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

• 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
• 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

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.

项目摘要 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SH 2结构域上鉴定了一个新发现的受调控的半胱氨酸 （SHP-1）作为衣康酸盐的靶标。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依赖于 这个关键的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（Cys 102）是巨噬细胞细胞因子产生的关键近端抑制剂， 化学修饰这个网站提供了一个前所未有的机会，以调节生产 炎性细胞因子我们将通过三个目标来测试我们的假设：目标1）确定结构 基于SHP-1半胱氨酸调节及其在巨噬细胞活化中的作用，我们将联合收割机结合我们的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， 确定SHP-1及其Cys 102残基在巨噬细胞活化中的作用。目的2）鉴定有效的衣康酸- 模拟化学型，可以选择性地靶向SHP-1激活半胱氨酸在巨噬细胞，我们将使用 结构指导的药物化学来提高我们衣康酸铅模拟物的效力和特异性。的 新合成的化合物将在小鼠和人类中使用表型和MS分析进行测试 巨噬细胞目的3）确定SHP-1在半胱氨酸激活后的相互作用组和下游效应物， 我们将使用MS来识别SHP-1结合伴侣，并通过CRISPR/Cas9进行遗传修饰来验证 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 已知的SHP-1结合配偶体IRAK和STAT 3在巨噬细胞失活中的作用。如果成功，我们的项目将为 首次明确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细胞毒活性的潜力的新化合物， 特异性和有效性。这将是我们在理解 巨噬细胞生物学的基本机制和化合物的开发， 高度特异性的生物工具，并具有转化潜力的病理特征是慢性 炎症