High content analgesic screening from human nociceptors

项目摘要 阿片类药物危机促使人们需要新的非成瘾性镇痛药来治疗严重的急性和 慢性疼痛在过去的25年里，对这种新型镇痛药的研究只有少数成功， 许多失败。现代镇痛药筛查有两个缺点，我们的目标是通过筛查来纠正 比色法首先，新型镇痛药的筛选通常依赖于易于检测的生化读数。 高通量筛选（HTS）。虽然这种方法具有优点（例如，速度），它不占 伤害感受器感觉神经元生物学的复杂性和异质性。第二，许多新的镇痛靶点 HTS的疗效研究依赖于啮齿动物行为和细胞生理学。出于明确的道德原因，啮齿动物 有效性研究已经完全有意义，然而，人类和小鼠之间的差异 生物体导致无法将令人兴奋的临床前靶点转化为人类疼痛。这些机制 随着多个研究小组，包括我们自己的研究小组， 原代人背根神经节（DRG）的神经元生理学和药理学。表达差异 小鼠、大鼠和人类之间的“标准”伤害感受器标记物说明了进入人类的重要性。 在开发新型镇痛药的过程中尽早使用细胞。在本提案中，我们解决了上述两个问题 通过对天然产物级分进行筛选，以确定它们降低人类诱导的过度兴奋性的能力， 多能干细胞衍生的伤害感受器。在过去，衍生性伤害感受器的承诺没有实现。 人类伤害感受器复制根据我们的初步和已发表的数据，我们相信我们的细胞，RealDRG， 真的是“伤害感受器”在这个提议中，我们将联合收割机这一丰富的、可复制的和可靠的人类来源 “伤害感受器”与尖端的多电极阵列（MEA）技术，以筛选天然产物馏分， 镇痛样作用。在过去的几年里，我们使用小鼠DRG和MEA来证明- 概念（CRH）镇痛剂筛选，包括记录和验证Z '因子测定质量评估 过程我们将使用R61部分来确定（1）RealDRG铺板后的最佳稳定基线， 估计活性电极产率，（2）外源性致敏后放电速率的稳定性，（3）Z'因子， （4）已知伤害感受器抑制剂的筛选。在达到我们的量化里程碑后，我们将 R33阶段的进展：（1）从国家癌症研究所筛选天然产物级分，（2）最终 在RealDRG和人类原发性DRG中检测亚组分。

PROJECT SUMMARY The opioid crisis has driven home the need for new, non-addictive analgesics to treat both severe acute and chronic pain. Over the past 25 years, the search for such novel analgesics has had only a few successes and many failures. There are two drawbacks of modern analgesic screening that we aim to correct with our screening assay. First, screening for novel analgesics typically relies on a biochemical readout that is readily amenable to high throughput screening (HTS). While such an approach has advantages (e.g., speed), it does not account for the complexity and heterogeneity of nociceptor sensory neuron biology. Second, many new analgesic targets and efficacy studies from HTS rely on rodent behavior and cellular physiology. For clear ethical reasons, rodent efficacy studies have made complete sense, and yet, the differences between homo sapiens and murine organisms have led to a failure to translate exciting preclinical targets to human pain. The mechanisms for these failures are now being illuminated as multiple research groups, including our own, have begun to study sensory neuron physiology and pharmacology in primary human dorsal root ganglia (DRG). Differences in expression of “standard” nociceptor markers between mice, rats, and humans illustrate the importance of moving into human cells as early as possible in the development of novel analgesics. In this proposal, we address both issues above by performing screening of natural product fractions for their ability to reduce hyperexcitability in human induced pluripotent stem cell-derived nociceptors. In the past, the promise of derived nociceptors has fallen short of true human nociceptor replication. Based on our preliminary and published data, we believe that our cells, RealDRGs, truly are “nociceptors.” In this proposal, we will combine this abundant, replicable, and reliable source of human “nociceptors” with cutting-edge multi-electrode array (MEA) technology to screen natural product fractions for analgesic-like effects. Over the last several years, we have used mouse DRGs with MEAs to show proof-of- concept (PoC) analgesic screening including documenting and validating a Z’-factor assay quality assessment process. We will use the R61 portion to determine (1) the optimal stable baseline after plating of RealDRGs to estimate the active electrode yield, (2) the stability in firing rate after exogenous sensitization, (3) the Z’ factor, and (4) PoC screening of known nociceptor inhibitors. Upon reaching our quantitative milestones, we will then progress in the R33 phase to (1) screen natural product fractions from the National Cancer Institute with (2) final testing of subfractions in RealDRGs and in human primary DRGs.