Development of a phenotypic screening assay for novel compounds that inhibit peripheral pain-sensing neurons

开发抑制外周痛觉神经元的新型化合物的表型筛选试验

基本信息

批准号：
10650640
负责人：
WILLIAM P CLARKE
金额：
$ 43.8万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-17 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10650640
关键词：
Adult Adverse effects Affect Afferent Neurons Analgesics Biological Biological Assay Calcitonin Gene-Related Peptide Calcitonin-Gene Related Peptide Receptor Calcium Cells Chemical Structure Chemicals Chemistry Coculture Techniques Collaborations Development Developmental Therapeutics Program Drug Screening Effectiveness Engineering Fluorescence Fractionation Genetic Engineering Goals Grant Human Human Resources Inflammatory Libraries Life Ligands Local Anesthetics Measurement Mediating Medical Methods Monitor Motor Motor Neurons National Center for Complementary and Integrative Health Natural Compound Natural Products Neurons Neuropeptides Nociceptors Non-Steroidal Anti-Inflammatory Agents Opioid Pain Peripheral Persons Pharmaceutical Preparations Phase Phenotype Physiological Process Rattus Reporting Screening procedure Sensory Sensory Ganglia Specific qualifier value Specificity Spinal Ganglia Stimulus System Testing Training United States National Institutes of Health Work antinociception assay development base cell type counterscreen daily pain density experience high throughput screening in vitro Assay in vivo in vivo evaluation inflammatory pain innovation interest novel pain inhibition pain model peripheral pain research clinical testing response screening

Adult Adverse effects Affect Afferent Neurons Analgesics Biological Biological Assay Calcitonin Gene-Related Peptide Calcitonin-Gene Related Peptide Receptor Calcium Cells Chemical Structure Chemicals Chemistry Coculture Techniques Collaborations Development Developmental Therapeutics Program Drug Screening Effectiveness Engineering Fluorescence Fractionation Genetic Engineering Goals Grant Human Human Resources Inflammatory Libraries Life Ligands Local Anesthetics Measurement Mediating Medical Methods Monitor Motor Motor Neurons National Center for Complementary and Integrative Health Natural Compound Natural Products Neurons Neuropeptides Nociceptors Non-Steroidal Anti-Inflammatory Agents Opioid Pain Peripheral Persons Pharmaceutical Preparations Phase Phenotype Physiological Process Rattus Reporting Screening procedure Sensory Sensory Ganglia Specific qualifier value Specificity Spinal Ganglia Stimulus System Testing Training United States National Institutes of Health Work antinociception assay development base cell type counterscreen daily pain density experience high throughput screening in vitro Assay in vivo in vivo evaluation inflammatory pain innovation interest novel pain inhibition pain model peripheral pain research clinical testing response screening

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项目摘要

A promising approach to treat pain is to inhibit peripheral pain-sensing neurons (nociceptors) that are activated in response to noxious (pain-causing) stimuli. For example, local anesthetics are extremely efficacious at blocking pain elicited by a variety of pain conditions that arise in the periphery. Unfortunately, local anesthetics inhibit all sensory, as well as motor, neurons. Our long-term goal is to identify new compounds that specifically inhibit nociceptors but spare other sensory and motor neurons. Such compounds, especially those that are peripherally restricted (do not enter the CNS), would be effective and safer analgesics. The immediate goal of this project, in response to the “Assay Development and Neurotherapeutic Agent Identiﬁcation” R61/R33 FOA, is to develop an in vitro assay to screen natural product libraries for novel and selective nociceptor inhibitory compounds with much higher throughput than is currently available. We will engineer calcitonin gene-related peptide (CGRP) receptor expressing “sniffer cells” able to detect nociceptor activity. Using co- cultures of nociceptors (derived from adult rat sensory ganglia from an inflammatory pain model) and CGRP sniffer cells, we will screen natural product libraries for compounds that inhibit stimulated nociceptor activity. The use of nociceptors themselves, that are the direct target for newly identified compounds, provides a strong biological rationale with high likelihood that compounds identified will be efficacious analgesic agents in vivo. Further, due to high congruence between mammalian nociceptors, the assay will have high translational validity for efficacy at human nociceptors. The Aims for this application are: Aim 1(R61), engineer sniffer cells that are highly sensitive to the neuropeptide, CGRP; Aim 2 (R61), develop, validate and optimize a co-culture assay (CGRP sniffer cells with sensory neurons derived from rats treated with CFA) to assess known compounds that inhibit nociceptor activity stimulated by a physiological inflammatory soup; and Aim 3 (R33), screen a pre- fractionated natural product library from the Developmental Therapeutics Program (DTP) of the NCI to identify novel compounds that inhibit nociceptor activity. Pre-fractions from the DTP natural products extract library, which currently consists of over 326,000 fractions, will be tested for selective inhibition of activated nociceptors using the assay developed in the R61 phase. Positive hits will be those that reduce nociceptor activity by ≥ 25% without altering fluorescence in sniffer cells (±CGRP) alone. Positive hits will be further refined for nociceptor selectivity by performing a counter screen for fractions that also inhibit the activity of SH-SY5Y neurons. Bioactive compounds will be identified from hit fractions through a process of bioassay-guided fractionation in collaboration with the DTP. These pure compounds will then be assessed for their potency, efficacy and nociceptor specificity in our assay. We expect to screen ~100,000 fractions (limited only by the budgetary restrictions of the grant) to identify 5-10 novel compounds with diverse chemical structures that will enter the NIH Blueprint Neurotherapeutics Network (PAR-20-122) for further development leading to phase 1 clinical testing.

一种有前途的治疗疼痛的方法是抑制被激活的外周疼痛神经元（伤害感受器）响应有害的（引起疼痛）刺激。例如，局部麻醉效率非常有效在周围出现的多种疼痛状况引起的疼痛。不幸的是，局部麻醉剂抑制所有感觉以及运动神经元。我们的长期目标是确定专门的新化合物抑制伤害感受器，但要避免其他感觉和运动神经元。这样的化合物，尤其是那些外围受限（不要进入中枢神经系统）将是有效且安全的镇痛药。直接目标该项目回应“测定开发和神经治疗剂识别” R61/R33 FOA是为了开发一种体外测定，以筛选自然产品库的新颖和选择性伤害感受器吞吐量的抑制性化合物比当前可用。我们将设计降钙素表达“嗅探细胞”的基因相关肽（CGRP）受体可以检测伤害感受器的活性。使用共同伤害感受器的培养物（源自炎症模型的成年大鼠感觉神经节）和CGRP Sniffer细胞，我们将筛选自然产物文库的化合物，以抑制受刺激的伤害感受器活性。使用伤害感受器本身，这是新鉴定的化合物的直接目标，可提供强大鉴定出的化合物的生物学原理具有很高的可能性是体内有效的镇痛药。此外，由于哺乳动物伤害感受器之间的一致性很高，该测定法将具有较高的翻译有效性在人类的伤害感受器上轻松。该应用程序的目的是：AIM 1（R61），工程师Sniffer单元格对神经肽CGRP高度敏感； AIM 2（R61），开发，验证和优化共培养测定法（CGRP嗅探细胞的感官神经元来自用CFA处理的大鼠），以评估已知化合物通过身体炎症汤刺激的伤害感受器的活性；和AIM 3（R33），屏幕 NCI的发展性治疗计划（DTP）的分离天然产品库以识别新型化合物抑制伤害感受器活性。 DTP天然产品提取文库的fr骨前二十一座目前由326,000多个分数组成，将测试以选择性抑制激活的伤害感受器使用在R61阶段开发的测定。积极的命中将是那些将伤害感受器活动降低≥25％的命中不用仅改变嗅觉细胞（±CGRP）的荧光。对于伤害感受器，将进一步精制命中率通过对隔离率进行计数器屏幕的选择性，从而抑制SH-SY5Y神经元的活性。生物活性通过合作的生物测定指导分馏的过程，将从HIT分数中识别化合物与DTP。然后，将评估这些纯化合物的效力，效率和伤害感受器的特异性在我们的测定中。我们预计将筛选约100,000个分数（仅受赠款的预算限制限制）识别5-10种具有多种化学结构的新型化合物，将进入NIH蓝图神经疗法网络（PAR-20-122）用于进一步开发，导致1阶段临床测试。