國立成功大學「邁向頂尖大學計畫」
延攬優秀人才「參與研究」計畫書
一、延攬理由（擬參與工作之重要性、其專長與計畫之配合程度、擬參與研究之工作內涵。如 為續聘申請案，請繳交前一聘期之工作報告。）
It is essential to have a postdoctoral fellow to optimize and design the integrin drug, to offer assistance to me, and to set up the new integrin-based cell adhesion assay.
In order to perform these experiments, I need a well-trained biologist who is familiar with my project to meet these requirements. Dr. Chang has completed his Ph.D. in my lab and worked on exploring the structure-activity relationships of the RGD loop, linker region, and C-terminus of disintegrin protein in the recognition of integrins. He has great expertise in protein expression and purification, drug design, protein structure determination, and cell assay manipulation. With his capacity for hard work and commitment for research career, he will be a good asset for my lab and for this project.
二、受聘人之工作內容。
In this study. Dr. Chang will
1. optimize and design the di-specific (αVβ3 and αVβ5, αVβ3 and α5β1, or αVβ5 and α5β1), tri-specific (αVβ3, αVβ5, and α5β1), and multi-specific (αVβx or αVβx and α5β1) integrin drugs;
2. set up the new integrin-based cell adhesion assay, such as integrin αVβ3, αVβ1, α8β1, and α9β1, for the design of the specific integrin drug;
3. determine the complex structure of integrin and integrins-specific disintegrins or FN-III 9 / 9-10 variants by X-ray crystallography.
三、預期完成之工作項目及具體成果。
In our previous study we have successfully designed selective and potent integrins (αVβ3- and αVβx-specific disintegrins and integrin α5β1-specific FN-III 9-10 using Rho and FN-III as protein scaffolds. We anticipate that the development of di-specific (αVβ3 and αVβ5, or αVβ3 and α5β1), tri-specific (αVβ3, αVβ5, and α5β1), and multi-specific (αVβx or αVβx and α5β1) integrin drug will be done within three years. We also found that integrins αV, β1, and β5 were highly expressed in clinical HCC patients. In xenogragh animal model, we also found that integrins αVβx and α5β1 -specific disintegrin can reduce tumor growth. Therefore, we anticipate that one of the integrin-specific drugs will be a drug candidate for liver cancer treatment. We anticipate that the drug candidate will be in preclinical trial within three years and in clinical trial within five years. Three papers with SCI > 5 will be published, and one patent will be applied. We will make exclusive license and development agreement or co-develop our drug candidate with biotechnology
company after finishing this study.
國立成功大學「邁向頂尖大學計畫」
延攬優秀人才工作報告表
NCKU’s “Aim for the Top University Project”
Work Report Form for Distinguished Scholars
□續聘continuation of employment ■離職resignation
受聘者姓名
Name of the Employee 張耀宗 ■男 □女
Male Female
聘 期 Period of Employment
From 107 年(y) 01 月(m) 01 日(d) To 107 年(y) 07 月(m) 31 日(d) 研究或教學或科技研發與
管理計畫名稱 The project title of research,
teaching, technology development and management
利用整合蛋白拮抗劑與天然物 發展抗癌藥物
計畫主持人
（申請單位主管）
Project Investigator (Head of Department/Center)
莊偉哲
補助延聘編號
Grant Number HUA107 -3-7-050
一、 研究、教學、科技研發與管理工作全程經過概述。（由受聘人填寫）
Please summarize the entire research, teaching, or science and technology R&D and management work process (To be completed by the employee)
According to previous research on mutagenesis of RGD motif of Rho, we have found that the ARLDDL mutant is a potent integrin αvβ3-specific antagonist. In vitro cell assays and animal model study has demonstrated that it has great therapeutic efficacy in inhibiting tumor metastasis. In order to develop ARLDDL as a protein drug and prolong its half-life in serum circulation, the long lasting types of ARLDDL were designed by fusing with HSA (C34S) and conjugating to PEG (30kDa PEG). Due to the pharmaceutical application of ARLDDL, HSA(C34S)-ARLDDL and PEGylated ARLDDL, their structure, stability, and state of aggregation should be examined in detail. On the other hand, to establishment of a suitable storage conditions is important to keep the physicochemical properties and to maintain the stability of the molecules for developing protein therapeutics in pharmaceutical application. Hence, several formulations will be screened for HSA(C34S)-ARLDDL to obtain the optimal condition.
So far, the work I have done as below:
A. Determination of the crystal structure of ARLDDL
Figure 1. Crystal structure of ARLDDL.
Overall structure of ARLDDL was refined to 1.35 Å resolution. Three β-sheets were shown in magentas, and the side chain of ARLDDL sequence was shown. This structure contains only one molecule of ARLDDL in the asymmetric unit having 638 atoms and 102 water molecules. The RMS deviations in bond lengths and bond angles are 0.026 Å and 2.28 Å , respectively.
Figure 2. The 2Fo-Fc electron density map at 1.35 Å resolution of ARLDDL loop.
Due to the cloning site of expression vector in Pichia pastoris, the expression of ARDDL exhibited inclusion of extra sequences EAEF at the N-terminal region and this made N-terminus of ARLDDL presenting a unique helix-like structure. High resolution structure of ARLDDL showed that all the residues were clearly visible in the electron densities, and even the side-chains of the flexible RGD motif also revealed well-resolved electron densities.
B. Thermal stability test by differential scanning calorimetry (DSC)
Figure 3. Thermal denaturation curves of Rho and ARLDDL.
Wild type Rho and matant ARLDDL were compared the thermal stability in PBS buffer by DSC method. The melting temperature (Tm) of ARLDDL is nearly 7℃ higher than Rho, indicating apparently increased thermal stability of ARLDDL.
Figure 4. Thermal denaturation curves of ARLDDL, PEG and PEG-ARLDDL.
ARLDDL, PEG and PEG-ARLDDL were compared the thermal stability in PBS buffer by DSC method. The Tm of PEGylated ARLDDL was only 56℃, and this is a large decrease compared to ARLLDL (87℃). Moreover, the Tm of unreacted PEG was only 44℃, indicating that ARLDDL may be destabilized by the conjugation with the large portion of unstructured 30KDa PEG.
Figure 5. Thermal denaturation curves of HSA, HSA-ARLDDL, HSA(C34S)-ARLDDL, and ARLDDL.
Fusing HSA or C34S-HSA was designed to prolong the half-life in serum. Also, the HSA,
HSA-ARLDDL, HSA(C34S)-ARLDDL, and ARLDDL were analyzed the thermal stability by DSC approach. The result showed that the HSA(C34S)-ARLDDL fusion protein exhibited two transition temperatures, Tm of 67 and 75°C. The similar result of two separated peaks for Tm was confirmed by HSA-ARLDDL (wild type HSA fused to ARLDDL). However, the Tm of the pure HSA only exhibited single peak, revealing that two transition peaks of HSA(C34S)-ARLDDL may come from two protein moieties, C34S-HSA and ARLDDL. They may be independent or have weak interacts between each other.
A. Melting temperature of HSA(C34S)-ARLDDL in different formulation buffers
Figure 6. Thermal denaturation curves of HSA(C34S)-ARLDDL in different formulation buffers.
HSA(C34S)-ARLDDL in the presence of several buffer species to understand the optimal condition were analyzed by DSC approach. The DSC thermograms and the melting temperatures were
determined and shown in figure above. The results were found that the thermal transitions of HSA(C34S)-ARLDDL in buffer A to E all contained two melting temperatures. Their Tm1 values were in the range of 66-69°C, and Tm2 values were in range of 73-79°C. HSA(C34S)-ARLDDL in buffer B possessed highest Tm1 and Tm2 values among these five formulations. The most
interesting finding was that HSA(C34S)-ARLDDL in buffer F displayed a single transition peak and had a much higher Tm of 88.6°C, indicating that the components of buffer F may interact with HSA(C34S)-ARLDDL and enhance ins melting temperature. Thus, buffer F could be a stabilized
二、研究或教學或科技研發與管理成效評估（由計畫主持人或單位主管填寫）
Please evaluate the performance of research, teaching or science and technology R&D and management Work: (To be completed by Project Investigator or Head of Department/Center)
(1)是否達到延攬預期目標？
Has the expected goal of recruitment been achieved?
受聘人利用整合蛋白拮抗劑與天然物發展抗癌藥物之研究，達預期目標，未來可供實驗室繼 續進一步探討。
(2)研究或教學或科技研發與管理的方法、專業知識及進度如何？
What are the methods, professional knowledge, and progress of the research, teaching, or R&D and management work?
受聘人對於瞭解整合蛋白拮抗劑與天然物發展抗癌藥物的機制與未來可行性之探討有達到期 望之進度，未來可繼續聘用執行研究目標。
(3)受延攬人之研究或教學或科技研發與管理成果對該計畫(或貴單位)助益如何？
How have the research, teaching, or R&D and management results of the employed person given benefit to the project (or your unit)?
受聘人在此期間對該計畫進行專業學術研究，對於其個人及受聘單位均有所助益。
(4)受延攬人於補助期間對貴單位或國內相關學術科技領域助益如何？
How has the employed person, during his or her term of employment, benefited your unit or the relevant domestic academic field?
受聘人在此聘期內的結果可供實驗室對於整合蛋白拮抗劑與發展抗癌藥物的領域繼續深入探 討，其對國內相關學術科技領域及未來醫學也有助益。
(5)具體工作績效或研究或教學或科技研發與管理成果：
Please describe the specific work performance, or the results of research, teaching, or R&D and management work:
受聘人在此期間對於整合蛋白拮抗劑發展為抗肝藥物的範疇有達成一定的成果，其工作結果
完成對 αvβ3 專一的整合蛋白拮抗劑 (ARLDDL) 結構與熱穩定性之分析。之後將延續上述的成果
做更進一步的探討整合蛋白拮抗劑之穩定性與影響。綜合這些成果期望未來能發展出有效的抗癌 藥物。
(6)是否續聘受聘人? Will you continue hiring the employed person? □續聘Yes ■不續聘No
※ 此報告表篇幅以三～四頁為原則。This report form should be limited to 3-4 pages in principle.
※ 此表格可上延攬優秀人才成果報告繳交說明網頁下載。
※ This report form can be downloaded in http://scholar.lib.ncku.edu.tw/explain/
