細菌細胞壁是細菌的重要結構，其中肽聚醣是細菌細胞壁主要的構成單位。其中Lipid II是肽聚醣的生物合成中最小之生物分子，同時也是天然物抗生素之作用標靶，至少四種不同類別之天然物抗生素以Lipid II為作用標靶，如: 萬古黴素(vancomycin)、替考拉寧(teicoplanin)、雷莫拉寧(ramoplanin)和乳鏈菌肽(nisin)。我們因此根據Lipid II及肽聚醣之結構，設計了三種分子探針，包括: 五肽(pentapeptide)，雙醣(GlcNAc-MurNAc-oligopeptide -monophosphate)及核苷酸(Park’s nucleotide)作為主骨架，以用於噬菌體展示篩選(phage display screening)，以期得到相對應之高親和力胜肽小分子。
這三種分子探針分別固定於磁珠上，以方便篩選之操作。經由三輪放大篩選，並利用高通量定序(high-throughput sequencing)得到有高度辨識分子探針能力之胜肽小分子。
最後，我們使用生物薄膜干涉技術(biolayer interferometry)來檢測有潛力之胜肽小分子與分子探針(核苷酸、雙醣)之相互作用力。實驗結果顯示胜肽小分子(C7CP2)與核苷酸有較佳之相互作用力，且略低於天然物萬古黴素與核苷之相互作用力。胜肽小分子(C7CP3’及C7CP6’)則與雙醣有較佳之相互作用力。同時，我們應用NMR實驗尋找胜肽小分子(C7CP2)與核苷酸相互作用之原子基團位置。期待此結果能幫助我們進一步修飾及改良胜肽小分子，以期增強有潛力之胜肽小分子與肽聚醣之相互作用力。

Peptidoglycan (PGN) is a huge biomolecule present in bacterial cell wall, which plays an essential role for bacteria growth and survival. Lipid II is the basic biomolecule in the biosynthesis of peptidoglycan, and interestingly, it is also a target of naturally occurring antibiotic, such as the vancomycin, teicoplanin, ramoplanin, and lantibiotic nisin.
Based on the structure of Lipid II and peptidoglycan, we designed three molecular probes, including pentapeptide, GlcNAc-MurNAc-oligopeptide- monophosphate and Park’s nucleotide as the main structure. Three molecular probes were applied in phage display screening to generate the low molecular weight peptides (M.W. < 1500 Da), which highly recognize with our desired molecular probes.
Three molecular probes were individually immobilized on the magnetic beads to facilitate phage display biopanning. After three rounds of screening and amplification, followed by high-throughput sequencing, with a high affinity to the molecular probes were found.
Next, our binding affinity study between the low molecular weight peptides and the molecular probes (GlcNAc-MurNAc- oligopeptide-monophosphate and Park’s nucleotide) was performed by biolayer interferometry (BLI). The results revealed that the low molecular weight peptide (C7CP2) has the better interactions with Park’s nucleotide and is slightly lower than the interactions between vancomycin and Park’s nucleotide. The low molecular weight peptides (C7CP3’ and C7CP6’) have the better interactions with GlcNAc-MurNAc-oligopeptide-monophosphate. Meanwhile, NMR experiments were applied to identify the interaction moieties and modes between the low molecular weight peptide (C7CP2) and Park’s nucleotide.
Our current finding and analytical methods might help further structural modifications to enhance the interactions between the potential low molecular weight peptides and peptidoglycans.

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