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

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.

治疗疼痛的一种有前途的方法是抑制被激活的外周疼痛感知神经元（伤害感受器）例如，局部麻醉剂对有害（引起疼痛）的刺激非常有效。不幸的是，局部麻醉剂可以阻止由周围出现的各种疼痛状况引起的疼痛。抑制所有感觉神经元和运动神经元我们的长期目标是识别特异性的新化合物。抑制伤害感受器但不影响其他感觉和运动神经元，尤其是那些外周限制（不进入中枢神经系统），将是有效且更安全的镇痛药的近期目标。该项目响应“检测开发和神经治疗药物鉴定”R61/R33 FOA，旨在开发一种体外测定法来筛选天然产物库中的新型选择性伤害感受器我们将设计具有比目前高得多的通量的抑制化合物。基因相关肽（CGRP）受体表达的“嗅探细胞”能够使用辅助检测伤害感受器活性。伤害感受器（源自炎症性疼痛模型的成年大鼠感觉神经节）和 CGRP 培养物嗅探细胞，我们将筛选天然产物库，寻找抑制刺激的伤害感受器活性的化合物。使用伤害感受器本身，作为新发现的化合物的直接目标，提供了强有力的生物学原理很可能鉴定出的化合物将是体内有效的镇痛剂。此外，由于哺乳动物伤害感受器之间的高度一致性，该测定将具有较高的翻译有效性该应用的目标是：目标 1(R61)，设计嗅探细胞对神经肽 CGRP Aim 2 (R61) 高度敏感，开发、验证和优化共培养测定（CGRP 嗅探细胞与源自 CFA 处理的大鼠的感觉神经元）评估已知化合物抑制生理炎症汤刺激的伤害感受器活性；和目标3（R33），筛选预- 来自 NCI 发展治疗计划 (DTP) 的分级天然产物库，用于鉴定抑制伤害感受器活性的新型化合物，来自 DTP 天然产物提取物库，目前由超过 326,000 个组分组成，将测试对激活的伤害感受器的选择性抑制使用 R61 阶段开发的测定法，阳性命中将是那些使伤害感受器活性降低 ≥ 25% 的结果。不改变嗅探细胞中的荧光（±CGRP），将进一步细化伤害感受器的阳性命中。通过对也抑制 SH-SY5Y 生物活性神经元活性的组分进行计数器筛选来实现选择性。将通过合作生物测定引导的分级分离过程从命中级分中鉴定化合物然后将评估这些纯化合物的效力、功效和伤害感受器特异性。在我们的分析中，我们期望筛选约 100,000 个分数（仅受资助的预算限制）确定 5-10 种具有不同化学结构的新化合物，这些化合物将进入 NIH 蓝图神经治疗网络 (PAR-20-122) 用于进一步开发以进行 1 期临床测试。