A High Throughput Protein Complementation Assay for Inhibitors of NEMO-K13 Intera

描述（由申请人提供）：人类疱疹病毒8 （HHV8，也称为KSHV）是世界部分地区年轻人中最常见的恶性肿瘤之一，与卡波西氏肉瘤、原发性积液性淋巴瘤（PEL）和多中心Castleman病（MCD）有关。hhv8相关淋巴细胞增生性疾病患者由于免疫功能低下，预后极差，迫切需要毒性较小的治疗方法来治疗这些恶性肿瘤。我们发现HHV8编码的小蛋白K13直接与IkB激酶（IKK）复合物的NEMO/IKK3亚基相互作用，激活NF-kB通路，并利用该通路促进细胞存活、增殖、转化和细胞因子分泌。以上研究都确立了NF-kB通路作为治疗hhv8相关恶性肿瘤的重要治疗靶点。然而，由于NF-kB通路在正常的免疫和炎症反应中起关键作用，该通路的全局抑制剂可能导致严重的免疫抑制，从而限制了其在hhv8感染患者中的潜在临床应用。为了解决这个问题，我们建议开发一种高通量筛选（HTS）方法来分离K13-NEMO相互作用的小分子抑制剂。希望这些抑制剂能够特异性阻断k13诱导的NF-kB，而不会干扰正常免疫和炎症反应中该途径的生理激活。此外，K13- nemo相互作用的特异性抑制剂将作为有用的药理学探针来了解K13的各种生物活性。

DESCRIPTION (provided by applicant): Human Herpesvirus 8 (HHV8, also known as KSHV) is one of the commonest causes of malignancies among young adults in parts of the world and has been associated with Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). The prognosis of patients with HHV8-associated lymphoproliferative disorders is extremely poor due to their immunocompromised status and there is an urgent need for less toxic therapies for the treatment of these malignancies. We have discovered that K13, a small protein encoded by HHV8, directly interacts with the NEMO/IKK3 subunit of the IkB kinase (IKK) complex to activate the NF-kB pathway and utilizes this pathway to promote cellular survival, proliferation, transformation and cytokine secretion. The above studies have established NF-kB pathway as an important therapeutic target for the treatment of HHV8-associated malignancies. However, since NF-kB pathway plays a key role in normal immune and inflammatory response, global inhibitors of this pathway are likely to lead to severe immunosuppression, thus limiting their potential clinical utility in HHV8-infected patients. To circumvent this problem, we propose to develop a high throughput screening (HTS) assay for isolating small molecule inhibitors of K13-NEMO interaction. It is hoped that such inhibitors will specifically block K13-induced NF-kB without interfering with the physiological activation of this pathway during normal immune and inflammatory response. Furthermore, specific inhibitors of K13-NEMO interaction will serve as useful pharmacological probes to understand the various biological activities of K13. PUBLIC HEALTH RELEVANCE: Infection with the Human Herpesvirus 8 (HHV8) has been linked to a number of human cancers. In this project, we propose to develop a high throughput screening assay for compounds that can block the interaction of K13, a small protein encoded by HHV8, with the cellular regulatory protein NEMO. It is hoped that such compounds will not only lead to a better understanding of the biological functions of K13 but also serve as lead compounds for the development of targeted therapies for HHV8-associated malignancies.