세포 내 prostaglandin E₂ 농도 조절을 위한 새로운 benzylidenethiazolidine-2,4-dione 계열 15-PGDH 활성 억제제 설계, 합성 및 활성 평가

Abstract

The lipid signaling molecule prostaglandin E₂ (PGE₂) plays important roles in cell regeneration, such as gut protection, bone formation, vasodilation, and wound healing. During wound healing, PGE₂ is synthesized from arachidonic acid (5,8,11,14-eicosatetraenoic acid), which exists in an esterified form in the cell membrane, by the action of cyclooxygenase (COX-1/2). The synthesized PGE₂ activates the Wnt signaling pathway to induce hematopoietic stem cell differentiation, thus directly contributing to cell regeneration. However, PGE₂ is rapidly oxidized to 15-keto-PGE₂ by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), thereby losing its activity. Therefore, in this study, we aimed to develop a drug with a new structure that could inhibit 15-PGDH activity to increase cellular PGE₂ concentration, in order to promote cell regeneration. Our research team has synthesized various derivatives of ciglitazone, a suiTable drug molecule that was shown in previous studies to inhibit 15-PGDH. In this study, 66 drug molecules were synthesized based on CT-8 and TD-203 structures, both of which exhibited the greatest inhibitory effects compared to previously synthesized derivatives, and the effects of the structural change on activity were analyzed. For experimental analysis, 15-PGDH was expressed in Escherichia coli BL-21 DE3 by using pGEX-2T vector, and was purified using a GST column. To assess the inhibitory activity of the drugs, different concentrations of the purified 15-PGDH enzyme were treated with the drugs and the optical density of NADH, which is produced in addition to 15-keto-PGE₂, was measured. Moreover, ELISA (enzyme-linked immunosorbent assay) was used to determine changes in cellular PGE₂ concentration. Among the synthesized molecules, seven lead compounds (compound 3, 9, 11, 12, 13, 14, and 25) that resulted in the highest PGE₂ concentration were selected. Scratch wound healing assay using HaCaT cell line was conducted to verify their effects on cell regeneration. Lead compounds (compound 3, 9, 11, 12, 13, 14, and 25) resulted in 50% inhibition of 15-PGDH at concentrations between 0.07 and 0.2 μM, and increased PGE₂ levels from 300% to over 600% in A549 cells. Analysis of the scratch wound healing assay showed that all lead compounds (compound 3, 9, 11, 12, 13, 14, and 25) had greater wound regeneration effects than the cell growth factor, TGF-β1, which was used as positive control. In particular, (Z)-N-benzyl-4-((2,4-dioxothiazolidin-5-ylidene)methyl)benzamide (compound 14) showed the highest wound closer rate, which was 360% higher compared to that of the control. Using in vitro experiments, newly synthesized 15-PGDH inhibitors were shown to effectively regulate cellular PGE₂ concentration and to stimulate cell growth. Therefore, 15-PGDH inhibitors could be used to treat various diseases that arise from PGE₂ deficiency.