Identifying the determinants of cell-penetrant miniproteins

鉴定细胞渗透性小蛋白的决定因素

• 批准号: 10752455
• 负责人: Claire M Phoumyvong
• 金额: $ 4.29万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752455
• 关键词: Antibodies; Binding; Biological Products; Cell membrane; Cells; Charge; Complex; Cytosol; Diffuse; Disease; Endosomes; Enzymes; FDA approved; Gel; Goals; Human; Hydrophobicity; Incubated; Individual; Libraries; Mass Spectrum Analysis; Measures; Methods; Penetration; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Plasma Cells; Property; Protein Engineering; Protein Inhibition; Proteins; Publishing; Reaction; Recovery; Research; Sampling; Specificity; Structure; Surface; System; Tertiary Protein Structure; Testing; Therapeutic; Visualization; Work; cellular targeting; covalent bond; design; extracellular; human disease; inhibitor; novel drug class; novel strategies; peptide drug; protein protein interaction; small molecule; success; therapeutic protein

Project Summary Up to 75% of human-disease causing targets are considered “undruggable” using the current drug classes available: small molecules and biologics. Many of these “undruggable” targets are involved in intracellular protein-protein interactions (PPIs), which are challenging to inhibit with small molecules and antibodies. Small molecules require hydrophobic pockets for binding, which PPIs generally lack. Proteins are capable of disrupting PPIs, but they generally cannot cross the cell's plasma membrane. Miniproteins are small proteins (<10 kDa) with defined tertiary structure. They are a promising class of protein therapeutics that have shown great success at inhibiting extracellular targets and several cell-penetrant miniproteins have been identified. However, we still do not know the design rules needed to develop miniproteins with intracellular delivery. The main bottleneck to developing cell-penetrating miniproteins is that measuring cytosolic delivery is slow and expensive because each protein must be purified and tested individually. To overcome this experimental limitation, we will develop an entirely new multiplexed approach to measure the cytosolic delivery of many candidate cell-penetrant miniproteins at once. We hypothesize that a high-throughput approach will rapidly uncover new cell-penetrant miniproteins to study the determinants of cytosolic delivery. In Aim 1, we will develop a cytosolic capture and pull-down approach to screen cell-penetrant miniproteins. We will create an entirely new approach that quantifies the cytosolic abundance of each miniprotein in a mass spectrometry-based approach. The goal of this aim is to extensively validate this method and see enrichment of our known cell-penetrant protein from a mixture of inactive proteins added to cells. In Aim 2, we will define the protein features that contribute to the cytosolic delivery of miniproteins. We will perform a second round of protein design to validate the features found in our initial screen. The goal of this aim is to apply a multiplexed approach for measuring cytosolic delivery and identify the protein features of successful cell-penetrant miniproteins. The long-term goal of the proposed work is to determine the protein features needed for cytosolic delivery to develop new cell-penetrant miniproteins capable of inhibiting PPIs implicated in disease.

项目摘要 使用当前的药物类别，高达75%的人类致病目标被认为是“不可用药的” 可用：小分子和生物制剂。这些“不可治疗”的靶点中有许多涉及细胞内的 蛋白质-蛋白质相互作用（PPI），这是具有挑战性的抑制与小分子和抗体。小 分子需要疏水口袋来结合，而PPI通常缺乏疏水口袋。蛋白质能够破坏 PPI，但它们通常不能穿过细胞的质膜。微蛋白是小蛋白（<10 kDa） 具有明确的三级结构。它们是一类很有前途的蛋白质疗法，已取得巨大成功 在抑制细胞外靶点和几种细胞渗透微蛋白方面已经被鉴定。不过我们还是 不知道开发具有细胞内递送的微蛋白所需的设计规则。主要瓶颈是 开发细胞穿透微蛋白的一个重要问题是，测量胞质递送是缓慢和昂贵的，因为每一个 蛋白质必须单独纯化和测试。为了克服这个实验限制，我们将开发一个 一种全新的多重方法来测量许多候选细胞渗透剂的胞质递送 蛋白质在一次。我们假设，高通量的方法将迅速发现新的细胞渗透剂， 微蛋白研究胞质传递的决定因素。在目标1中，我们将开发胞质捕获， pull-down方法筛选细胞渗透微蛋白。我们将创造一种全新的方法， 在基于质谱的方法中每个微蛋白的胞质丰度。这一目标的目的是 广泛地验证了这种方法，并看到我们已知的细胞渗透蛋白从混合物中的富集， 添加到细胞中的非活性蛋白质。在目标2中，我们将定义有助于细胞溶质的蛋白质特征。 微蛋白的递送。我们将进行第二轮的蛋白质设计，以验证我们在 初始屏幕。本研究的目的是应用一种多重方法来测量胞质递送，并鉴定 成功的细胞渗透微蛋白的蛋白质特征。拟议工作的长期目标是 确定胞质传递所需的蛋白质特征，以开发新的细胞穿透微蛋白， 抑制与疾病有关的PPI。