in MTT assay to test for cell toxicity, and finally we selected two best compounds, SC78 and SC80.
In order to understand the efficacy and the application of the candidate compounds, we tested SC78 and SC80 against the other bacteria, the result showed that SC78 and SC80 inhibit the growth of 45 clinically-isolated MRSAs and other non-Staphylococcus bacteria, but they are ineffective against Gram negative bacteria. Also, we have compared SC78 and SC80 to common antibiotics used in clinical treatment, the efficacy of SC78 is more effective than other antibiotics against MRSA and Vancomycin-Resistant Enterococci (VR-E).
To further understand the action mechanism of SC78 and SC80, we proceeded time-kill assay and discovered that four times and eight times the MIC of SC78 and SC80 killed 99.9% bacteria within 24 hours, but two times the MIC of SC78 and SC80 could not cause the same outcome, thus this indicated that it is bactericidal antibiotics in high dose and bacteriostatic antibiotics in low dose.
On the other hand, in order to estimate whether SC78 has potential in clinical application, we have given SC78 to mice infected by MRSA for animal experiment. Although there is no significant recovery in this condition, but SC78, the lead compound which inhibits the growth of MRSA efficaciously, has a lot of potential in the research on antibiotics.

Chapter II
Genome-wide Identification of the Action Mechanism of Novel Celecoxib Derived Agents against Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) possesses multi-drug resistance and increase difficulty in treatment, and the development of novel antibiotics is an important issue.
In our previous study, our laboratory had successfully developed two potent anti-MRSA drugs, named Compound-36 (Cpd-36) and Compound-46 (Cpd-46).
To examine the effects and applications of the compounds, we test the susceptibility of several Gram positive bacteria, including Staphylococci, Enterococci, standard MRSA strains as well as 35 clinically-isolated MRSAs. Gram negative bacteria such as E. coli, Salmonella, and A. baumannii were also tested. The results showed that Gram positive bacteria tested are susceptible to these two drugs. In contrast, for the Gram negative bacteria tested, they are not susceptible to the drugs.
To further understand the action mechanism of these two drugs, we cultured Staphylococcus aureus in the presence of compounds for a long period of time to select resistant mutant strains. The genomic DNA of resistant strains were sequenced by using Next-generation Sequencing (NGS) technique to identify the potential mutations associated with compounds resistance.
The NGS result has exclusively pointed out several mutation spots, and some of them are located within essential genes. We used conventional sequencing method to confirm and identified one possible mutated gene, yidC2. From the protein product of the yidC2 gene, we found that amino acid alterations are indeed present; therefore we presumed that there is a relationship between the change of the YidC2 protein and the rise of resistance.
We transformed plasmid carrying mutated yidC2 gene into wild-type S. aureus, and found that the resistance against compounds was increased, thus we suppose that the yidC2 gene is the target of compounds.
In previous study, YidC2 has been reported to influence the ability of bacteria to exclude hydrogen. If YidC2 protein lost its function, bacteria will become sensitive to acidic enviroment. Our result indicated that while S. aureus were treated with Cpd-46 and Cpd-36, the growth of S. aureus in acidic environment was reduced. It implicated that the compounds might act on the YidC2 protein.
On the other hand, to evaluate whether Cpd-36 and Cpd-46 have potential for clinical application, we infect mice with MRSA via intraperitoneal injection followed by orally administrated of Cpd-46. The result showed that oral Cpd-46 can suppress the MRSA and reduce the mortality rate, proving the feasibility of this drug in infection treatment. Altogether, Cpd-46 represent a promising lead agent to develop more potent anti-MRSA agents by targeting to YidC2.

並列關鍵字

S.aureus ； MRSA ； novel development of antibiotics ； resistance of antibiotics

參考文獻

1. Gordon, R.J. and F.D. Lowy, Pathogenesis of methicillin-resistant Staphylococcus aureus infection. Clin Infect Dis, 2008. 46 Suppl 5: p. S350-9.

2. Goss, C.H. and M.S. Muhlebach, Review: Staphylococcus aureus and MRSA in cystic fibrosis. J Cyst Fibros, 2011. 10(5): p. 298-306.

3. Diekema, D.J., et al., Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997-1999. Clin Infect Dis, 2001. 32 Suppl 2: p. S114-32.

4. Appelbaum, P.C., Microbiology of antibiotic resistance in Staphylococcus aureus. Clin Infect Dis, 2007. 45 Suppl 3: p. S165-70.

5. Johnson, A.P., A. Pearson, and G. Duckworth, Surveillance and epidemiology of MRSA bacteraemia in the UK. J Antimicrob Chemother, 2005. 56(3): p. 455-62.