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

基本信息

批准号：
10907362
负责人：
James Inglese
金额：
$ 69.33万
依托单位：
NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/10907362
关键词：
2019-nCoV Active Sites Adenylate Cyclase Affinity Agonist Allosteric Site Anabolism Analytical Chemistry Antibiotic Resistance Antineoplastic Agents Applications Grants Aromatic Amino Acids Attenuated Binding Biochemical Biochemistry Biological Assay Brain natriuretic peptide Carbapenems Cell Differentiation process Cells Cessation of life Chemicals Cholesterol Chorismate Mutase Clinical Collaborations Colorado Complement Complex Coupling Cyclic AMP Cyclic Peptides Development Disease Doctor of Philosophy Embryo Enzyme Inhibitor Drugs Enzymes Erinaceidae Evaluation Extramural Activities G-Protein-Coupled Receptors GTP-Binding Proteins Gene Expression Guanylate Cyclase Interferons Laboratories Lactamase Lactams Lead Link Malignant Neoplasms Mammals McCune-Albright Syndrome Messenger RNA Methods Missense Mutation Modeling Molecular Conformation Morphology Mutation National Center for Advancing Translational Sciences National Institute of Child Health and Human Development National Institute of Dental and Craniofacial Research Natriuretic Peptides Natural Products Nerve Nucleic Acids Osteitis Fibrosa Disseminata Pathway interactions Peptide Library Peptide Receptor Peptides Peripheral Pharmaceutical Preparations Pharmacological Treatment Phenylalanine Plants Process Protein Precursors Proteins Pruritus Public Health Quality of life SARS coronavirus SARS-CoV-2 inhibitor Scientist Series Signal Pathway Signal Transduction Source Spectrophotometry Spinal Cord Technology Therapeutic Thinness Tokyo Transferase Translations Tyrosine United States National Institutes of Health Universities Validation Viral Viral Nonstructural Proteins Virulence Factors Virus Work antagonist antibiotic resistant infections antimicrobial drug antimicrobial resistant infection assay development atrial natriuretic factor receptor B beta-Lactamase chronic itch design dietary drug discovery embryo cell high throughput screening inhibitor medical schools method development microorganism mutant novel palmitoylation peptide analog pharmacologic programs prototype receptor research and development response screening small molecule small molecule libraries synthetic peptide transmission process vaccine trial virology

项目摘要

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, program objectives 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. These compounds are also anticipated to have value as structural, functional, and pharmacological probes. 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., in press). Most recently, in collaboration with the NCATS ASPIRE program and Mytide Therapeutics we are assessing synthetic peptide analogs of the receptors natural cyclic peptide agonist as novel modulators of Npr1 signaling. Chorismate mutase inhibitors. This project seeks to identify and develop 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. Assay methods for CM have not progressed for decades and have been hampering the discovery of novel CM inhibitors. Thus, this project will serve as a model for the application of mass spectrophotometry-based screening with the RapidFire technology in collaboration with the Dingyin Tao in Analytical Chemistry. In collaboration with the Suga lab, mRNA-encoded macrocyclic peptide library-based affinity selection has resulted in the first novel active site and allosteric site macrocyclic peptide inhibitors. 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. SARS CoV-2 Nsp1 Inhibitors. The nonstructural viral protein, nsp1 is a virulence factor of SARS-CoV2 that inhibits gene expression in infected cells. The resultant inhibition of host gene expression restricts antiviral signaling and the interferon response. In the closely related virus SARS-CoV1, mutations in Nsp1 create highly attenuated viruses, which have been used in vaccine trials. Here in collaboration with the Structural Virology Laboratory of Prof. Jeffrey Kieft at the University of Colorado School of Medicine, Prof. Anna-Lena Steckelberg, Ph.D. at Columbia University, and Prof. Hiro Suga, Ph.D. of The University of Tokyo we are developing a compound screening and validation assay series to identify inhibitors of SARS-CoV2 Nsp1 function. Metallo beta-lactamases. With 2.8 million acquired antibiotic-resistant infections yearly in the US, leading to >35,000 deaths, antibiotic resistance remains a significant and increasing public health challenge. -lactamases which hydrolyze and thereby inactivate a broad range of clinically used -lactam drugs are a major global cause of antibiotic resistance. New Delhi metallo--lactamases (NDM) can hydrolyze the last-resort carbapenems. In collaboration with Prof. Michael W. Crowder, Ph.D. and colleagues we are developing machine leaning-enabled screening paradigms to discovery novel metallo--lactamases inhibitors chemotypes. Inhibition of Hedgehog autoprocessing and cholesteroylation. The Hedgehog (Hh) signaling pathway is essential to embryonic cell differentiation as Hh protein concentration gradients are critical to proper embryonic morphologic development, where pathway malfunction can result in cancer. In a collaboration with Dr. Callahan, an expert in the biochemistry of Hh cholesteroylation, which occurs via the self-catalyzed endo-proteolysis of the hedgehog precursor protein by activated cholesterol bound to the precursor C-terminus, we are developing, optimizing, and validating cell-based qHTS assays for the identification of Hh autoprocessing inhibitors. Inhibitors of this process may have potential as anticancer agents .

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、计划目标和原型项目。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。已链接。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。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 等人，出版中）。最近，我们与 NCATS ASPIRE 计划和 Mytide Therapeutics 合作，正在评估受体天然环肽激动剂的合成肽类似物作为 Npr1 信号传导的新型调节剂。分支酸变位酶抑制剂。该项目旨在鉴定和开发分支酸变位酶（CM）抑制剂。 CM 是植物和微生物中发现的一种重要酶，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。 CM 的测定方法几十年来一直没有进展，并且一直阻碍着新型 CM 抑制剂的发现。因此，该项目将与分析化学系的陶鼎银合作，作为基于质谱分光光度法的RapidFire技术筛选应用的模型。与 Suga 实验室合作，基于 mRNA 编码的大环肽库的亲和力选择产生了第一个新型活性位点和变构位点大环肽抑制剂。用小分子靶向 G 蛋白。骨纤维性发育不良（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 突变形式进行定量高通量筛选测定，以鉴定能够拮抗 R201C Gs 腺苷酸环化酶激活构象的小分子。 SARS CoV-2 Nsp1 抑制剂。非结构病毒蛋白 nsp1 是 SARS-CoV2 的毒力因子，可抑制受感染细胞中的基因表达。由此产生的宿主基因表达抑制限制了抗病毒信号传导和干扰素反应。在密切相关的病毒 SARS-CoV1 中，Nsp1 的突变产生了高度减毒的病毒，已用于疫苗试验。 Anna-Lena Steckelberg 教授与科罗拉多大学医学院 Jeffrey Kieft 教授的结构病毒学实验室合作，开展了这项研究。哥伦比亚大学教授和 Hiro Suga 教授博士东京大学的我们正在开发一种化合物筛选和验证测定系列，以鉴定 SARS-CoV2 Nsp1 功能的抑制剂。金属β-内酰胺酶。美国每年有 280 万人获得抗生素耐药性感染，导致超过 35,000 人死亡，抗生素耐药性仍然是一个重大且日益严重的公共卫生挑战。 β内酰胺酶可水解并从而使多种临床使用的β内酰胺药物失活，是全球抗生素耐药性的主要原因。新德里金属内酰胺酶（NDM）可以水解最后的碳青霉烯类药物。与 Michael W. Crowder 教授、博士合作。我们和同事正在开发支持机器学习的筛选范例，以发现新的金属内酰胺酶抑制剂化学型。抑制 Hedgehog 自动加工和胆固醇化。 Hedgehog (Hh) 信号通路对于胚胎细胞分化至关重要，因为 Hh 蛋白浓度梯度对于正常的胚胎形态发育至关重要，其中通路故障可能导致癌症。 Hh 胆固醇化的生物化学专家 Callahan 博士与 Callahan 博士合作，Hh 胆固醇化是通过与前体 C 末端结合的活化胆固醇对刺猬前体蛋白进行自催化内蛋白水解而发生的。验证基于细胞的 qHTS 测定法以鉴定 Hh 自动加工抑制剂。这一过程的抑制剂可能具有作为抗癌剂的潜力。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A fission yeast platform for heterologous expression of mammalian adenylyl cyclases and high throughput screening.

用于哺乳动物腺苷酸环化酶异源表达和高通量筛选的裂殖酵母平台。

DOI：
10.1016/j.cellsig.2019.04.010
发表时间：
2019
期刊：
Cellular signalling
影响因子：
4.8
作者：
Getz,RachelA;Kwak,Grace;Cornell,Stacie;Mbugua,Samuel;Eberhard,Jeremy;Huang,ShengXiang;Abbasi,Zainab;deMedeiros,AnaSantos;Thomas,Rony;Bukowski,Brett;Dranchak,PatriciaK;Inglese,James;Hoffman,CharlesS
通讯作者：
Hoffman,CharlesS

qHTSWaterfall: 3-dimensional visualization software for quantitative high-throughput screening (qHTS) data.