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有关的特定蛋白质或核酸靶标的生化和目标测定的开发。在分析和进步策略的背景下，考虑了使用高吞吐量筛选技术评估各种化合物类别的测定设计。非常重视方法开发研究以提高测定和铅发现效率。与这些活动相辅相成，我们还探索并设计了对复杂化学文库审问的方法（例如，天然产物提取物，mRNA显示）。该程序的工作用于支持一系列赠款应用程序，计划目标和原型项目。以下正在进行：用小分子靶向蛋白质棕榈酰化。与A. Banerjee（NICHD，NIH）合作，我们开发了1536孔兼容的蛋白质棕榈酰酰基转移酶测定酶，以评估化学文库的潜在酶抑制剂，作为可能的疾病的潜在抑制剂，用于大量的疾病，这类酶已连接。这些化合物还预计将具有结构，功能和药理探针的价值。测定开发以发现受体鸟苷酸环化酶NPR1的新型小分子拮抗剂。与Hoon M. hoon（NIDCR，NIH）合作，我们开发了B型Natriaretic肽（BNP）受体NPR1的测定法。最近，激动剂被证明是在周围和脊髓神经之间传播痒感所必需的。 NPR1测定在大规模的化学文库筛查中采用，以鉴定新型的脂肪肽受体拮抗剂，以研究慢性瘙痒的药理学治疗的潜力，这种疾病导致长期不持续的急切划痕以显着降低患者的生活质量（Solinski HJ等人）。最近，我们正在与NCATS ASPIRE程序和mytide Therapeutics合作评估自然环状肽激动剂的合成肽类似物，作为NPR1信号传导的新型调节剂。绒毛膜突变酶抑制剂。该项目旨在识别和开发乳腺突变酶（CM）的抑制剂。 CM是在芳香氨基酸，苯丙氨酸和酪氨酸的生物合成所需的植物和微生物中发现的重要酶。哺乳动物不能进行芳香氨基酸的从头生物合成，并且必须依靠饮食来源。因此，CM的有效药物样抑制剂将是一种有价值的抗菌剂，尤其是对于抗菌素耐药性感染。数十年来，CM的测定方法一直没有进展，并且一直在阻碍新型CM抑制剂的发现。因此，该项目将作为与Dingyin Tao合作在分析化学中与RapidFire技术一起应用基于质量分光光度法的模型。与Suga Lab合作，基于mRNA编码的大环肽库的亲和力选择导致了第一个新颖的活性位点和变构位点的大环肽抑制剂。用小分子靶向G蛋白。骨骼（McCune-Albright综合征）的纤维发育不良是由G蛋白的小含量突变引起的，GS导致细胞cAMP的水平升高，导致的内分泌性过度过度。该项目的目的是开发适合评估G蛋白与其GPCR和效应子腺苷酸环化酶的活性和偶联的生化和基于细胞的测定（Getz Ra等，2019）。例如，通过使用R201C突变体GS的定量高通量筛选测定法，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。非结构性病毒蛋白NSP1是SARS-COV2的毒力因子，可抑制感染细胞中的基因表达。最终的抑制宿主基因表达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中的突变产生了高度减毒的病毒，这些病毒已用于疫苗试验中。在这里与科罗拉多大学医学院杰弗里·基夫（Jeffrey Kieft）教授的结构病毒学实验室合作，Anna-Lena Steckelberg教授，博士哥伦比亚大学和Hiro Suga教授博士在东京大学，我们正在开发一个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。在美国，每年有280万采集抗生素的感染，导致35,000人死亡，抗生素耐药性仍然是一项重大且日益严重的公共卫生挑战。 - 乳酸酶酶水解并因此使广泛的临床使用的-LACTAM药物灭活是抗生素耐药性的主要原因。新德里金属 - 乳糖酶（NDM）可以水解最后的碳青霉烯。与Michael W. Crowder教授合作和同事我们正在开发基于机器倾斜的筛选范例，以发现新型的金属 - 乳糖酶抑制了化学型。抑制刺猬自动化和胆固醇的抑制作用。刺猬（HH）信号通路对于胚胎细胞的分化至关重要，因为HH蛋白浓度梯度对于适当的胚胎形态发育至关重要，在这种形态发展中，途径故障会导致癌症。 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. 该过程的抑制剂可能具有抗癌药的潜力。

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.