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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点（1）。然而，其他估计表明，只有不到10%的人类蛋白质组可被传统的小分子药物化，并且这两种情况的交叉使得只有2%至5%的人类蛋白质既参与疾病又可被药物化。这些因素突出了对能够扩大用于治疗当前难治性疾病的可药用靶点范围的新型药物分子的需求。通常认为具有挑战性的一类靶标是蛋白质-蛋白质相互作用（3）。直觉上，肽将是破坏特定蛋白质-蛋白质相互作用的理想分子。事实上，筛选肽文库的高通量方法，例如噬菌体、核糖体和酵母展示，已经成功地鉴定了可以在体外破坏蛋白质-蛋白质相互作用的肽（4-6）。然而，作为药物分子，肽具有缺点，包括对蛋白水解的敏感性和构象灵活性（7）。然而，尽管如此，自然界已经进化出基于肽的生物活性小分子的生产机制。核糖体合成和后修饰肽（RiPP）是目前正在研究用于治疗从细菌和寄生虫感染到癌症的疾病的一大类天然产物（8）。研究最多的一类RIPP是羊毛硫肽，其含有羊毛硫肽或甲基羊毛硫肽硫醚交联（9）。这些交联可以提供对蛋白水解切割的抗性，并赋予羊毛硫肽构象稳定性（10-13）。用于安装这些交联的一种途径涉及一般命名为LanM的双功能酶，其可以使丝氨酸或苏氨酸残基脱水并催化半胱氨酸残基在这些脱水残基上的迈克尔型加成以产生硫醚交联（9）。研究已经鉴定了在嗜盐海洋食蟹猴细菌原绿球藻MIT 9313中的一种特定LanM酶，称为ProcM，其能够加工29种内源性序列多样性羊毛硫肽前体（14）以及来自不同细菌属的羊毛硫肽前体（15），这表明它对其底物具有相当的耐受性。由于羊毛硫肽是遗传编码的，它们有助于通过组合DNA文库合成来容易地合成大文库。该提案将集中于开发lanthipeptides的酵母展示系统，设计能够被ProcM环化的肽库，以及lanthipeptides向破坏特定蛋白质-蛋白质相互作用的方向进化，这些蛋白质-蛋白质相互作用与使用荧光激活细胞分选（FACS）的人类疾病有关。