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: 10683014
• 负责人: James Inglese
• 金额: $ 45.05万
• 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/10683014
• 关键词: 2019-nCoV; ATP-Binding Cassette Transporters; Acids; Adenylate Cyclase; Agonist; Amino Acids; Anabolism; Analytical Chemistry; Antibiotic Resistance; Antineoplastic Agents; Applications Grants; Aromatic Amino Acids; Attenuated; Autoimmune Diseases; Binding; Biochemical; Biochemistry; Biological Assay; Biological Sciences; Brain natriuretic peptide; Carbapenems; Carbon; Cell Differentiation process; Cells; Cessation of life; Chemicals; Cholesterol; Chorismate Mutase; Clinical; Collaborations; Colorado; Complement; Complex; Coupling; Crowding; Cyclic AMP; Cyclic Peptides; DNA biosynthesis; Development; Dihydrofolate Reductase; Dihydrofolate Reductase Inhibitor; Disease; Doctor of Philosophy; Embryo; Enzyme Inhibitor Drugs; Enzymes; Erinaceidae; Evaluation; Extramural Activities; Folic Acid; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Guanylate Cyclase; Hydrolysis; Interferons; Intracellular Transport; Laboratories; Lactamase; Lactams; Lead; Link; Malignant Neoplasms; Mammals; McCune-Albright Syndrome; Messenger RNA; Metabolism; Methotrexate; Missense Mutation; Modeling; Molecular Conformation; Morphology; Mutate; 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; Peripheral; Pharmaceutical Preparations; Pharmacological Treatment; Pharmacology; Phenylalanine; Plants; Process; Prodrugs; Protein Precursors; Proteins; Proteolysis; Pruritus; Public Health; Purines; Quality of life; Reaction; Resort; Rheumatoid Arthritis; SARS coronavirus; SARS-CoV-2 inhibitor; Scientist; Series; Signal Transduction; Source; Specificity; Spectrophotometry; Spinal Cord; Technology; Tetrahydrofolates; Therapeutic; Thinness; Tokyo; Transferase; Translations; Tyrosine; United States National Institutes of Health; Universities; Validation; Viral Nonstructural Proteins; Virulence Factors; Virus; Work; analog; antagonist; antibiotic resistant infections; antimicrobial drug; antimicrobial resistant infection; assay development; atrial natriuretic factor receptor B; base; beta-Lactamase; blastomere structure; chronic itch; cofactor; design; dietary; drug discovery; gamma-Glutamyl Hydrolase; high throughput screening; improved; inhibitor; insight; medical schools; member; method development; methotrexate analog; microorganism; mutant; novel; nucleobase; overexpression; palmitoylation; peptide analog; polyglutamates; programs; prototype; receptor; research and development; resistance mechanism; response; screening; side effect; small molecule; small molecule inhibitor; small molecule libraries; smoothened signaling pathway; synthetic peptide; therapy resistant; thymidylate; transmission process; vaccine trial; virology

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. 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. Due to assay conditions not amenable to standard qHTS processes, 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 CM is being used to enrich an mRNA-encoded macrocyclic peptide library to identify novel CM modulators. 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. Targeting enzymes in one carbon metabolism. Dihydrofolate reductase (DHFR) is an essential enzyme in the folate pathway that catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid (THF), a cofactor important in several one-carbon transfer reactions which participate in amino acid and nucleobase synthesis. Methotrexate (MTX), a potent DHFR inhibitor, has been used for decades as a potent anti-cancer agent, as well as to treat autoimmune diseases like rheumatoid arthritis. MTX is a prodrug having a complex pharmacology, leading to reduced intracellular levels of THF cofactors, which results in inhibition of thymidylate, purine, and DNA biosynthesis. Mechanistically, MTX undergoes an enzyme catalyzed polyglutamylation which leads to irreversible intracellular accumulation of these analogs. Reduced THF cofactor concentrations result in DNA replication arrest. Unfortunately, therapeutic resistance to MTX is a significant clinical barrier. Mechanisms of resistance include DHFR amplification, decreased intracellular transport of MTX, decreased retention of MTX (due to lack of polyglutamylation), increased export of MTX (due to overexpression of some members of the ATP binding cassette transporters), mutated DHFR (resulting in weak binding to MTX), increased levels of lysosomal gamma-glutamyl hydrolase (leading to hydrolysis of MTX polyglutamates), and others. While many MTX analogs have been made over the years to improve its specificity and side-effect profile significantly improved versions have been elusive. New insights and technology is being employed to develop novel modulators of one carbon metabolism.

该项目包括基于与疾病相关的特定蛋白质或核酸靶标的生化和靶向检测的发展。分析设计是在分析和进展策略的背景下考虑的，以评估使用高通量筛选技术的大范围化合物类别。有一个强烈的强调方法开发研究，以提高分析和领先的发现效率。作为这些活动的补充，我们还探索和设计了复杂化学文库（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。以下是正在进行的：