초록 ▼

본 연구과제는 암세포 이동을 억제하는 MD-164 화합물의 표적 발굴 및 암세포 사멸을 유도하는 HSF1 저해제 KRIBB31의 항암기전을 규명하였으며, 약물 작용점 분석시스템 최적화 및 약물들에 대한 작용점 탐색을 수행함

1. 암세포의 이동을 저해하는 화합물 MD-164
- 현재 사용중인 의약품 라이브러리로부터 암세포이동 저해제 MD-164(진해거담제로 사용 중인 약물)를 발굴하였으며 이의 작용기전을 암세포 및 모델동물 수준에서 분석함

- biotin-MD-164를 합성하여 affinity chro

본 연구과제는 암세포 이동을 억제하는 MD-164 화합물의 표적 발굴 및 암세포 사멸을 유도하는 HSF1 저해제 KRIBB31의 항암기전을 규명하였으며, 약물 작용점 분석시스템 최적화 및 약물들에 대한 작용점 탐색을 수행함

1. 암세포의 이동을 저해하는 화합물 MD-164
- 현재 사용중인 의약품 라이브러리로부터 암세포이동 저해제 MD-164(진해거담제로 사용 중인 약물)를 발굴하였으며 이의 작용기전을 암세포 및 모델동물 수준에서 분석함

- biotin-MD-164를 합성하여 affinity chromatography 방법으로 MD-164의 분자표적 ARPC2를 확인함

2. 천연물유래 HSF1 저해화합물 KRIBB31
- HSF1 저해제 KRIBB31를 2,000여종의 의약품/개발약물/천연물 라이브러리로부터 발굴
- KRIBB31은 HSF1 전사조절인자의 hsp70 promoter 결합을 저해하여 항암활성을 보임

3. HCS 약물 작용점 시스템 최적화 및 약물작용점 탐색
- Bar-code 불량 균주 수선을 통한 약물 작용점 분석 시스템 개량 및 reference DB 구축

- 기업의뢰 항선충제 1종, 항암제 2종, 항진균제 1종 약물작용점 분석
- 장수와 관련된 유전자 군의 발굴 및 기전 연구

(출처 : 요약서 3p)

Abstract ▼

Results

1. Identification of MD-164 as an inhibitor of DLD1/PRL-3 migration
To search for migration inhibitors, we used transwell migration assay for PRL-3-overexpressing DLD-1 cells that had treated with 783 medicinal drugs and 57 herbal medicines. From this, MD-164 was discovered as a mi

Results

1. Identification of MD-164 as an inhibitor of DLD1/PRL-3 migration
To search for migration inhibitors, we used transwell migration assay for PRL-3-overexpressing DLD-1 cells that had treated with 783 medicinal drugs and 57 herbal medicines. From this, MD-164 was discovered as a migration inhibitor. MD-164 is widely used as a cough suppressant in non-productive cough. In our results, treatment of MD-164 effectively suppressed migration and invasion of PRL-3-overexpressing DLD-1 cells using trans-well migration assay, trans-well invasion assay. These results suggest that MD164 inhibits the migration and invasion of PRL-3-overexpressing DLD-1 cells and can be good candidate for the therapeutic control of cancer cell invasion or metastasis.

2. Cantharidin inhibits HSF1 activity and induces cancer cell apoptosis.
To identify inhibitors of HSF1 activity, we performed cell-based screening with a library of marketed and experimental drugs and identified cantharidin as an HSF1 inhibior. Cantharidin is a potent antitumor agent from traditional Chinese medicine. Cantharidin inhibited heat shock-induced luciferase activity with an IC50 of 4.2μM. In contrast, cantharidin did not inhibit NF-κB luciferase reporter activity, demonstrating that cantharidin is not a gteneral transcription inhibitor. When the HCT-116 colorectal cancer cells were exposed to heat shock in the presence of cantharidin, the induction of HSF1 downstream target proteins, such as HSP70 and BAG3 (Bcl-2-associated athanogene domain 3), was suppressed. HSP70 and its co-chaperone BAG3 have been reported to protect cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. As expected, treating HCT-116 cancer cells with cantharidin significantly decreased the amounts of BCL-2, BCL-xL, and MCL-1 protein and induced apoptotic cell death. Chromatin immunoprecipitation analysis showed that cantharidin inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent p-TEFb recruitment. Therefore, the p-TEFb-dependent phosphorylation of the C-terminal domain of RNA polymerase II was blocked, arresting transcription at the elongation step. PP2A inhibition with PP2CA siRNA or okadaic acid did not block HSF1 activity, suggesting that cantharidin inhibits HSF1 in a PP2A-independent manner. We show for the first time that cantharidin inhibits HSF1 transcriptional activity.

3. Development of drug-target screening technique using haploinsufficiency
Gene-targeted mutants were constructed using a host-exchange method. 350 defective mutants were removed and replaced with new mutants and pool libraries containing all mutants in equal number of cells.

40 conventionally used drugs in different categories were selected for setting up the reference DB. Half of them, targets of which are already known, were used to verify the accuracy of the experiments. Determination of effective concentrations for drug treatments (IC50 experiments). Building the reference DB following drug target screening for the selected 40 drugs Setting up a gene list to exclude multi-drug target and haploinsufficient genes.

In addition, we analyzed targets for two anticancer drugs requested by the company. 6 genes except nematocide A1 (a tentative name) and cct1 that are involved in tubulin assembly, were selected as candidate target genes (chaperonin-containing T-complex cct5, cct8, cct7, cct4, cct6, cct1).

We examined the doxorubicin-sensitivity of V-ATPase subunit and RNAP I subunits in the cancer cell line, which are new doxorubicin candidate targets selected from heterozygotes diplod S. pombe mutant model system. In addition, we conducted microarray with heterozygotes diploid S. pombe mutant pool and selected tamoxifen-sensitive genes. Further more, we analyzed targets for several bioactive compounds such as CB class, Obovatol, Honokiol, Ginkgetin, Sciadopitysin, MD-164 and autophage-inducing reagent.


(출처 : SUMMARY 7p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 4
• SUMMARY ... 9
• 목차 ... 12
• 제1장 연구개발과제의 개요 ... 13
• 제 1절 연구개발의 목적 ... 13
• 제 2절 연구의 필요성 ... 13
• 제2장 국내외 기술개발 현황 ... 16
• 제 1절 암전이와 PRL-3 유전자 ... 16
• 제 2절 Heat Shock Factor 1 및 Non-oncogene Addiction ... 18
• 제 3절 약물작용점 분석 Chip 시스템 개량 및 표적 발굴 ... 21
• 제3장 연구개발수행 내용 및 결과 ... 22
• 제 1절 암전이 저해제 발굴 및 기전 규명 ... 22
• 제 2절 HSF1 저해제 Cantharidin 발굴 및 항암기전 규명 ... 30
• 제 3절 약물작용점 분석 Chip 시스템 개량 및 표적 발굴 ... 38
• 제4장 목표달성도 및 관련분야에의 기여도 ... 45
• 제5장 연구개발결과의 활용계획 ... 49
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 50
• 제 1절. 관련특허 조사결과 ... 50
• 제 2 절. 핵심논문 조사결과 ... 52
• 제7장 연구시설ㆍ장비 현황 ... 54
• 제8장 참고문헌 ... 55
• 끝페이지 ... 57