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

描述(申请人提供)：人类疱疹病毒8型(HHV8，又称KSHV)是世界部分地区年轻人中最常见的恶性肿瘤原因之一，与卡波西氏肉瘤、原发渗出性淋巴瘤(PEL)和多中心Castleman病(MCD)有关。由于免疫功能受损，HHV8相关淋巴增生性疾病患者的预后非常差，迫切需要毒性较低的治疗方法来治疗这些恶性肿瘤。我们发现，HHV8编码的小分子蛋白K13直接与IKB激酶(IKK)复合体的Nemo/IKK3亚基相互作用，激活核因子-kB途径，促进细胞的存活、增殖、转化和细胞因子的分泌。上述研究已经确立了核因子-kB通路作为治疗HHV8相关恶性肿瘤的重要靶点。然而，由于核因子-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很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。为了避免这个问题，我们建议建立一种高通量筛选(HTS)方法来分离K13-NEMO相互作用的小分子抑制剂。在正常的免疫和炎症反应过程中，人们希望这些抑制剂能特异性地阻断K13诱导的核因子-kB，而不干扰这一通路的生理激活。此外，K13-NEMO相互作用的特异性抑制剂将作为有用的药理探针来了解K13的各种生物学活性。 公共卫生相关性：人类疱疹病毒8型(HHV8)感染与许多人类癌症有关。在这个项目中，我们建议建立一种高通量筛选试验，以阻止K13(一种由HHV8编码的小蛋白)与细胞调节蛋白NEMO的相互作用。希望这些化合物不仅有助于更好地了解K13的生物学功能，而且还将作为先导化合物，用于开发针对HHV8相关恶性肿瘤的靶向治疗。

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.