New Hybrid Molecular Modalities Comprised of DNA-Origami and Interfering Peptides as Inhibitors of Protein-Protein Interactions

由 DNA 折纸和干扰肽组成的新混合分子模式作为蛋白质-蛋白质相互作用的抑制剂

• 批准号: 10378010
• 负责人: Amanda Nicole Haymond Still
• 金额: $ 16.17万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10378010
• 关键词: 3-Dimensional; 4T1; Adverse effects; Affinity; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Antibodies; Area; Automobile Driving; Binding; Biological Assay; Breast Cancer Model; Cancer Patient; Cell Line; Cell Surface Receptors; Cells; Complex; Coupled; Custom; Cyclization; DNA; Development; Dimensions; Ensure; Entropy; Flow Cytometry; Funding; Generations; Hot Spot; Human; Hybrids; Immune; Immune response; Immune system; Immunohistochemistry; Immunosuppression; Immunotherapy; In Vitro; Interleukin-1 Receptors; Length; Ligands; Malignant Neoplasms; Membrane Proteins; Modality; Modeling; Molecular; Molecular Conformation; Mus; Myeloid-derived suppressor cells; Penetration; Peptides; Pharmaceutical Preparations; Protein Inhibition; Proteins; Receptor Signaling; Reporting; Shapes; Site; Specificity; Surface; Surface Plasmon Resonance; Techniques; Technology; Therapeutic; Tissues; Toxic effect; Vertebral column; Work; base; cancer cell; cancer immunotherapy; cancer therapy; cost; design; ds-DNA; feasibility testing; flexibility; hybrid protein; immunogenicity; in vitro testing; inhibitor; interleukin-1 receptor accessory protein; new therapeutic target; novel; protein complex; protein protein interaction; public health relevance; receptor; scaffold; small molecule; structural biology; tool; tumor; tumor microenvironment

PROJECT SUMMARY/ABSTRACT: New molecular modalities to target sites of protein-protein interaction with high affinity and specificity are desperately needed in cancer treatment. Blocking protein-protein interactions between cell surface receptors on immune cells and cancer cells is the basis for the design of a new generation of cancer immunotherapy therapeutics. However, targeting protein-protein interactions with small molecules has proven difficult due to the large area of interaction between proteins and the dearth of small molecule binding pockets. Additionally, targeting these site with antibodies can prove difficult due to lack of tumor pentration and immune related adverse effects. We propose to overcome these difficulties with a novel class of multivalent inhibitors designed to perfectly interact with a protein surface by combining two exciting technologies. Protein painting, an in-house IMAT-funded structural biology technique designed to discover hotspots of protein interaction, will be used to identify protein sequences that drive affinity between interacting proteins. DNA Origami will be used to prepare a size-scalable, semi-rigid scaffold for the interfering peptides identified with protein painting. This scaffold can be precisely tuned for ideal interaction with the 3D topology of the target protein and will spatially orient the interfering hot spot targeting peptides for interaction with the protein partner. Additionally, multivalency provides increases in affinity and specificity over interfering peptides alone. For proof- of-principle, we will develop a multivalent inhibitor designed to target myeloid-derived suppressor cells (MDSCs). These cells express a receptor called ST2, which when bound to IL-33 and its co-receptor IL-1RAcP, allow the MDSCs to exert an immunosuppressive function in the tumor microenvironment. Disrupting the IL-33/ST2/IL- 1RACP protein complex represents a new avenue for cancer immunotherapy. Under Aim 1, we will optimize interfering peptide inhibitors we have previously discovered targeting the hotspots of protein-protein interaction between IL-1RAcP and IL-33/ST2. Following truncation, cyclization, and amino acid substitution, we will have three potent interfering peptide inhibitors of the IL-33/ST2/IL-1RAcP complex. Under Aim 2, we will construct a DNA origami scaffold designed for precise interaction with the three-dimensional topology IL-33/ST2 surface, and will attach the interfering peptides generated in Aim 1 to this scaffold to synthesize a multivalent polyligand inhibitor of the IL-33/ST2/IL-1RAcP complex. Immunogenicity will be examined for both the scaffold alone, and for the multivalent inhibitor after interfering peptides are attached to the DNA origami scaffold. Under Aim 3, we will first examine affinity of the multivalent inhibitor as compared to the interfering peptides alone via surface plasmon resonance. Second, we will determine the functional potency of the multivalent polyligand inhibitor at reducing ST2 receptor signaling by using a HEKBLue IL-33 ligand cell line assay. Finally, we will verify the activity of the multivalent inhibitor using myeloid derived suppressor cells derived from the murine 4T1 breast cancer model using flow cytometry.

项目摘要/摘要：针对蛋白质 - 蛋白质相互作用的靶点位点的新分子方式 在癌症治疗中迫切需要高亲和力和特异性。阻断蛋白质 - 蛋白质相互作用 在免疫细胞和癌细胞上的细胞表面受体之间是新一代设计的基础 癌症免疫疗法疗法。但是，靶向蛋白质 - 蛋白质与小分子的相互作用的靶向 由于蛋白质与小分子结合的缺乏，因此被证明很难 口袋。此外，由于缺乏肿瘤五牙和 免疫相关的不良反应。我们建议通过一系列新颖的多价克服这些困难 通过结合两种激动人心的技术，旨在与蛋白质表面完美相互作用的抑制剂。蛋白质 绘画，一种内部IMAT资助的结构生物学技术，旨在发现蛋白质的热点 相互作用将用于鉴定驱动相互作用蛋白质之间亲和力的蛋白质序列。脱氧核糖核酸 折纸将用于准备一个可鉴定的干扰肽的可尺寸尺寸的半刚性支架 蛋白质绘画。可以精确调整此脚手架，以与目标的3D拓扑相互作用 蛋白质并将在空间上定位干扰靶向肽的热点以与蛋白质伴侣相互作用。 此外，多价性提供了仅干扰肽的亲和力和特异性的提高。进行证明 - 本质上，我们将开发一种旨在靶向髓样衍生的抑制细胞（MDSC）的多价抑制剂。 这些细胞表达一个称为ST2的受体，当与IL-33及其共受体IL-1RACP结合时，该受体允许 MDSC在肿瘤微环境中发挥免疫抑制功能。破坏IL-33/ST2/IL- 1RACP蛋白复合物代表了癌症免疫疗法的新途径。在AIM 1下，我们将优化 干扰肽抑制剂我们以前已经发现针对蛋白质 - 蛋白质相互作用的热点 在IL-1RACP和IL-33/ST2之间。截断，环化和氨基酸取代后，我们将拥有 IL-33/ST2/IL-1RACP复合物的三种有效干扰肽抑制剂。在AIM 2下，我们将构建一个 DNA折纸支架设计用于与三维拓扑IL-33/ST2表面的精确相互作用， 并将将AIM 1中产生的干扰肽连接到该脚手架上，以合成多价poligand IL-33/ST2/IL-1RACP复合物的抑制剂。将对单独的支架进行检查，并将检查免疫原性 对于干扰肽后的多价抑制剂，附着在DNA折纸支架上。在AIM 3下，我们 与单独通过表面干扰的肽相比，将首先检查多价抑制剂的亲和力 等离子体共振。其次，我们将确定多价poligand抑制剂在 通过使用Hekblue IL-33配体细胞系测定法降低ST2受体信号传导。最后，我们将验证 使用源自鼠4T1乳房的髓样衍生的抑制细胞的多价抑制剂的活性 使用流式细胞仪的癌症模型。