Uniquely constrained peptides for modulating TNF receptor activity

项目总结 生物制品，特别是单抗的临床应用正在稳步增加。我们呈现的是 套索多肽在单抗之间化学空间的低杠杆区域内作为潜在的药物形式 和小分子。套索肽代表一类核糖体合成和翻译后的多肽 修饰多肽(RIPP)。套索肽的生物合成涉及基因编码的前体多肽和 成熟的酶，催化一个大内酰胺环，并将多肽配置成一个独特的套索结。套索 多肽概括了合成环肽所需的性质，但具有关键的优点：(I)套索肽 是唯一受约束的、高度稳定的球状结构，具有正确的形状以进行肽结合 靶标，(Ii)套索多肽完全是基因编码的，直接产生大的和序列- 不同的文库，(Iii)酶耐受性支持对整个暴露在溶剂中的表面进行修饰，非 天然氨基酸和翻译后修饰，以及(Iv)C末端提供了一种简单的接合 站点用于多肽展示，并进行高通量筛选。由于其独特的拓扑结构，套索多肽不能 化学合成，因此我们依靠生物合成酶来缠绕多肽。这项提议将 设计一个套索多肽的生物合成信息库，作为概念验证，使用这个库来 为结合OX40的套索肽选择此选项。OX40是肿瘤坏死因子的免疫刺激成员 受体(TNFR)，研究发现天然的OX40配体、OX40L和激动剂抗体可以激活 OX40能刺激T细胞的增殖和激活。迫切需要验证免疫 靶点，从而可以开发治疗方法，补充抗PD1(即Keytruda)和抗PD1的成功 肿瘤坏死因子α(即Humira)生物制品。我们团队开发了一种高通量的无细胞生物合成方法 (Cfb)生产套索多肽文库。在Aim I中，我们将CFB和mRNA展示相结合来鉴定文库 成功循环的成员。AIM II将选择与OX40结合的套索肽。我们将评估 通过验证OX40结合变异体的三维结构和 进行结合亲和力研究和体外信号分析，以评估其活性。与OX40相关的基序 结合和活性将通过结构和突变数据进行鉴定。更好地理解 受体结合-活性关系将显著地指导药物的努力。这一奖学金将提供 为我的学术生涯做好准备的必要培训，包括指导研究人员和教授学生。 我的目标是通过改善沟通来发展我的能力，成为一名更全面的科学家 在口头和书面形式，强烈的合作精神，并指导本科生和研究生 学生们。与我的赞助人道格拉斯·米切尔合作，道格拉斯·米切尔是Ripps生物合成和资源方面的专家 可在伊利诺伊大学厄巴纳-香槟分校、化学系和 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.