Molecular screens for pVHL-associated isopeptidase VDU1

描述(由申请人提供)：在常染色体显性遗传性von Hippel-Lindau病(VHL)患者中，100%观察到泛素E3连接酶成分pVHL的突变，并使个人易患各种肿瘤，包括肾脏透明细胞癌。此外，在更常见的散发性肾透明细胞癌患者中，可观察到50-80%的pVHL基因突变。随着蛋白酶体抑制剂Bortezomib的临床成功，泛素途径酶被认为是筛选比Bortezomib更具选择性的抑制剂的有吸引力的靶点，从而实现了更好的治疗指数。两种相关的脱辅酶--VDU1，现在称为USP33，和VDU2，现在称为USP20--是pVHL E3连接酶复合体的底物，因此与癌症的发生有关。肿瘤抑制基因pVHL对USP20和USP33蛋白水平的严格调控支持了USP20和USP33蛋白在肿瘤发生中起作用的假说。大约两年前，在第一阶段的授权申请中，有人提议建立一种方法来筛选USP33(VDU1)异肽酶的活性，该方法使用一种需要游离N末端才能发挥催化活性的报告酶。随后，纯化了重组USP33，并建立了用于筛选抑制剂的高通量试验，实现了第一阶段赠款的主要目标(见第一阶段最终报告)。在这个第二阶段的应用中，相关的脱泛素酶VDU2(USP20)将被纯化，并将利用异肽酶试验来筛选多个化合物文库，以鉴定USP33和USP20的抑制剂。HITS将通过额外的二次化验来确认，以确定抑制剂的特异性和有效性。在导联优化之后，将进行初步的临床前评估。鉴定USP33和/或USP20的特异性抑制剂是开发针对pVHL突变肿瘤的新型靶向治疗的第一步。据预测，这种靶向治疗在治疗多种恶性肿瘤方面是有效的，例如肾透明细胞癌。在第一阶段SBIR赠款资助的项目中，Progenra，Inc.开发了一种检测方法，可以用来找到阻止一种名为VDU1(或USP33)的酶的化合物，这种酶与某些肾癌有关。在第二阶段，有人建议使用这种检测方法来筛选大量化合物，以找到通过阻断这种酶或其亲属VDU2(现在称为USP20)来治疗肾癌的药物的最佳候选者。在拟议的第二阶段工作计划中，将提纯USP20，并将USP20和USP33都放入筛选中，以寻找对酶具有强烈阻断剂的化合物(其中之一，或两者都)。寻找VDU1或VDU2的有效抑制剂是开发新抗癌药物的第一步。

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.