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：
8783145
负责人：
Mark Chalfant Walker
金额：
$ 5.15万
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/8783145
关键词：
Affinity Agglutinins Antineoplastic Agents Apoptosis Bacteria Bacteriophages Binding Biological Factors Biological Process Cell Separation Cell Wall Cell division Cells Cysteine DNA Library Dehydration Development Dimerization 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 Nature Parasites Partner in relationship Peptide Hydrolases Peptide Library Peptides Pharmaceutical Preparations Pharmacologic Substance Play Predisposition Process Prochlorococcus Production Protein Binding Proteins Proteolysis Proteome Randomized Resistance Ribosomes Roche brand of trastuzumab Role Route Saccharomyces cerevisiae Serine Signal Transduction Sulfhydryl Compounds Surface System Therapeutic Threonine Yeasts angiogenesis base combinatorial crosslink design directed evolution 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％的基因可能是治疗疾病的靶标（1）。然而，其他估计表明，不到10％的人蛋白质组可以被常规的小分子药物吸毒，并且这两种疾病的相交剩下的人类蛋白质的2％至5％均参与疾病和毒品（2）。这些因素强调了需要扩大可药物靶标范围以治疗当前棘手疾病的新型药物分子的需求。蛋白质相互作用是一类通常被认为具有挑战性的靶标（3）。直觉上，肽将是破坏特定蛋白质相互作用的理想分子。实际上，筛选肽库（例如噬菌体，核糖体和酵母显示）的高吞吐量方法已成功地识别可以破坏体外蛋白质蛋白质相互作用的肽（4-6）。但是，由于药物分子的肽具有缺点，包括对蛋白水解和构象柔韧性的敏感性（7）。尽管如此，自然已经发展了基于肽的生物活性小分子生产的机制。核糖体合成和翻译后修饰的肽（RIPP）是一大类天然产物，目前正在研究以治疗从细菌和寄生虫感染到癌症的疾病（8）。研究最多的Ripps是灯笼肽，其中含有灯笼或甲基二硫代硫代硫代交叉链接（9）。这些交联可以对蛋白水解裂解和赋予型型构肽构象稳定性（10-13）提供抗性。安装这些交联的一条途径涉及一种通常命名为LANM的双功能酶，该酶可以脱水丝氨酸或苏氨酸残基，并催化迈克尔型在这些脱水残基上的半胱氨酸残基添加，以产生硫代交叉链接（9）。研究已经确定了浮游生物cynaobacterium cynaobacterium procm的特定LANM酶，称为Procm，该酶能够处理29个内源性序列多样性多样性肽前体（14）的内源性序列多样性多样性多样性，以及从不同属的遗传植物（15）中的lanthipepters（15），这是对其属于不同属（15）的尊重。随着植物肽的遗传编码，它们通过组合DNA库的合成来使自己合成大型库。该提案将着重于开发用于灯笼二肽的酵母显示系统，能够被PROCM环绕的肽库的设计，以及使用荧光激活的细胞（FACS）与人类疾病中有关的特定蛋白质蛋白质相互作用的破坏型蛋白质蛋白质相互作用（FACS）。