세포 내의 PGE2 농도 조절을 위한 새로운 ((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl 유도체 합성 및 구조-활성 상관관계 분석

Abstract

염증 반응의 생성에 Prostaglandin (PG)은 중요한 역할을 지니며 급성 염증의 주요 반응에 관여하여 다양한 생리적 기능을 갖는다. PG 종류 중 하나인 Prostaglandin E2는 염증 효과 및 항염증 효과에 모두 관여함으로써 과도한 염증반응을 억제하여 정상적인 상처 치유 회복을 향상시키는 물질로 알려져 있다. 하지만 PG 생합성 과정으로 생성된 PGE2는 NAD+ 의존형 15-PGDH에 의해 15-keto PGE2로 빠르게 산화되어 PGE2가 지니는 생리활성을 잃게 된다. 따라서 본 연구에서는 생체 내 PGE2의 농도를 증가시키기 위한 15-PGDH에 대한 강력한 억제제 개발을 목표로 진행하였다. 약물 설계를 위해 이전 연구에서 Ciglitazone을 기반으로 합성해 온 여러 TD 계열 유도체 중 가장 효능이 좋았던 TD203의 구조 변화를 통해 39개의 새로운 유도체를 합성하였다. 합성한 약물들로부터 15-PGDH의 억제 효능을 측정하기 위해 먼저 15-PGDH의 DNA를 pGEX-2T expression vector에 삽입한 후 E coli. BL-21 DE3에서 형질 전환하여 높은 순도의 15-PGDH 단백질을 얻었다. 정제된 15-PGDH에 각 화합물을 처리하여 생성된 NADH의 농도를 Fluorescence spectrophotometer로부터 측정한 후 IC50값을 각각 도출하였다. 이 후 각 화합물에 따라 A549 cell로부터 생성된 PGE2 농도를 ELISA (Enzyme Linked Immunosorbent Assay)로 측정하였으며 Control 대비 100% 이상의 PGE2 농도 증가율을 보인 화합물 4, 22, 23, 38, 39를 Lead compound로 선정하였다. 이들이 실제 세포 치유에 효과를 주는지 확인하기 위해 Scratch wound healing assay를 실시하였다. 화합물 4, 22, 38, 39는 Positive control인 TGF β1 보다 더 높은 상처 치유 효과를 보였으며 이들 모두 동일한 Phenylpropyl 치환기를 지니고 있어 Phenylpropyl기는 PGE2 농도 증가를 촉진시키는 중요한 구조적 요인이라 판단된다. 또한 4-((2,4-dioxothiazolidin-5-yl)methyl)phenyl 4-phenylbutanoate (Compound 39)는 200% 이상의 가장 높은 상처 치유 회복율을 보였으며 이로 인해 앞으로의 연구에서 39 화합물은 새로운 유도체 개발의 출발점이 될 수 있다. 따라서 lead compound (4, 22, 38, 39)는 PGE2 결핍으로 인한 다양한 질병의 치료 관리에 유용할 것이다.|Prostaglandin (PG) plays a crucial role in generating inflammatory reactions. It is also involved in key reactions in acute inflammation where it performs various physiological functions. Prostaglandin E2 is a type of PG related to both the inflammatory and anti-inflammatory effects that suppress excessive inflammatory reactions and enhance normal wound healing processes. However, when PGE2 is produced during PG biosynthesis, it is rapidly oxidized to 15-keto PGE2 by the NAD+-dependent 15-PGDH, which reduces the physiological activity of PGE2. Thus, the present study focused on developing a powerful inhibitor of 15-PGDH to increase the concentration of physiologically active PGE2. To design the novel drug, 39 new derivatives were synthesized by altering the structure of TD203, one of several TD derivatives synthesized based on ciglitazone that exhibited good inhibitory effects. To estimate the inhibitory effects of these synthetic compound against 15-PGDH, DNA encoding 15-PGDH was inserted into the pGEX-2T expression vector and transformed into Escherichia coli BL-21 DE3 cells to produce highly pure 15-PGDH protein. Purified 15-PGDH was treated with the different compounds, and the concentration of the produced NADH was measured with a fluorescence spectrophotometer to obtain IC50 values for each compound. Subsequently, the concentration of PGE2 produced from A549 cells depending on each compound was measured by enzyme-linked immunosorbent assay. Compounds 4, 22, 23, 38, and 39 were selected as lead compounds, as they led to an increase in PGE2 concentration of greater than 100% compared to that observed for the control. To verify their effects on cellular healing, scratch wound healing assays were carried out. Compounds 4, 22, 38, and 39 showed far greater wound healing effects than the positive control transforming growth factor-β1. These results show that phenylpropyl substituent of compound was considered essential for increasing the PGE2 concentration. Furthermore, the strongest effects on wound recovery of greater than 200% was exhibited by 4-((2,4-dioxothiazolidin-5-yl)methyl)phenyl 4-phenylbutanoate (compound 39), indicating that this compound can be used to develop novel derivatives in future studies. Therefore, lead compounds (4, 22, 38, 39) will be valuable for the therapeutic management of various diseases caused by PGE2 deficiency.