Uniquely constrained peptides for modulating TNF receptor activity

项目摘要 生物制剂，特别是单克隆抗体（mAb）的临床使用稳步上升。我们提出 套索肽作为mAb之间化学空间的低杠杆区域内的潜在药物形式 和小分子。Lasso肽代表一类核糖体合成和后降解的蛋白质。 修饰肽（RiPP）。套索肽的生物合成涉及遗传编码的前体肽， 成熟酶，其催化大环内酰胺环并将肽配置成独特的乳链结。Lasso 肽概括了合成环肽的所需性质，但具有关键优点： 具有独特的约束力，高度稳定的球形结构，具有正确的形状，可与肽结合 目标，（ii）套索肽是完全遗传编码的，直接产生大的和序列- 多样化的库，（iii）酶耐受性支持整个溶剂暴露表面的修饰，非- 天然氨基酸和翻译后修饰，和（iv）C-末端提供了一个容易的缀合 用于肽展示和高通量筛选的位点。由于其独特的拓扑结构，套索肽不能被 化学合成，因此我们依靠生物合成酶来连接肽。这项建议会 设计套索肽的生物合成信息库，并作为概念验证，使用该库 选择结合OX 40的套索肽。OX 40是肿瘤坏死因子的免疫刺激成员 研究发现，天然OX 40配体、OX 40 L和激动剂抗体可以激活TNFR受体， OX 40刺激T细胞的增殖和活化。迫切需要验证免疫 靶点，以便开发治疗方法，补充抗PD 1（即Keytruda）和抗PD 1（即Keytruda）的成功。 TNFα（即修美乐）生物制剂。我们的小组已经开发了一种高通量的无细胞生物合成方法 (CFB)以产生套索肽文库。目的一：将联合收割机CFB与mRNA展示技术相结合， 成功循环的成员。目的II将选择结合0X 40的套索肽。我们将评估 通过验证OX 40结合变体的三维结构来确定结构-活性关系， 进行结合亲和力研究和体外信号传导测定以评估其活性。与OX 40相关的主题 结合和活性将通过结构和诱变数据来鉴定。更好地了解 受体结合-活性关系将显著地为药学工作提供信息。该奖学金将提供 必要的培训，以准备我在指导研究人员和教学学生的学术生涯。 我的目标是通过改善沟通来发展我的能力，成为一个更全面的科学家 在口头和书面形式，一个强大的合作职业道德，并指导本科生和研究生 学生与我的赞助商道格拉斯米切尔，在RIPPs生物合成的专家，和资源， 可在伊利诺伊大学厄巴纳-香槟分校，化学系，和研究所 基因组生物学，将提供一个无与伦比的机会，成功的研究和培训计划的目标。

PROJECT SUMMARY There is a steady rise in the clinical use of biologics, particularly monoclonal antibodies (mAbs). We present lasso peptides as a potential drug modality within the underleveraged region of chemical space between mAbs and small molecules. Lasso peptides represent a class of ribosomally-synthesized and post-translationally modified peptide (RiPP). Biosynthesis of lasso peptides involves a genetically encoded precursor peptide and maturation enzymes that catalyze a macrolactam ring and configure the peptide into a unique lariat knot. Lasso peptides recapitulate the desired properties of synthetic cyclic peptide but with key advantages: (i) lasso peptides are uniquely constrained, highly stable globular structures with the correct shape to engage peptide-binding targets, (ii) lasso peptides are entirely genetically encoded, straightforward generation of large and sequence- diverse libraries, (iii) enzymatic tolerance supports modification of the entire solvent-exposed surface, non- natural amino acids, and post-translational modifications, and (iv) the C-terminus provides a facile conjugation site for peptide display, and high-throughput screening. Due to their unique topology, lasso peptides cannot be chemical synthesized, thus we rely on the biosynthetic enzymes to thread the peptide. This proposal will design a biosynthesis-informed library of lasso peptides and as a proof-of-concept, use this library to select for lasso peptides that bind OX40. OX40 is an immunostimulatory member of the tumor necrosis factor receptors (TNFR), studies have found that the native OX40 ligand, OX40L, and agonist antibodies can activate OX40 to stimulate the proliferation and activation of T cells. There is a compelling need to validate immune targets so that therapeutics can be developed, complementing the success of anti-PD1 (ie Keytruda) and anti- TNFα (ie Humira) biologics. Our group has developed a high-throughput method with cell-free biosynthesis (CFB) to produce lasso peptide libraries. In Aim I we will combine CFB with mRNA display to identify library members that are successfully cyclized. Aim II will select for lasso peptides that bind OX40. We will assess structure-activity relationships by validating the three-dimensional structure of the OX40-binding variants and conduct binding affinity studies and in vitro signaling assays to evaluate their activity. Motifs related to OX40 binding and activity will be identified through structural and mutagenesis data. Improved understanding of receptor binding-activity relationship will significantly inform pharmaceutical efforts. This fellowship will provide the necessary training to prepare me for an academic career in mentoring researchers and teaching students. My goal is to develop my abilities to become a more comprehensive scientist through improving communication in both oral and written form, a strong collaborative work ethic, and mentorship of undergraduate and graduate students. Working with my sponsor Douglas Mitchell, an expert in RiPPs biosynthesis, and the resources available at the University of Illinois at Urbana-Champaign, the Department of Chemistry, and the Institute for Genomic Biology, will provide an unrivaled chance to succeed in aims of the research and the training plan.