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.

项目总结/摘要：靶向蛋白质-蛋白质相互作用位点的新分子模式， 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 免疫细胞和癌细胞上的细胞表面受体之间的联系是设计新一代 癌症免疫疗法的最新进展然而，用小分子靶向蛋白质-蛋白质相互作用， 由于蛋白质之间的大面积相互作用和小分子结合的缺乏， 口袋此外，由于缺乏肿瘤渗透，用抗体靶向这些位点可能证明是困难的， 免疫相关的不良反应。我们建议用一类新的多价化合物来克服这些困难。 通过结合两种令人兴奋的技术，设计出与蛋白质表面完美相互作用的抑制剂。蛋白 绘画，一个内部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的亲和力的蛋白质序列。DNA Origami将用于制备尺寸可缩放的半刚性支架，用于用以下方法鉴定的干扰肽： 蛋白质绘画该支架可以被精确地调整，以实现与目标的3D拓扑结构的理想相互作用。 蛋白质，并将空间定向干扰热点靶向肽以与蛋白质配偶体相互作用。 另外，多价性提供了相对于单独的干扰肽的亲和力和特异性的增加。为了证明- 原则上，我们将开发一种多价抑制剂，旨在靶向髓源性抑制细胞（MDSC）。 这些细胞表达一种称为ST 2的受体，当与IL-33及其辅助受体IL-1 RAcP结合时， MDSC在肿瘤微环境中发挥免疫抑制功能。破坏IL-33/ST 2/IL-10 1 RACP蛋白复合物代表了癌症免疫治疗的新途径。在目标1下，我们将优化 干扰肽抑制剂，我们以前发现的目标蛋白质-蛋白质相互作用的热点 IL-1 RAcP和IL-33/ST 2之间的关系。在截短、环化和氨基酸取代之后，我们将得到 IL-33/ST 2/IL-1 RAcP复合物的三种有效干扰肽抑制剂。根据目标2，我们将构建一个 设计用于与三维拓扑IL-33/ST 2表面精确相互作用的DNA折纸支架， 并将Aim 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/ST 2/IL-1 RAcP复合物的抑制剂。将检查单独支架的免疫原性， 在将干扰肽连接到DNA折纸支架上之后，根据目标3，我们 将首先通过表面活性剂检测多价抑制剂与单独的干扰肽相比的亲和力， 等离子共振第二，我们将确定多价多配体抑制剂的功能效力， 通过使用HEKBLue IL-33配体细胞系测定减少ST 2受体信号传导。最后，我们将验证 使用来源于鼠4 T1乳腺的髓源性抑制细胞的多价抑制剂的活性 使用流式细胞术的癌症模型。