Targeting protein-protein interactions through directed evolution of lanthipeptid

通过羊毛肽的定向进化靶向蛋白质-蛋白质相互作用

基本信息

批准号：
9120381
负责人：
Mark Chalfant Walker
金额：
$ 5.8万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-16 至 2017-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9120381
关键词：
Affinity Agglutinins Antineoplastic Agents Apoptosis Bacteria Bacteriophages Binding Binding Proteins Biological Process Cell Separation Cell Wall Cell division Cells Cysteine DNA Library Dehydration Development Dimerization Directed Molecular Evolution Disease Disulfides ERBB2 gene Endoplasmic Reticulum Enzymes Evolution Fluorescence Fluorescence-Activated Cell Sorting Gene Library Generations Genes Genome Human Human Genome Hydrocarbons In Vitro Infection Label Length Libraries Malignant Neoplasms Marines Methods Names Natural Products Nature Parasites Partner in relationship Peptide Hydrolases Peptide Library Peptides Pharmaceutical Preparations Pharmacologic Substance Play Predisposition Process Prochlorococcus Production Proteins Proteolysis Proteome Randomized Resistance Ribosomes Role Route Saccharomyces cerevisiae Serine Signal Transduction Sulfhydryl Compounds Surface System Therapeutic Threonine Trastuzumab Yeasts angiogenesis base combinatorial crosslink design flexibility human disease lacticin 481 lanthionine novel protein protein interaction public health relevance screening small molecule success thioether tool

项目摘要

DESCRIPTION (provided by applicant): It has been estimated that between 10 and 30 percent of genes in the human genome may be targets for the treatment of disease (1). However, other estimates suggest that less than 10 percent of the human proteome is druggable by conventional small molecule drugs and that the intersection of these two conditions leaves as little as 2 to 5 percent of human proteins both involved in disease and druggable (2). These factors highlight the need for new classes of drug molecules capable of expanding the scope of druggable targets for the treatment of currently intractable diseases. One class of targets that is generally considered challenging to perturb is protein- protein interactions (3). Intuitively, peptides would be ideal molecules for disrupting specific protein- protein interactions. Indeed, high throughput methods of screening peptide libraries, such as phage, ribosome, and yeast display, have been successful at identifying peptides that can disrupt protein- protein interactions in vitro (4-6). However, as drug molecules peptides have drawbacks including susceptibility to proteolysis and conformational flexibility (7). Nevertheless, nature has evolved mechanisms for the production of biologically active small molecules based on peptides. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of natural products that are currently being investigated for the treatment of conditions ranging from bacterial and parasite infections to cancer (8). The most studied class of RiPPs is the lanthipeptides, which contain lanthionine or methyllanthionine thioether crosslinks (9). These crosslinks can provide resistance to proteolytic cleavage and confer conformational stability on lanthipeptides (10-13). One route for the installation of these crosslinks involves a bifunctional enzyme generically named LanM, which can dehydrate serine or threonine residues and catalyze the Michael-type addition of a cysteine residue onto these dehydrated residues to produce the thioether crosslink (9). Studies have identified a particular LanM enzyme in the planktonic marine cynaobacterium Prochlorococcus MIT9313, called ProcM, which is capable of processing 29 endogenous sequence-diverse lanthipeptide precursors (14) as well as a lanthipeptide precursor from a different genera of bacteria (15), suggesting that it is quite tolerant with respect to its substrate. As lanthipeptides are genetically encoded, they lend themselves to the facile synthesis of large libraries through combinatorial DNA library synthesis. This proposal will focus on the development of a yeast display system for lanthipeptides, the design of peptide libraries that are capable of being cyclized by ProcM, and the evolution of lanthipeptides towards the disruption of specific protein-protein interactions that have been implicated in human disease using fluorescent-activated cell sorting (FACS).

描述（由申请人提供）：据估计，人类基因组中 10% 至 30% 的基因可能是疾病治疗的靶标 (1)。然而，其他估计表明，只有不到 10% 的人类蛋白质组可通过常规小分子药物进行药物治疗，而这两种情况的交集使得只有 2% 至 5% 的人类蛋白质既涉及疾病又可进行药物治疗 (2)。这些因素凸显了对新型药物分子的需求，这些分子能够扩大治疗当前难治性疾病的药物靶点范围。通常认为难以干扰的一类目标是蛋白质-蛋白质相互作用 (3)。直观上，肽是破坏特定蛋白质-蛋白质相互作用的理想分子。事实上，筛选肽库的高通量方法（例如噬菌体、核糖体和酵母展示）已成功鉴定出可在体外破坏蛋白质-蛋白质相互作用的肽 (4-6)。然而，作为药物分子，肽具有一些缺点，包括对蛋白水解敏感和构象灵活性 (7)。尽管如此，大自然已经进化出生产基于肽的生物活性小分子的机制。核糖体合成和翻译后修饰肽 (RiPP) 是一大类天然产物，目前正在研究用于治疗从细菌和寄生虫感染到癌症等疾病 (8)。研究最多的 RiPP 类别是羊毛硫肽，其含有羊毛硫氨酸或甲基羊毛硫氨酸硫醚交联 (9)。这些交联可以提供对蛋白水解裂解的抗性，并赋予羊毛硫肽构象稳定性 (10-13)。安装这些交联的一种途径涉及一种双功能酶，一般称为 LanM，它可以使丝氨酸或苏氨酸残基脱水，并催化半胱氨酸残基以迈克尔型加成到这些脱水残基上以产生硫醚交联 (9)。研究在浮游海洋蓝藻原绿球菌 MIT9313 中发现了一种特殊的 LanM 酶，称为 ProcM，它能够处理 29 种内源序列多样化的羊毛脂肽前体 (14) 以及来自不同细菌属的羊毛脂肽前体 (15)，这表明它对其基质具有相当的耐受性。由于羊毛硫肽是基因编码的，因此它们可以通过组合 DNA 文库合成轻松合成大型文库。该提案将重点关注羊毛硫肽酵母展示系统的开发、能够被 ProcM 环化的肽库的设计，以及羊毛硫肽朝着破坏与人类疾病有关的特定蛋白质-蛋白质相互作用的方向发展。使用荧光激活细胞分选（FACS）。