Targeting SHP-1 through a newfound metabolite-regulated cysteine activation site

通过新发现的代谢物调节的半胱氨酸激活位点靶向 SHP-1

基本信息

批准号：
10802649
负责人：
Edward Thomas Chouchani
金额：
$ 86.19万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-25 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10802649
关键词：
Aconitic Acid Acute Anti-Inflammatory Agents Antiinflammatory Effect Binding Biochemistry Biological Biological Assay Biology CRISPR/Cas technology Carboxy-Lyases Cell physiology Cells Chemicals Chronic Citric Acid Cycle Crystallography Cysteine Data Development Electron Transport Genetic Human IL18 gene IRAK1 gene Immunity Impairment Inflammasome Inflammation Inflammatory Interferon Type I Interleukin-6 Laboratories Lead Libraries Literature MAPK8 gene Macrophage Macrophage Activation Maps Mediating Methods Modeling Modification Mus NF-kappa B NR0B2 gene Neutrophil Activation Pathology Peptides Pharmaceutical Chemistry Pharmaceutical Preparations Phenocopy Phenotype Process Production Protein Tyrosine Phosphatase Proteins Proteome Regulation Research Role STAT3 gene Shunt Device Site Specificity Stimulus Structure Succinate Dehydrogenase Testing Time cytokine cytotoxic immune activation improved inhibitor innate immune function interest loss of function mimetics novel p38 Mitogen Activated Protein Kinase prevent response scaffold small molecule src Homology Region 2 Domain tool translational potential

项目摘要

PROJECT SUMMARY Itaconate is a major regulator of macrophage inflammatory activity through its ability to directly alkylate cysteines on critical target proteins. Using our newly developed mass spectrometric (MS) method (CPT-MS), we identified a newfound regulated cysteine on the SH2 domain containing protein tyrosine phosphatase-1 (SHP-1) as a target of itaconate in both mouse and human macrophages. We developed a library of highly potent itaconate-mimetics and found that the anti-inflammatory activity of these molecules was dependent on this critical SHP-1 cysteine modification. Together, this led us to hypothesize that the newfound SHP-1 regulatory cysteine (Cys102) is a critical proximal inhibitor of macrophage cytokine production, and that chemical modification of this site presents an unprecedented opportunity to modulate the production of inflammatory cytokines. We will test our hypothesis through three aims: Aim 1) To determine the structural basis of SHP-1 cysteine regulation and its role in macrophage activation, we will combine our CPT-MS platform, protein crystallography and biochemistry, loss-of-function models, and cytokine production assays to define the role of SHP-1 and its Cys102 residue in macrophage activation. Aim 2) To identify potent itaconate- mimetic chemotypes that can selectively target the SHP-1 activation cysteine in macrophages, we will use structure guided medicinal chemistry to improve potency and specificity of our lead itaconate-mimetics. The newly synthesized compounds will be tested using phenotypic and MS assays in both mouse and human macrophages. Aim 3) To define the interactome and downstream effectors of SHP-1 upon cysteine activation, we will use MS to identify the SHP-1 binding partners, and genetic modification via CRISPR/Cas9 to validate the role of these interactions in macrophage activity and cytokine production. We will also examine the role of known SHP-1 binding partners, IRAK and STAT3, in macrophage deactivation. If successful, our project will for the first time 1) define the role of SHP-1 cysteine activation in itaconate-mediated inhibition of macrophage activity and 2) identify new compounds with potential to inhibit macrophage cytotoxic activity with high specificity and efficacy. This will represent an important step forward in both our understanding of the fundamental mechanisms of macrophage biology and development of compounds that can serve as both highly specific biological tools and have translational potential for pathologies characterized by chronic inflammation.

项目概要衣康酸通过其直接烷基化的能力是巨噬细胞炎症活性的主要调节剂关键靶蛋白上的半胱氨酸。使用我们新开发的质谱（MS）方法（CPT-MS），我们在含有蛋白酪氨酸磷酸酶-1的SH2结构域上发现了一个新发现的调节半胱氨酸 (SHP-1) 作为小鼠和人类巨噬细胞中衣康酸的靶标。我们开发了一个高度有效的衣康酸模拟物，并发现这些分子的抗炎活性取决于这种关键的 SHP-1 半胱氨酸修饰。总之，这使我们假设新发现的 SHP-1 调节性半胱氨酸 (Cys102) 是巨噬细胞细胞因子产生的重要近端抑制剂，并且该位点的化学修饰为调节生产提供了前所未有的机会炎症细胞因子。我们将通过三个目标来检验我们的假设：目标 1) 确定结构基于 SHP-1 半胱氨酸调节及其在巨噬细胞激活中的作用，我们将结合我们的 CPT-MS 平台、蛋白质晶体学和生物化学、功能丧失模型和细胞因子产生测定定义 SHP-1 及其 Cys102 残基在巨噬细胞激活中的作用。目标 2) 鉴定有效的衣康酸- 可以选择性地靶向巨噬细胞中 SHP-1 激活半胱氨酸的模拟化学型，我们将使用结构引导药物化学，以提高我们的主要衣康酸酯模拟物的效力和特异性。这新合成的化合物将在小鼠和人类中使用表型和 MS 检测进行测试巨噬细胞。目标 3) 定义半胱氨酸激活后 SHP-1 的相互作用组和下游效应子，我们将使用 MS 来鉴定 SHP-1 结合伴侣，并通过 CRISPR/Cas9 进行基因修饰来验证这些相互作用在巨噬细胞活性和细胞因子产生中的作用。我们还将研究以下角色的作用：已知 SHP-1 结合伙伴 IRAK 和 STAT3 在巨噬细胞失活中的作用。如果成功的话，我们的项目将首次1）定义SHP-1半胱氨酸激活在衣康酸介导的巨噬细胞抑制中的作用活性和 2) 鉴定具有抑制巨噬细胞细胞毒活性潜力的新化合物特异性和有效性。这将代表我们在理解巨噬细胞生物学的基本机制以及可作为两者的化合物的开发高度特异性的生物工具，对以慢性为特征的病理学具有转化潜力炎。