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

• 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
• 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

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.

一种有希望的治疗疼痛的方法是抑制被激活的外周疼痛感受神经元（伤害感受器） 对有害（引起疼痛）刺激的反应。例如，局部麻醉剂在 阻断由外周出现的各种疼痛状况引起的疼痛。不幸的是局部麻醉剂 抑制所有感觉和运动神经元。我们的长期目标是确定新的化合物， 抑制伤害感受器，但不影响其他感觉和运动神经元。这些化合物，特别是那些 外周限制（不进入中枢神经系统），将是有效和更安全的镇痛药。这一近期目标 本项目，响应“检测开发和神经治疗剂鉴定”R61/R33 FOA的目的是开发一种体外试验来筛选天然产物库中的新型和选择性伤害感受器 抑制性化合物具有比目前可用的高得多的通量。我们会制造降钙素 基因相关肽（CGRP）受体表达“嗅探细胞”能够检测伤害感受器活性。使用co- 伤害感受器（来自炎性疼痛模型的成年大鼠感觉神经节）和CGRP的培养物 嗅探细胞，我们将筛选天然产物库的化合物，抑制刺激伤害感受器的活动。的 伤害感受器本身是新鉴定的化合物的直接靶标， 生物学原理，所鉴定的化合物很可能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，该测定将具有高翻译有效性 对人类伤害感受器的功效。该应用程序的目标是：目标1（R61），工程嗅探器细胞， 对神经肽CGRP; Aim 2（R61）高度敏感，开发、验证和优化共培养试验 (CGRP用来自CFA处理的大鼠的感觉神经元的嗅探细胞）来评估已知的化合物， 抑制由生理炎症汤刺激伤害感受器活性;和Aim 3（R33），筛选前 来自NCI的开发治疗学计划（DTP）的分级天然产物库，以鉴定 抑制伤害感受器活性的新化合物。来自DTP天然产物提取物文库的前级分， 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作用。 使用在R61阶段开发的测定法。阳性命中将是使伤害感受器活性降低≥ 25%的那些命中 而不改变单独的嗅探细胞（±CGRP）中的荧光。正命中将进一步完善伤害感受器 通过对也抑制SH-SY 5 Y神经元活性的级分进行计数筛选来确定选择性。生物活性 将通过生物测定指导的分馏过程，从命中馏分中鉴定化合物 在DTP。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 在我们的试验中。我们预计筛选~ 100，000个片段（仅受赠款预算限制的限制）， 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 神经治疗学网络（PAR-20-122）进行进一步开发，以进行1期临床试验。