Molecular screens for pVHL-associated isopeptidase VDU1

描述（由申请人提供）：在100%的常染色体显性遗传性von Hippel-Lindau病（VHL）患者中观察到泛素E3连接酶组分pVHL的突变，并使个体易患各种肿瘤，包括肾脏透明细胞癌。此外，在50-80%患有更常见的散发性肾透明细胞癌的患者中观察到pVHL突变。随着蛋白酶体抑制剂硼替佐米的临床成功，泛素途径酶已被认为是筛选比硼替佐米更具选择性的抑制剂的有吸引力的靶标，从而实现改善的治疗指数。两种相关的脱泛素酶-VDU 1，现在称为USP 33，和VDU 2，现在称为USP 20-是pVHL E3连接酶复合物的底物，因此与致癌作用有关。肿瘤抑制因子pVHL对USP 20和USP 33蛋白水平的严格调节支持了USP 20和USP 33蛋白在癌发生中起作用的假设。大约两年前，在一项I期资助申请中，提出建立一种使用报告酶筛选USP 33（VDU 1）异肽酶活性的测定法，该报告酶需要游离N末端以获得催化活性。随后，纯化了重组USP 33，并建立了筛选抑制剂的高通量测定法，实现了I期资助的主要目标（见I期最终报告）。在该II期申请中，将纯化相关的去泛素化酶VDU 2（USP 20），并利用异肽酶试验筛选多个化合物库，以鉴定USP 33和USP 20的抑制剂。将通过额外的二次试验确认命中，以确定抑制剂的特异性和有效性。电极导线优化后，将进行初始临床前评价。USP 33和/或USP 20特异性抑制剂的鉴定是开发pVHL突变型肿瘤新型靶向治疗的第一步。预计这种靶向疗法在治疗多种恶性肿瘤如肾透明细胞癌中是有效的。在SBIR资助的I期项目中，Progenra公司开发了一种检测方法，可用于寻找阻断一种称为VDU 1（或USP 33）的酶的化合物，这种酶与某些肾癌有关。在第二阶段，建议使用这种检测方法来筛选大量化合物，以找到可以通过阻断这种酶或其相对物VDU 2（现在称为USP 20）来治疗肾癌的药物的最佳候选物。在II期拟定的工作计划中，将纯化USP 20，并将USP 20和USP 33进行筛选，以寻找作为酶（一种或另一种，或两者）强阻断剂的化合物。找到VDU 1或VDU 2的强效抑制剂是开发新癌症药物途径的沿着第一步。

DESCRIPTION (provided by applicant): Mutations in the ubiquitin E3 ligase component pVHL are observed in 100% of patients with the autosomal dominant von Hippel-Lindau disease (VHL) and predisposes individuals to a variety of tumors including clear cell carcinomas of the kidneys. Furthermore, mutations in pVHL are observed in 50-80% of patients with the more common sporadic renal clear cell carcinoma. Following the clinical success of the proteasome inhibitor bortezomib, ubiquitin pathway enzymes have been considered attractive targets to screen for inhibitors more selective than bortezomib thereby achieving an improved therapeutic index. Two related deubquitinating enzymes -- VDU1, now called USP33, and VDU2, now called USP20 -- are substrates of the pVHL E3 ligase complex and hence are implicated in carcinogenesis. The tight regulation of USP20 and USP33 protein levels by the tumor suppressor pVHL support the hypothesis that USP20 and USP33 proteins play a role in carcinogenesis. Approximately two years ago, in a phase I grant application it was proposed to establish an assay to screen USP33 (VDU1) isopeptidase activity using a reporter enzyme that required a free N terminus for catalytic activity. Subsequently, recombinant USP33 was purified and a high throughput assay for screening for inhibitors was established, fulfilling the main objective of the phase I grant (see phase I final report). In this phase II application the related deubiquitinase VDU2 (USP20) will be purified and the isopeptidase assay will be utilized to screen multiple libraries of compounds to identify inhibitors of USP33 and USP20. The hits will be confirmed by additional secondary assays to establish the specificity and efficacy of the inhibitors. Following lead optimization, initial preclinical evaluation will be performed. The identification of specific inhibitors of USP33 and/ or USP20 is the first step in the development of novel targeted therapies for pVHL mutant tumors. Such targeted therapies are predicted to be efficacious in the treatment of multiple malignancies such as renal clear cell carcinomas. In a Phase I SBIR grant funded project, Progenra, Inc developed an assay that can be used to find compounds that block an enzyme called VDU1 (or USP33), which is related to certain kidney cancers. In Phase II, it is proposed to use this assay to screen a large number of compounds to find the best candidates for a drug that can treat kidney cancer by blocking this enzyme or its relative, VDU2 (now called USP20). In the proposed work plan for Phase II, USP20 will be purified, and both USP20 and USP33 will be put into the screen to find compounds that are strong blockers of the enzymes (one or the other, or both). Finding powerful inhibitors of VDU1 or VDU2 is the first step along the pathway to develop new cancer drugs.