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：以蛋白质-蛋白质相互作用位点为靶点的新分子模式 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 细胞表面受体对免疫细胞和癌细胞的影响是设计新一代细胞的基础 癌症免疫治疗疗法的专家。然而，以小分子为靶点的蛋白质相互作用 事实证明，由于蛋白质之间相互作用的面积很大，而且缺乏小分子结合，因此很难实现 口袋里。此外，用抗体靶向这些部位可能被证明是困难的，因为缺乏肿瘤侵袭和 免疫相关不良反应。我们建议用一类新的多价化合物来克服这些困难 通过将两种令人兴奋的技术相结合，设计出与蛋白质表面完美相互作用的抑制剂。蛋白 绘画，一项由IMAT资助的内部结构生物学技术，旨在发现蛋白质的热点 相互作用，将用于识别推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 折纸将被用来为经鉴定的干扰肽制备尺寸可缩放的半刚性支架 蛋白质涂装。这种支架可以精确地调整，以便与目标的3D拓扑进行理想的交互 并将空间定向干扰热点靶向多肽以与蛋白质伙伴相互作用。 此外，多价性提供了比单独干扰肽更高的亲和力和特异性。作为证据- 原则上，我们将开发一种针对髓系抑制细胞(MDSCs)的多价抑制剂。 这些细胞表达一种名为ST2的受体，当它与IL-33及其辅助受体IL-1RAcP结合时，允许 MDSCs在肿瘤微环境中发挥免疫抑制功能。干扰IL-33/ST2/IL- 1RACP蛋白复合体是肿瘤免疫治疗的新途径。在目标1下，我们将优化 我们之前发现的针对蛋白质相互作用热点的干扰肽抑制剂 IL-1RAcP和IL-33/ST2之间的差异。在截断、环化和氨基酸替换之后，我们将拥有 IL-33/ST2/IL-1RAcP复合体的三种强有力的干扰肽抑制剂。在目标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表面精确相互作用， 并将在目标1中产生的干扰肽连接到该支架上以合成多价多配体 IL-33/ST2/IL-1RAcP复合体的抑制剂。仅对支架和支架进行免疫原性检测 对于多价抑制剂，干扰后的多肽被附着在DNA折纸支架上。在目标3下，我们 我将首先通过表面比较多价抑制物与干扰多肽的亲和力 等离子激元共振。其次，我们将在以下位置确定多价多配体抑制剂的功能效力 使用HEKBLue IL-33配基细胞系分析减少ST2受体信号。最后，我们将验证 用小鼠4T1乳房髓系来源的抑制细胞研究多价抑制物的活性 采用流式细胞术建立肿瘤模型。