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.

项目概要 使用当前药物类别，高达 75% 的人类疾病致病靶点被认为是“无法成药”的 可用：小分子和生物制剂中有许多“不可成药”的靶标涉及细胞内。 蛋白质-蛋白质相互作用（PPI），用小分子和小抗体很难抑制。 分子需要疏水性口袋才能结合，而蛋白质通常缺乏这种疏水性口袋。 PPI，但它们通常不能穿过细胞质膜。微蛋白是小蛋白质（<10 kDa）。 它们是一类有前途的蛋白质疗法，已取得巨大成功。 然而，我们仍然鉴定出抑制细胞外靶标和几种细胞渗透性小蛋白的能力。 不知道开发细胞内递送微型蛋白所需的设计规则是主要瓶颈。 开发细胞穿透性微型蛋白的一个缺点是测量胞质传递缓慢且昂贵，因为每个 蛋白质必须单独纯化和测试，为了克服这一实验限制，我们将开发一种方法。 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 我们勇敢地说，高通量方法将迅速发现新的细胞渗透剂。 在目标 1 中，我们将开发一种胞质捕获和微蛋白。 我们将创建一种全新的定量方法来筛选细胞渗透性微蛋白。 通过基于质谱的方法确定每种微型蛋白的胞质丰度。 广泛验证该方法并观察从混合物中富集我们已知的细胞渗透蛋白 添加到细胞中的非活性蛋白质在目标 2 中，我们将定义有助于胞质的蛋白质特征。 我们将进行第二轮蛋白质设计以验证我们发现的特征。 该目的的目标是应用多重方法来测量细胞质递送并识别。 成功的细胞渗透性小蛋白的蛋白质特征拟议工作的长期目标是 确定胞质递送所需的蛋白质特征，以开发新的细胞渗透性小蛋白 抑制与疾病有关的 PPI。