DRUG DESIGINS OF INHIBITORS TARGETED TO REPLICASE AND REPLICATION OF HEPATITIS C VIRUS (HCV)

针对丙型肝炎病毒（HCV）复制和复制的抑制剂的药物设计

• 批准号: 12558084
• 负责人: MURAKAMI Seishi
• 金额: $ 8.77万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12558084/
• 关键词: HCV NS5B; RNA dependent RNA polymerase (RdRP); Alanine scanning; HCV replication; NS5A; Template; primer binding; Oligomerization; Nucleolin; RNA複製; RdRP活性; アラニンスキャンニング

As inhibition of HCV replication is expected to reduce or even block incidence of HCV-associated hepatocellular carcinoma, HCV NS5B, aRNA-dependent RNA polymerase, might be a strong target for drug designs to inhibit HCV replication. The main results are followings. 1) 5 residues of NS5B were newly identified to be critical for RNA-dependent RNA synthesis (RdRP) activity. 2)Two residues among them located far from the RdRP catalytic center were specified to be indispensable ones for oligomerization of NS5B. The oligomerization of NS5B was found to be prerequisite to RdRP activity of NS5B by demonstrating that the two residues are residue-specific and exchangeable for oligomerization and RdRP activities. 3)Specific interaction between NS5A and NS5B was characterized, and we found that NS5A can modulate RdRP activity through the direct interaction in vitro. 4) Nucleolin, was found to bind directly to NS5B and affected subcellular localization of NS5B when the C-terminal membrane-attachment domain was truncated. These interaction surfaces of NS5B would be putative targets for drug designs to inhibit HCV replication.

由于抑制 HCV 复制有望减少甚至阻止 HCV 相关肝细胞癌的发生，因此 HCV NS5B（aRNA 依赖性 RNA 聚合酶）可能成为抑制 HCV 复制的药物设计的有力靶点。主要结果如下。 1) 新发现 NS5B 的 5 个残基对于 RNA 依赖性 RNA 合成 (RdRP) 活性至关重要。 2)其中远离RdRP催化中心的两个残基被确定为NS5B寡聚反应中不可缺少的残基。通过证明这两个残基具有残基特异性并且可交换寡聚化和 RdRP 活性，发现 NS5B 的寡聚化是 NS5B RdRP 活性的先决条件。 3)表征了NS5A和NS5B之间的特异性相互作用，我们发现NS5A可以在体外通过直接相互作用调节RdRP活性。 4) 核仁素被发现直接与 NS5B 结合，当 C 端膜连接结构域被截断时，会影响 NS5B 的亚细胞定位。 NS5B 的这些相互作用表面将成为抑制 HCV 复制的药物设计的假定目标。

项目成果

K. Masutomi et al.: "Telomerase Activity Reconstituted in Vitro with Purified Human Telomerase Reverse Transcriptase and Human Telomerase RNA Component."J. Biol. Chem.. 275-29. 22568-22573 (2000)

K. Masutomi 等人：“用纯化的人端粒酶逆转录酶和人端粒酶 RNA 成分在体外重建端粒酶活性”。

K. Masutomi et al.: "Identification of serum anti-human telomerase reverse Transcriptase (hTERT) auto-antibodies during progression to hepato-cellular carcinoma."Oncogene. 21. 5946-5950 (2002)

K. Masutomi 等人：“在肝细胞癌进展过程中血清抗人端粒酶逆转录酶 (hTERT) 自身抗体的鉴定。”癌基因。

Arai, K., et al.: "Two independent regions of human telomerase reverse transcriptase(hTERT) are important for their oligomerization and telomerase activity"J. Biol. Chem.. (In press ). (2002)

Arai, K., 等人：“人端粒酶逆转录酶 (hTERT) 的两个独立区域对于寡聚化和端粒酶活性非常重要”J.

D. Dorjsuren et al.: "A novel protein, RMP, which functionally counteracts"Experimental Medicine. 18. 1137-1141 (2000)

D. Dorjsuren 等人：“一种新的蛋白质，RMP，它在功能上抵消”实验医学。

K. Masutomi et al.: "Expression and Purification Method of Human Telomerase Reverse Transcriptase with Baculovirus Expresssion System"Bio Industry. 17. 35-42 (2000)

K. Masutomi等：“杆状病毒表达系统的人端粒酶逆转录酶的表达和纯化方法”生物工业。