Covalent Inhibition of Essential Deubiquitinases of Leishmania

背景：泛素蛋白酶体系统是蛋白质降解和细胞信号转导的重要机制。泛素通过E1、E2和E3缀合酶的顺序作用与靶蛋白偶联。同时，去泛素化酶（DUBs）从靶蛋白中去除泛素并分解多聚泛素链。在20个已鉴定的DUB基因中，有4个在L. mexicana是必需的，并且编码的DUB是属于泛素特异性蛋白酶和泛素-羧基末端水解酶家族的半胱氨酸蛋白酶。在这里，我们建议筛选库的半胱氨酸反应片段，以确定那些形成共价加合物的DUBs。之前约克和我们的工业合作者之间的合作工作已经证明了这种方法的前景，目标是含溴结构域的蛋白质与一组240个片段与磺酰氟弹头。目的：筛选针对L.利用质谱和活性分析Crystallise DUB：片段复合物指导开发更高效的靶标开发一种方法，使用基于LC-MSMS的定量分析来测量寄生虫中的靶向接合使用抑制剂作为化学工具来探测DUB抑制的下游生物学影响，并识别底物蛋白和相互作用物新颖性：利什曼原虫属于动质体-一个分支很深的真核生物群，其分子生物学不同于模式生物和人类宿主。DUBs的特异性抑制剂有可能作为化学工具来阐明这些生物体中的调控途径。墨西哥成立了。与此同时，我们的工业合作者的化学生物学小组一直在开发一套反应性片段平台，用于有效识别共价抑制剂，这些平台已成功应用于利什曼原虫含溴结构域的蛋白质。实验方法：我们构建了表达三种靶DUB的结构，第一个目标是开发表达第四种的结构。学生将纯化四种重组酵素以筛选共价片段库。这将涉及DUB与片段的平行孵育，然后通过完整蛋白质LCMS进行测定，以鉴定特定的质量变化。将通过加合物的胰蛋白酶解酶的LC-MSMS分析基于其反应性和选择性的感兴趣片段，以鉴定共价连接位点。将进行一轮命中扩展，以探索新兴命中的SAR并提高效力。这些片段与DUB的结合将在生物物理测定中以及通过结晶和结构测定来表征。它们抑制DUB活性的能力可以用泛素-荧光团缀合物在荧光测定中测量。将评估鉴定的共价片段与活利什曼原虫体内所选DUB接合的能力。这些测量可以通过用游离和共价抑制剂复合的DUB的质量标记混合物掺入所得寄生虫裂解物来定量进行。

Background: The ubiquitin proteasome system is an important mechanism for protein degradation and cell signaling. Ubiquitin is coupled to target proteins through the sequential action of E1, E2 and E3 conjugating enzymes. Meanwhile, deubiquitinases (DUBs) remove ubiquitin from target proteins and breakdown polyubiquitin chains. Four of the 20 identified DUB genes in L. mexicana are essential and the encoded DUBs are cysteine proteases belonging to the ubiquitin-specific protease and ubiquitin-carboxyl terminal hydrolase families. Here we propose to screen a library of cysteine reactive fragments to identify those that form covalent adducts with the DUBs. Previous collaborative work between York and our industrial collaborator has demonstrated the promise of this approach targeting bromodomain-containing proteins with a panel of 240 fragments with sulfonyl fluoride warheads. These reacted with binding site tyrosine residues and displayed selectivity between targets.Objectives:Screen a library of covalent fragments against the four essential DUBs of L. mexicana using mass spectrometry and activity assaysCrystallise DUB:fragment complexes to guide the development of higher potency inhibitorsDevelop a method to measure on-target engagement in parasites using an LC-MSMS based quantitative assay Use inhibitors as chemical tools to probe the downstream biological impact of DUB inhibition and identify substrate proteins and interactorsNovelty: Leishmania belong to the kinetoplastida - a deeply branched group of eukaryotes whose molecular biology differs from that of model organisms and the human host. Specific inhibitors of DUBs have the potential to serve as chemical tools to elucidate pathways of regulation in these organisms.Timeliness: The essential components of the ubiquitination and deubiquitination system of L. mexicana have been established. Meanwhile, the Chemical Biology group of our industrial collaborator has been developing a suite of reactive fragment platforms for the efficient identification of covalent inhibitors and these have been applied successfully to bromodomain-containing proteins from Leishmania.Experimental Approach: We have constructs expressing three of the target DUBs and the first goal will be to develop a construct expressing the fourth. The student will purify the four recombinant enzymes for screening against a covalent fragment library. This will involve parallel incubation of the DUBs with the fragments followed by assay by intact protein LCMS to identify specific mass shifts. Fragments of interest based on their reactivity and selectivity will be analysed by LC-MSMS of tryptic digests of the adducts to identify the site of covalent attachment. A round of hit expansion will be performed to explore the SAR of emerging hits and improve potency. Binding of these fragments to the DUBs will be characterised in biophysical assays and through crystallisation and structure determination. Their capacity to inhibit DUB activity can be measured in fluorimetric assays with ubiquitin-fluorophore conjugates. The capacity of identified covalent fragments to engage with the selected DUB inside living Leishmania parasites will be assessed. These measurements can be made quantitative by spiking the resulting parasite lysates with a mass-labelled mixture of the free and covalent inhibitor complexed DUB.