Targeting Multiple Aspects of Alzheimer's Disease Pathology with a Novel Small Molecule = 알츠하이머병의 다양한 신경병리학적 측면을 타깃으로 하는 새로운 저분자 화합물 개발

국문 초록 (Abstract)

알츠하이머병(AD)은 복잡하고 다양한 신경병리학적 요인들에 의해 발생하는 퇴행성 신경 질환이다. 이 때문에 알츠하이머병의 임상 개입에 있어서는 병리학적 요인들의 다양한 측면을 고려한 저분자 화합물(small compound)의 개발이 필요하다는 점이 강조되고 있다. 스핑고지질 대사를 조절하는 효소인 ‘산성 스핑고미엘리나제 (acid sphingomyelinase, ASM)’ 활성화가 알츠하이머병의 신경병리학적 특징에 영향을 미치는 것으로 알려져 있으며, 이를 바탕으로 ASM 억제제의 활용이 잠재적 치료 전략으로서 주목받고 있다. 본 연구에서는 COMPOUND 2이라는 새로운 물질을 개발하고, 이 COMPOUND 2이 직접적인 ASM 억제제로서 기능하는 것을 확인하였다. COMPOUND 2은 ASM에 대해 매우 선택적으로 작용하며, 특히 뇌 내 분포에 있어 뛰어난 약동학적 특성을 보인다. 더욱 놀라운 것은, COMPOUND 2이 그렐린 수용체(ghrelin receptor)에 대한 작용제(agonist)로서의 부가적인 기능을 가지고 있음을 발견하였으며, 이는 알츠하이머병 치료에 있어 유망한 잠재력을 가짐을 시사한다. 뉴런에서 COMPOUND 2의 이중 기능은 알츠하이머병 모델 마우스의 신경병리학적 특징, 즉 아밀로이드-베타 침착, 자가포식 손상, 신경염증, 시냅스 손실, 해마 신경 생성 및 시냅스 가소성의 감소를 효과적으로 개선하였으며, 이는 궁극적으로 기억 기능의 향상을 가져왔다. 이런 발견들은 COMPOUND 2이 알츠하이머병 치료를 위한 혁신적이고 다기능적인 약물로서의 잠재력을 보여준다.

다국어 초록 (Multilingual Abstract)

Alzheimer's disease (AD) is characterized by complex and diverse neuropathological factors, suggesting that the development of small molecules targeting various aspects of this neuropathology may be necessary for effective clinical intervention. Acid sphingomyelinase (ASM) activation has been recognized as a significant contributor to the neuropathological features in AD, leading to the exploration of ASM inhibitors as a potential therapeutic strategy. Here I present the identification of COMPOUND 2, a direct ASM inhibitor evaluated for AD treatment. COMPOUND 2 exhibits highly selective inhibition of ASM, demonstrating remarkable pharmacokinetic properties, particularly in terms of its distribution in the brain. Surprisingly, I have also identified an additional function of COMPOUND 2 as an agonist of the ghrelin receptor, which also presents promising therapeutic potential for AD treatment. The dual functionality of COMPOUND 2 in neurons effectively ameliorated neuropathological hallmarks in AD mice, including the accumulation of amyloid-beta, impaired autophagy, neuroinflammation, synaptic loss, and reduced hippocampal neurogenesis and synaptic plasticity, ultimately leading to improved memory function. These findings highlight the clinical potential of COMPOUND 2 as an innovative and multifunctional therapeutic agent for the treatment of AD.

목차 (Table of Contents)

• ABSTRACT 1
• ABBREVIATIONS 2
• INTRODUCTION 3
• 1. Alzheimer's Disease (AD) 3
• 2. Acid Sphingomyelinase (ASM) and Its Relation to Alzheimer's Disease 4
• 3. Acid Sphingomyelinase (ASM) Inhibitors and Their Development Direction for Alzheimer's Disease Treatment 5
• 4. Significance Statement 6
• MATERIALS AND METHODS 8
• 1. Chemical compounds screening 8
• 2. Molecular docking 9
• 3. Surface plasmon resonance (SPR) spectroscopy 10
• 4. Biochemical IC50 determinations 12
• 5. Enzyme kinetics 12
• 6. ASM, NSM, AC, and SphK activity assays 13
• 7. Lipid extraction and ceramide/sphingomyelin/sphingosine/S1P quantification 14
• 8. PK and brain distribution of COMPOUND 5, COMPOUND 2 or COMPOUND 1 15
• 9. Microsomal stability 17
• 10. Cytochrome P450 inhibition 18
• 11. Kinase profiler, enzyme assay, and GPCR screening 19
• 12. Mice 19
• 13. Plasma collection 20
• 14. Histological analysis 20
• 15. Western blotting 22
• 16. ELISA 23
• 17. BrdU injection 24
• 18. Behavioral studies 24
• 19. Cell culture and treatment 25
• 20. Cell viability and apoptosis assay 27
• 21. Mouse and human hippocampal neuron culture 27
• 22. Mouse hippocampal neural stem cell (NSC) culture 28
• 23. Neurosphere (NS) formation assay and EdU staining 29
• 24. HEK 293T cell culture and transfection 30
• 25. Ca2+ immobilization assay 30
• 26. RNA seq 31
• 27. RNA isolation and real-time PCR analysis 33
• 28. Statistical analysis 36
• RESULT 37
• 1. ASM Inhibitor Compound Series Directly Inhibit ASM Activity and Show Potential as AD Therapeutics. 37
• 1.1 ASM Activity Increases with AD Progression and ASM Inhibitor Compound Series Directly Inhibit ASM Activity 37
• 1.2 COMPOUND 2 Is a Selective and Competitive Inhibitor of ASM with Excellent Pharmacokinetic Properties 47
• 2. COMPOUND 2 Improves Neuropathology in APP/PS1 Mice via Neuronal ASM Inhibition and Ghrelin Receptor Agonist 53
• 2.1 COMPOUND 2 Improves Neuropathological Features in APP/PS1 Mice 53
• 2.2 Neuronal ASM Inhibition by COMPOUND 2 Rescues Autophagy Dysfunction and Microglial Phagocytic Function in APP/PS1 Mice 58
• 2.3 COMPOUND 2 Is Also a Ghrelin Receptor Agonist, Contributing to Improvement of Neuropathological Features in Hippocampus of APP/PS1 Mice 63
• DISCUSSION 72
• FUTURE DIRECTIONS BASED ON THIS THESIS 77
• REFFERENCES 78
• ABSTRACT IN KOREAN 88