One-Bead-One-Compound 고리형 펩토이드 라이브러리를 이용한 사이클로필린 저해제 발굴

학위논문 (석사) -- 서울대학교 대학원 : 사범대학 과학교육과(화학전공), 2021. 2. 유재훈.Cyclophilins (Cyps) are peptidyl-prolyl isomerases (PPIases) which catalyze cis-trans isomerization of peptidyl-proline. Among 17 human Cyp isofoms, first identified and most abundant isoform, cyclophilin A is mainly expressed in cytosol, involved in several human diseases such as rheumatoid arthritis (RA), Alzheimers disease (AD), cancer and viral infection. Another important therapeutic target isoform, cyclophilin D (CypD) is located in mitochondrial matrix, known as a key player of Ca2+ induced opening of mitochondrial permeability transition pore (mPTP). Opening of mPTP causes the loss of mitochondrial inner membrane potential, releasing cytochrome c to cytosol, which leads to mitochondria-driven apoptosis. The well-known natural Cyp binder, cyclosporine A (CsA) inhibits the activities of the Cyps at low concentrations. However, the limited use of CsA owing to its undruggable properties such as high cytotoxicity, low solubility and poor BBB permeability suggests the necessity of new strategies or even new compounds that can replace CsA. Herein, One-bead-one-compound (OBOC) cyclic peptoid library was used for the identification of de novo cyclophilin inhibitors. Ten selected compounds were individually synthesized and their inhibitory activities against Cyps were monitored. Compared to CsA, cyclic peptoid I11 showed equipotent inhibition of CypD activity with lower cytotoxicity and better BBB permeability.Cyclophilin (Cyp) 은 펩타이드 프롤린의 시스-트랜스 이성질화에 관여하는 효소이다. 인간에게는 서열이 유사한 17 개의 Cyp 동형 단백질이 있는데, 그 중 가장 많이 발현되어 있고 가장 먼저 발견된 것은 cyclophilin A (CypA) 이다. CypA 는 주로 세포질에 위치하며 류마티스 관절염, 알츠하이머 병, 암 및 바이러스 감염과 같은 많은 인간 질병에 관여하고 있다. 최근에는 코로나바이러스감염증-19에 의한 팬데믹 상황을 가져온 바이러스인 SARS-CoV-2 에 Cyp 저해제를 적용하려는 연구도 이루어지고 있다. 또 다른 동형 단백질인 cyclophilin D (CypD) 는 미토콘드리아의 기질에 위치하며, Ca2+에 의해 유도되는 미토콘드리아 투과성 전이 기공 (mPTP, Mitochondrial permeability transition pore)의 개방에 중요한 역할을 하는 것으로 알려져 있다. 미토콘드리아 투과성 전이 기공이 열리면, cytochrome C 가 세포질로 방출되며 세포사멸을 유도하여 여러가지 질병을 일으킬 수 있다. 한편, cyclosporine A (CsA) 는 Cyp 의 천연 억제제로, 적은 농도로 사이클로필린 활성을 억제한다. 그러나 CsA 는 독성이 높은 편이고 용해도가 낮아서 약으로 쓰이기 어려운 특징을 가지며, 뇌혈관장벽을 잘 통과하지 못해서 중추신경계 약물로 사용하기 어렵다. 따라서 CsA 를 이용할 수 있는 새로운 전략이나, 비슷한 효과를 내는 새로운 화합물의 개발이 필요하다. 이 연구에서는 OBOC (One-bead-one-compound) 고리형 펩토이드 라이브러리를 이용하여 CypA 에 잘 결합하는 물질들을 얻고, 여러가지 실험을 통해 새로운 Cyp 억제제를 발굴하고자 하였다. 고리형 펩토이드 I11 은 CsA 와 비슷한 수준의 미토콘드리아 막 전위 회복 능력을 보이면서, 세포 독성은 더 낮고 뇌혈관장벽에 대한 투과성은 더 좋을 것으로 나타났다. 따라서 이 연구에서는 고리형 펩토이드 라이브러리의 구축 및 스크리닝을 통해, 고리형 펩토이드가 가지는 장점을 이용하여, CypD 가 관여하는 중추신경계 질환에 기존의 CsA 보다 더 적합한 물질을 발굴해 내었다는 의의가 있다.Abstract i Contents iii List of tables ⅴ List of figures ⅴ Introduction 1 Experimental Section 4 1. Peptoid synthesis 4 1.1 Synthesis of cyclic peptoids 1.2 Synthesis of TAMRA-labeled cyclic peptoids 1.3 Synthesis of Cy5.5-labeled I11 2. Anti-inflammatory activity (Real-time PCR) 7 3. Binding affinity measurement 8 3.1 Surface Plasmon Resonance (SPR) 3.2 Fluorescence Anisotropy 4. Blood-brain barrier permeability measurement (BBB-PAMPA) 10 5. Mitochondrial membrane potential recovery (JC-1) 10 5.1 Microplate reader 5.2 Confocal Laser Scanning Microscopy (CLSM) 6. Cell viability assay (WST-1) 11 7. Biodistribution 12 Results and Discussion 13 1. Design and Synthesis of macrocyclic peptoid library 13 2. Library screening and selection 14 3. Anti-inflammatory activity 29 4. Binding affnity to CypA 32 5. Optimization of I11 41 6. Binding affinity to CypD 45 7. Blood-brain barrier permeability 47 8. Mitochondrial membrane potential recovery effect 49 9. Cytotoxicity 51 10. Biodistribution 52 Conclusions 54 References 55 Abstract (in Korean) 62 List of tables Table 1. Names and sequences of 100 peptoids 20 Table 2. Sequence and mass data of cyclic peptoids 28 Table 3. Sequence and mass data of TAMRA-labeled peptoids 39 Table 4. Binding affinity to CypA measured by fluorescence anisotropy 40 Table 5. Binding affinity to CypD measured by fluorescence anisotropy 46 Table 6. Effective permeability (Pe) value from BBB-PAMPA 48 Table 7. Sequence and mass data of Cy5.5-I11 52 List of figures Figure 1. OBOC cyclic peptoid library construction and screening of CypA inhibitors 16 Figure 2. Amine monomers for peptoid library construction 17 Figure 3. General scheme for the construction of sequenceable OBOC cyclic peptoid library using bi-functional resin. 18 Figure 4. Synthetic details for the construction of sequenceable OBOC cyclic peptoid library using bi-functional resin 19 Figure 5. Sequences and structures of 10 selected peptoids 21 Figure 6. Synthetic scheme for cyclic peptoids 22 Figure 7. Synthetic details for cyclic peptoids . 23 Figure 8. Chromatograms of cyclic peptoids 24 Figure 9. Schematic model for the inhibition of anti-inflammatory activity of CypA by CsA or cyclic peptoids 30 Figure 10. Relative IL-2 inhibition of selected cyclic peptoids 30 Figure 11. Relative IL-2 inhibition of I11 31 Figure 12. Concept of SPR-based relative binding affinity screening 33 Figure 13. Representative SPR sensorgrams for the interaction of selected peptoids with CypA 33 Figure 14. Relative binding affinity of cyclic peptoids 34 Figure 15. Correlation of binding affinity and IL-2 inhibition 34 Figure 16. Synthetic details for TAMRA-labeled cyclic peptoids 35 Figure 17. Chromatograms of TAMRA-labeled cyclic peptoids 37 Figure 18. Fluorescence anisotropy binding curves for CsA-CypA and I11-CypA interaction 40 Figure 19. Glycine scanning of peptoid I11 42 Figure 20. IL-2 inhibition of glycine scanning mutants of I11 42 Figure 21. Modification of peptoid I11 43 Figure 22. Loss of IL-2 inhibition of Npip-to-Nleu substitution of I11 43 Figure 23. Improved IL-2 inhibition of modified I11 44 Figure 24. Fluorescence anisotropy binding curves for CsA-CypD and I11-CypD interaction 46 Figure 25. Concept of BBB-PAMPA 48 Figure 26. Analysis of mitochondrial membrane potential 50 Figure 27. Monitoring of mitochondrial membrane potential using CLSM 50 Figure 28. Effects of CsA and peptoids on viability of SH-SY5Y cells 51 Figure 29. Chromatogram of Cy5.5-I11 52 Figure 30. Biodistribution of Cy5.5-I11 53Maste