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

治疗疼痛的一种有希望的方法是抑制激活的外周痛觉神经元(伤害性感受器)。 对有害的(引起疼痛的)刺激作出反应。例如，局部麻醉剂在以下情况下非常有效 外周出现的各种疼痛状态引起的阻滞性疼痛。不幸的是，局麻药 抑制所有感觉神经元和运动神经元。我们的长期目标是确定新的化合物，特别是 抑制伤害性感受器，但保留其他感觉神经元和运动神经元。这类化合物，尤其是那些 外周受限(不要进入中枢神经系统)，将是有效和更安全的止痛药。眼前的目标是 本项目，响应于R61/R33《检测开发和神经治疗剂IDENTIfi阳离子》 FOA，正在开发一种体外试验来筛选新的和选择性的伤害感受器的天然产物文库 具有比目前可用的产量高得多的抑制化合物。我们将设计降钙素 表达“嗅觉细胞”的基因相关肽(CGRP)受体能够检测到伤害性感受器的活性。使用联合- 伤害性感受器(来源于炎性痛模型的成年大鼠感觉神经节)和CGRP的培养 对于嗅觉细胞，我们将筛选天然产物库中抑制刺激的伤害性感受器活动的化合物。这个 使用伤害性感受器本身，这是新发现的化合物的直接靶标，提供了一种强大的 生物学原理：所鉴定的化合物在体内是有效的止痛剂的可能性很高。 此外，由于哺乳动物伤害性感受器之间的高度一致性，该检测将具有很高的翻译有效性 对人类伤害性感受器的疗效。这项应用的目标是：目标1(R61)，工程嗅探器细胞， 对神经肽CGRP高度敏感；Aim 2(R61)，开发、验证和优化共培养试验 (来自CFA处理的大鼠感觉神经元的CGRP嗅探细胞)以评估已知的化合物 抑制由生理性炎症汤刺激的伤害性感受器活动；以及目标3(R33)，筛选出一种预 NCI开发治疗计划(DTP)的分级天然产物库以识别 抑制伤害感受器活性的新型化合物。DTP天然产物萃取库中的前级分， 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 使用在R61阶段开发的分析方法。积极命中是那些将伤害感受器活动减少≥25%的命中 不改变嗅觉细胞(±CGRP)的荧光。伤害性感受器的阳性命中将进一步细化 通过对也抑制SH-SY5Y神经元活性的部分进行计数器筛选来实现选择性。生物活性 将通过合作的生物测定指导的分级过程从HIT组分中鉴定出化合物 有了DTP。然后将对这些纯化合物的效力、有效性和伤害感受器特异性进行评估。 在我们的化验中。我们预计将筛选~100,000个部分(仅受拨款的预算限制) 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)的进一步发展，导致第一阶段的临床测试。

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.