Uniquely constrained peptides for modulating TNF receptor activity

用于调节 TNF 受体活性的独特限制肽

• 批准号: 10387498
• 负责人: Ashley M. Kretsch
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-16 至 2023-02-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387498
• 关键词: Affinity; Agonist; Anabolism; Anti-Bacterial Agents; Antibodies; Antigens; Area; Autoimmune; Autoimmune Diseases; Binding; Binding Sites; Biological; Biological Assay; Biological Products; Biology; CD19 gene; Cell Surface Receptors; Cells; Characteristics; Chemicals; Chemistry; Clinical; Communication; Complement; Consumption; Cyclic Peptides; Data; Development; Disease; Educational process of instructing; Endothelin; Enzymes; Exhibits; Fellowship; Generations; Genetic; Genome; Genomics; Glucagon Receptor; Goals; Health; Human; Humira; Illinois; Immune Targeting; Immunologic Receptors; In Vitro; Individual; Institutes; Lasso; Learning; Libraries; Ligands; Mentors; Mentorship; Messenger RNA; Methods; Modality; Modification; Monoclonal Antibodies; Mutagenesis; OX40; Oral; Peptide Conformation; Peptide Hydrolases; Peptide Library; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Positioning Attribute; Post-Translational Protein Processing; Property; Research Personnel; Research Training; Resistance; Resources; Ribosomes; Safety; Scientist; Shapes; Signal Transduction; Site; Solvents; Specificity; Structure; Structure-Activity Relationship; Students; Surface; T-Cell Activation; TNF gene; TNFSF4 gene; Therapeutic; Therapeutic Monoclonal Antibodies; Time; Toxic effect; Training; Tumor Necrosis Factor Receptor; Universities; Variant; Work; Work Ethic; adalimumab; analog; antagonist; anti-PD-1; anti-cancer; base; cancer therapy; career; design; effective therapy; graduate student; high throughput screening; improved; member; next generation; novel; pembrolizumab; peptide I; rational design; receptor binding; screening; small molecule; small molecule therapeutics; success; targeted agent; three dimensional structure; tumor necrosis factor ligand superfamily member 4; undergraduate student; unnatural amino acids

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.

项目概要 生物制剂，特别是单克隆抗体（mAb）的临床使用稳步增长。我们呈现 套索肽作为单克隆抗体之间化学空间未充分利用区域内的潜在药物形式 和小分子。套索肽代表一类核糖体合成和翻译后肽 修饰肽（RiPP）。套索肽的生物合成涉及基因编码的前体肽和 成熟酶催化大环内酰胺环并将肽配置成独特的套索结。套索 肽概括了合成环肽的所需特性，但具有以下主要优点：(i) 套索肽 是独特约束的、高度稳定的球状结构，具有正确的形状来接合肽结合 目标，(ii) 套索肽完全由基因编码，可直接生成大序列- 不同的文库，(iii) 酶耐受性支持对整个溶剂暴露表面的修饰，非 天然氨基酸和翻译后修饰，以及 (iv) C 末端提供了简便的缀合 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 化学合成，因此我们依靠生物合成酶来合成肽。该提案将 设计一个基于生物合成的套索肽库，并作为概念验证，使用该库 选择结合 OX40 的套索肽。 OX40是肿瘤坏死因子的免疫刺激成员 受体（TNFR），研究发现天然 OX40 配体、OX40L 和激动剂抗体可以激活 OX40刺激T细胞的增殖和活化。迫切需要验证免疫 从而开发出治疗方法，补充抗 PD1（即 Keytruda）和抗 PD1 药物的成功。 TNFα（即 Humira）生物制剂。我们课题组开发了无细胞生物合成的高通量方法 （CFB）产生套索肽库。在目标 I 中，我们将 CFB 与 mRNA 展示相结合来识别文库 成功循环的成员。 Aim II 将选择结合 OX40 的套索肽。我们将评估 通过验证 OX40 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 OX40 相关的图案 将通过结构和诱变数据来鉴定结合和活性。加深对以下内容的理解 受体结合活性关系将为制药工作提供重要信息。该奖学金将提供 必要的培训，让我为指导研究人员和教学学生的学术生涯做好准备。 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 以口头和书面形式，强烈的协作职业道德以及对本科生和研究生的指导 学生。与我的赞助商 Douglas Mitchell（RiPP 生物合成专家）和资源合作 伊利诺伊大学厄巴纳-香槟分校化学系和研究所提供 基因组生物学将为成功实现研究目标和培训计划提供无与伦比的机会。