Establishment of proven molecular evolution method PLM through the acquisition of Aβ42 aggregation-inhibitory/cytotoxicity-preventive peptides

• Material Type: 博士論文
• Title: Establishment of proven molecular evolution method PLM through the acquisition of Aβ42 aggregation-inhibitory/cytotoxicity-preventive peptides
• Author/Editor: Sunita, Ghimire Gautam
• Author Heading: Sunita, Ghimire Gautam
• Publication Date: 2014
• Publication Date (W3CDTF): 2014
• Alternative Title: Aβ42 重合阻害／細胞毒性阻止ペプチドの取得などを通じての分子進化戦略PLM法の確立
• Periodical title: 博士論文（埼玉大学大学院理工学研究科（博士後期課程））
• Degree grantor/type: 埼玉大学
• Date Granted: 2014-03-24
• Date Granted (W3CDTF): 2014-03-24
• Dissertation Number: 甲第954号
• Degree Type: 博士(学術)
• Conferring No. (Dissertation): 甲第954号
• Text Language Code: eng
• Target Audience: 一般
• Note (General): type:text; There is a great demand for the development of novel therapeutic molecules that combine the high specificity and affinity shown by biologics with the bioavailability and lower cost of small molecules. Since peptides (typically less than 100 amino acids in size) possess high binding affinity and specificity comparative to antibodies and have the high possibility to penetrate cells due to their smallness, peptides are ideal therapeutic candidates to bridge the gap between small molecules and biologics. Although many research studies are going on to develop the therapeutic peptides, there is a great deal of room to develop much better therapeutic peptides. This situation motivated us to develop the novel peptides with high binding affinity, specificity and functional activity towards different therapeutic targets. So, the objectives of this study are i) to develop novel peptides which have a high binding affinity and specificity towards beta amyloid 42 (Aβ42); ii) to screen the peptide-based inhibitors of Aβ42 aggregation and cytotoxicity so that these peptides can be therapeutic candidates for Alzheimer`s disease (AD) iii) to prove that progressive library method (PLM) can be an established molecular evolution method to engineer the peptides with high binding affinity, specificity and potential functional activity against different therapeutic targets, e.g. Aβ42, cathepsin E and etc.Several decades of cumulated research evidence has proven that Aβ42 is the main cause of neuronal death in the brain of the patient with AD. Therefore, inhibition of Aβ42 aggregation holds great promise for the prevention and treatment of AD. Although several drugs based on Aβ immunotherapy and small molecule have been developed and recently much effort has been given to develop drugs based on the peptides/peptide aptamers, none of them has yet translated into new medicines. To this end, it was aimed to develop the peptides which have high binding affinity and specificity to Aβ42.To achieve the first two objectives of this study, the mRNA (cDNA) display technique and the progressive library method (PLM) were used. Once evolved peptides by the second stage all-steps-all-combinations (ASCS) method were further evolved by the third stage paired peptide (PP) method. The PP library generated from the second library selection products was subjected to in vitro selection against Aβ42 by using cDNA display method and then cloning and sequence analysis was performed to select the most frequently occurred PPs in the library.By using surface plasmon resonance experiment, it was found that two PPs, P84 and P131 had higher binding affinity for the Aβ42 peptide (Kd value of 20 nM and 12 nM, respectively) than the previously reported Aβ42 binding peptides. To the best of our knowledge, this is the first report showing the development of randomly evolved peptide aptamers with the highest binding affinity to Aβ42. Then the functional characterization of P84 and P131 was performed to check their potential of inhibiting aggregation and cytotoxicity of Aβ42 peptide by using thioflavin T assay, atomic force microscopy assay and PC12 cells based cytotoxicity assay. It was found that both P84 and P131inhibited the aggregation of Aβ42, leading to the reduction of the cytotoxic effect of Aβ42 on PC12 cells. Therefore, it is believed that these PPs can be the potential therapeutic seeds for the AD.As of now, PLM consists of the evolution of the first, second and third library and each library selection can be assigned as module finding, module shuffling and module pairing, quite similar phenomena occurring in natural evolution of proteins. As the last step in PLM, in this study the fourth library was introduced based on the point mutation together with the DNA shuffling method to further improve the affinity and functional activity of the most improved paired peptide evolved from the third library. So, the P109 peptide, which showed the strongest binding affinity to cathepsin E at neutral pH (Kd value of 2 nM), was selected and a randomly base-substituted library (fourth library) was generated by inserting point mutations in the sequence of P109. One mutant containing single point mutation of P109, here after named SK1, showed 1.6 fold improved binding affinity to cathepsin E at neutral pH compared to the affinity of nonmutated P109. SK1 also showed significantly improved functional activity (5% increased functional activity compared to that of P109).In this study, highly functional peptides with a strong binding affinity towards the respective peptides were successfully developed from the third and fourth library, demonstrating the effectiveness of the progressive library method (PLM) in the evolution of peptides/proteins. Since the natural evolution of proteins is based on point mutation and recombination, the evolution strategy for proteins, i.e., PLM seems to be completed by the introduction of the fourth library of point mutation-based one following the recombination-based second and third libraries.; Dedication………………………………………………………………iAcknowledgements………………………………………………………iiAbstract…………………………………………………………………iiiList of figures………………………………………………………viList of tables…………………………………………………………viiiList of abbreviations………………………………………………………ixTable of contents…………………………………………………………xiChapter One: Introduction and literature review…………………11.1 Background………………………………………………………………………11.2 Peptides as therapeutic drugs………………………………………………………21.3 Directed evolution………………………………………………………………31.4 mRNA/cDNA display technology……………………………………………51.5 Evolutionary rapid panning and analysis system (eRPANSY)……………………61.6 Progressive library method (PLM)………………………………………………71.7 Research objectives and methodology………………………………………91.7 Thesis organization……………………………………………………………11Chapter Two: Acquisition of Aβ42 aggregation inhibitory/cytotoxicity-preventive peptides using PLM……………………132.1 Abstract………………………………………………………132.2 Introduction……………………………………………………142.2.1 Alzheimer`s Disease…………………………………………………142.2.2 Beta amyloid 42 (Aβ42) as a therapeutic target for AD…………………152.2.3 Objectives relevant to this study………………………………………172.3 Materials and Methods…………………………………………182.3.1 Generation of a paired peptide library and cDNA-peptide fusion product ……182.3.2 In vitro selection of novel peptides …………………………………………232.3.3 Preparation of Aβ42 aggregates………………………………………………242.3.4 Measurement of binding affinity……………………………………………252.3.5 Thioflavin T (ThT) binding assay……………………………………………262.3.6 Atomic force microscopy……………………………………………………262.3.7 Tricine-SDS-PAGE………………………………………………………272.3.8 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence analysis……………272.3.9 Cell-based assay……………………………………………………………282.4 Results……………………………………………………………………………292.4.1 In vitro selection of novel peptides from the PP library………………………292.4.2 Improved binding affinity of the novel peptides to Aβ42………………………322.4.3 P84 and P131 inhibit the fibrillization of Aβ42………………………………332.4.4 Effect of P84 and P131 on the oligomerization of Aβ42……………………362.4.5 Effect of P84 and P131 on the cytotoxicity of Aβ42…………………………372.5 Discussion……………………………………………………………………432.6 Conclusions……………………………………………………………………49Chapter Three. Improvement of binding affinity and functionalactivity of cathepsin E activator P109 peptide by the fourth librarymethod of PLM…………………………………………………………513.1 Abstract…………………………………………………………………………513.2 Introduction…………………………………………………………………523.3 Materials and Methods…………………………………………………………543.3.1 Preparation of protease cathepsin E and its substrate……………………………543.3.2 Generation of the fourth library (Mutated library)………………………………553.3.3 In vitro selection of cathepsin E activating peptides……………………………553.3.4 Affinity measurement……………………………………………………………573.3.5 Cathepsin E protease activity assay……………………………………………583.4 Results……………………………………………………………………………583.4.1 In vitro selection of cathepsin E binding peptides from the fourth library……583.4.2 Improved binding affinity and functional activity of SK1 to cathepsin E………593.5 Discussion………………………………………………………………………613.6 Conclusions……………………………………………………………………63Chapter Four: Overall conclusions and prospects to the futurestudy............................................................................................................654.1 Overall conclusions……………………………………………………………654.2 Prospects to the future study……………………………………………………66References……………………………………………………………………………67; 主指導教員 : 西垣功一
• DOI: 10.24561/00010321; https://doi.org/10.24561/00010321
• Persistent ID (NDL): info:ndljp/pid/8977199; https://dl.ndl.go.jp/pid/8977199
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• Date Accepted (W3CDTF): 2015-03-01T07:09:42+09:00
• Date Created (W3CDTF): 2015-02-16
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• Periodical Title (URI): http://dx.doi.org/10.24561/00010321; http://id.nii.ac.jp/1586/00010321/
• Data Provider (Database): 国立国会図書館 : 国立国会図書館デジタルコレクション; https://dl.ndl.go.jp

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