Target-based Assays and Screening Strategies for Chemical Probe and Therapeutic Lead Discovery

化学探针和治疗性先导化合物发现的基于靶标的测定和筛选策略

• 批准号: 10263805
• 负责人: James Inglese
• 金额: $ 42.06万
• 依托单位: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10263805
• 关键词: 2019-nCoV; Adenylate Cyclase; Agonist; Anabolism; Antibiotics; Antineoplastic Agents; Applications Grants; Aromatic Amino Acids; Biochemical; Biological Assay; Biological Sciences; Brain natriuretic peptide; CD47 gene; Cell Proliferation; Cells; Cellular Assay; Chemicals; Chorismate Mutase; Collaborations; Complement; Complex; Coupling; Cyclic AMP; Development; Diet; Disease; Dose-Limiting; Enzyme Inhibitor Drugs; Enzymes; Evaluation; Extramural Activities; Folic Acid; Folic Acid Antagonists; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Guanylate Cyclase; Hematology; Hepatotoxicity; Immunosuppression; Laboratories; Lead; Ligands; Link; Mammals; McCune-Albright Syndrome; Messenger RNA; Methods; Methotrexate; Missense Mutation; Molecular Conformation; Molecular Target; Monitor; National Institute of Child Health and Human Development; National Institute of Dental and Craniofacial Research; Natriuretic Peptides; Natural Products; Nerve; Normal tissue morphology; Nucleic Acids; Osteitis Fibrosa Disseminata; PTPNS1 gene; Peptide Receptor; Peripheral; Pharmaceutical Preparations; Pharmacological Treatment; Pharmacology; Phenylalanine; Plants; Proteins; Pruritus; Quality of life; RNA; Radiation therapy; Regulator Genes; Research; Rheumatoid Arthritis; Science; Scientist; Source; Spinal Cord; Structure; Technology; Therapeutic; Transferase; Translations; Tyrosine; United States National Institutes of Health; Untranslated RNA; Work; antimicrobial drug; antimicrobial resistant infection; assay development; atrial natriuretic factor receptor B; base; cancer therapy; chronic itch; design; drug discovery; folic acid metabolism; high throughput screening; inhibitor/antagonist; method development; microorganism; mutant; novel; novel strategies; palmitoylation; programs; protein protein interaction; prototype; receptor; research and development; screening; small molecule; small molecule inhibitor; small molecule libraries; transmission process; tumor

This project includes the development of biochemical and target-focused assays based on specific protein or nucleic acid targets implicated in disease. The assay designs are considered in the context of analysis and progression strategies for evaluation of a wide range of compound classes using high throughput screening technologies. There is a strong emphasis on methods development research to advance assay and lead discovery efficiency. Complementing these activities, we also explore and devise approaches for the interrogation of complex chemical libraries (e.g., natural product extracts, mRNA display). The work from this program is used to support a range of grant applications and prototype projects. The following are on-going: Targeting protein palmitoylation with small molecules. In collaboration with A. Banerjee (NICHD, NIH) we have developed 1536-well compatible protein palmitoyl acyl transferase assays to evaluate chemical libraries for potential inhibitors of the enzyme as possible therapeutic leads for the large number of diseases to which this class of enzyme have been linked, including proteins associated with SARS CoV-2. These compounds are also anticipated to have value as structural, functional, and pharmacological probes. Strategies for the discovery of small molecule ligands of RNA. In collaboration with Prof. N. Baird (U. Sciences) we are exploring assay designs to probe the interaction of small molecules with non-coding gene-regulatory mRNAs. The research has the potential of the discovery of novel antibiotics or anticancer agents. Assay development to enable discovery of novel small molecule antagonists of the receptor guanylate cyclase Npr1. In collaboration with M. Hoon (NIDCR, NIH) we have developed assays of the b-type natriuretic peptide (BNP) receptor, Npr1. Recently the agonist, BNP was shown to be required for the transmission of itch sensation between peripheral and spinal cord nerves. The Npr1 assays were employed in large-scale chemical library screening to identify novel natriuretic peptide receptor antagonists to investigate the potential of pharmacological treatments of chronic itch, a condition that results in long-term unremitting urge to scratch that significantly degrades the quality of life for sufferers (Solinski HJ et al., 2019). This project is being continued under the HEAL initiative. SIRPa-CD47 Protein-protein interaction. In collaboration with T. Miller (Paradigm Shift Therapeutics) and D. Roberts (NCI, NIH) our goal is to leverage the broad potential of CD47 as a molecular target in a number of tumors to create therapeutics that protect normal tissue from chemo and radiation therapy while differentially enhancing the effects of these therapies on the tumor. We designed and validated several biochemical SIRPa-CD47 protein-protein interaction (Miller T et al., 2019) and cellular assay formats (Burgess, T et al. 2020). Using these assays, we are investigating the potential SIRPa-CD47 complex disruption activity of several compounds identified from large-scale high throughput chemical library screening. Chorismate mutase inhibitors. In collaboration with J. Padia (PrimeTime Life Sciences) this project seeks to develop a quantitative high throughput screening (qHTS) assay for the identification of small molecule inhibitors of chorismate mutase (CM). CM is an important enzyme found in plants and microorganisms required for the biosynthesis of the aromatic amino acids, phenylalanine, and tyrosine. Mammals cannot carry out the de novo biosynthesis of aromatic amino acids and must rely on dietary sources. Thus, a potent and selective drug-like inhibitor of CM would be a valuable antimicrobial agent, particularly for antimicrobial resistant infections. Targeting G proteins with small molecules. Fibrous dysplasia of bone (McCune-Albright syndrome) is a hyperfunctioning endocrinopathy resulting from mis-sense mutations in the small -subunit of the G-protein, Gs leading to increased levels of cellular cAMP. The aim of this project is to develop biochemical and cell-based assays suitable for evaluating the activity and coupling of G proteins to their GPCRs and effector adenylyl cyclase (Getz RA et al., 2019). For example, by enabling a quantitative high throughput screening assay using the R201C mutant form of Gs to identify small molecules capable of antagonizing the R201C Gs adenylyl cyclase-activating conformation. Novel approaches to antifolate modulators of cell proliferation and immunosuppression. Methotrexate is a widely used chemotherapeutic for the treatment of cancer and rheumatoid arthritis. However, alternatives have been sought for decades due to the dose-limiting hematologic and hepatic toxicity of MTX, attributable in part to long-term cellular retention by polyglutamylation. This project is developing reduced folate utilizing enzyme inhibitors and assay methods to monitor cellular folate metabolism for more rapid evaluation of novel antifolates.

该项目包括基于与疾病相关的特定蛋白质或核酸靶点开发生化和靶向检测方法。 检测设计是在分析和进展策略的背景下考虑的，用于使用高通量筛选技术评估各种化合物类别。非常重视方法开发研究，以提高检测和先导化合物发现效率。 作为这些活动的补充，我们还探索和设计了复杂化学库（例如天然产物提取物、mRNA 展示）的询问方法。 该计划的工作用于支持一系列拨款申请和原型项目。 以下内容正在进行中： 用小分子靶向蛋白质棕榈酰化。我们与 A. Banerjee（NICHD、NIH）合作开发了 1536 孔兼容的蛋白质棕榈酰酰基转移酶测定法，以评估该酶的潜在抑制剂的化学库，作为此类酶所治疗的大量疾病的可能治疗方法。已被联系起来，包括与 SARS CoV-2 相关的蛋白质。 预计这些化合物也具有作为结构、功能和药理学探针的价值。 发现 RNA 小分子配体的策略。 我们与 N. Baird 教授（美国科学学院）合作，正在探索检测设计，以探测小分子与非编码基因调节 mRNA 的相互作用。 该研究具有发现新型抗生素或抗癌药物的潜力。 开发检测方法以发现受体鸟苷酸环化酶 Npr1 的新型小分子拮抗剂。我们与 M. Hoon（NIDCR、NIH）合作开发了 b 型利钠肽 (BNP) 受体 Npr1 的检测方法。 最近，BNP 激动剂被证明是外周神经和脊髓神经之间瘙痒感传递所必需的。 Npr1 检测用于大规模化学文库筛选，以鉴定新型利尿钠肽受体拮抗剂，以研究慢性瘙痒的药物治疗潜力，慢性瘙痒是一种导致长期不断抓挠的冲动，从而显着降低患者生活质量的疾病。患者（Solinski HJ 等，2019）。该项目正在 HEAL 倡议下继续进行。 SIRPa-CD47 蛋白质-蛋白质相互作用。 与 T. Miller（Paradigm Shift Therapeutics）和 D. Roberts（NCI、NIH）合作，我们的目标是利用 CD47 作为多种肿瘤分子靶点的广泛潜力，创造出保护正常组织免受化疗和放疗影响的治疗方法疗法，同时差异化地增强这些疗法对肿瘤的效果。 我们设计并验证了几种生化 SIRPa-CD47 蛋白质-蛋白质相互作用 (Miller T et al., 2019) 和细胞测定格式 (Burgess, T et al. 2020)。使用这些测定，我们正在研究从大规模高通量化学库筛选中鉴定出的几种化合物的潜在 SIRPa-CD47 复合物破坏活性。 分支酸变位酶抑制剂。该项目与 J. Padia (PrimeTime Life Sciences) 合作，旨在开发一种定量高通量筛选 (qHTS) 检测方法，用于鉴定分支酸变位酶 (CM) 的小分子抑制剂。 CM 是在植物和微生物中发现的重要酶，是芳香族氨基酸、苯丙氨酸和酪氨酸生物合成所需的。 哺乳动物不能进行芳香族氨基酸的从头生物合成，必须依赖饮食来源。 因此，一种有效的、选择性的 CM 类药物抑制剂将是一种有价值的抗菌剂，特别是对于抗菌素耐药性感染。 用小分子靶向 G 蛋白。骨纤维发育不良（McCune-Albright 综合征）是一种功能亢进的内分泌病，由 G 蛋白小亚基 Gs 的错义突变导致细胞 cAMP 水平升高。该项目的目的是开发适合评估 G 蛋白与其 GPCR 和效应腺苷酸环化酶的活性和偶联的生化和基于细胞的测定法（Getz RA 等人，2019）。例如，通过使用 Gs 的 R201C 突变体形式进行定量高通量筛选分析来鉴定 能够拮抗 R201C Gs 腺苷酸环化酶激活构象的小分子。 细胞增殖和免疫抑制的抗叶酸调节剂的新方法。甲氨蝶呤是一种广泛使用的化疗药物，用于治疗癌症和类风湿性关节炎。然而，由于 MTX 的剂量限制性血液学和肝脏毒性，几十年来人们一直在寻找替代品，部分原因是多谷氨酰化作用导致细胞长期滞留。 该项目正在开发利用酶抑制剂和测定方法来减少叶酸，以监测细胞叶酸代谢，以便更快速地评估新型抗叶酸剂。