High content analgesic screening from human nociceptors

从人类伤害感受器中筛选高含量镇痛剂

• 批准号: 10578042
• 负责人: GREGORY O DUSSOR
• 金额: $ 37.02万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10578042
• 关键词: Absence of pain sensation; Action Potentials; Acute Pain; Address; Afferent Neurons; Agonist; Analgesics; Anatomy; Animals; Baclofen; Basic Science; Behavior; Biochemical; Biological Assay; Biology; Capsaicin; Cell physiology; Cells; Chili Pepper; Data; Development; Electrodes; Electrophysiology (science); Ethics; Evaluation; Event; Failure; Genes; Heterogeneity; Home; Homo sapiens; Human; In Vitro; Inflammatory; Interleukin 6 Receptor; Interleukin-6; Libraries; Measures; Methods; Modeling; Modernization; Molecular Target; Mus; National Cancer Institute; Natural Products; Natural Source; Neurons; Nociception; Nociceptors; Operative Surgical Procedures; Organ Donor; Organism; Pain; Paper; Patients; Peripheral; Pharmacology; Phase; Phenotype; Physiological; Physiology; Plants; Process Assessment; Proteins; Publishing; Rattus; Research; Rodent; Rodent Model; Sorting; Source; Speed; Spinal Ganglia; Stimulus; TRPV1 gene; Technology; Testing; Therapeutic; Translating; Translations; antagonist; chronic pain; cytokine; efficacy study; falls; high throughput screening; in vivo; induced pluripotent stem cell; inflammatory pain; inhibitor; innovation; marine organism; mouse model; multi-electrode arrays; novel; opioid epidemic; pain model; patch clamp; pharmacologic; pharmacophore; pre-clinical; repository; screening; single cell sequencing; success; tool; transcriptome; transcriptome sequencing; transcriptomics

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.

项目概要 阿片类药物危机促使人们认识到需要新的、非成瘾性镇痛药来治疗严重急性和慢性疼痛。 在过去的 25 年里，对这种新型镇痛药的研究只取得了一些成功，并且 现代镇痛筛查有两个缺点，我们的目标是通过筛查来纠正这些缺点。 首先，新型镇痛药的筛选通常依赖于易于检测的生化读数。 虽然这种方法具有优势（例如速度），但它没有考虑到高通量筛选（HTS）。 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 的功效研究依赖于啮齿动物的行为和细胞生理学，出于明确的伦理原因，啮齿动物。 功效研究已经完全有意义，然而，智人和小鼠之间的差异 生物体导致无法将令人兴奋的临床前目标转化为人类疼痛。这些机制。 随着包括我们自己在内的多个研究小组开始研究感官，失败现在正在被阐明 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”伤害感受器标记说明了进入人类的重要性 在开发新型镇痛药时尽早使用细胞在这项提案中，我们解决了上述两个问题。 通过筛选天然产物组分来降低人类诱发的过度兴奋的能力 过去，衍生的伤害感受器的前景并未实现。 根据我们的初步和已发表的数据，我们相信我们的细胞，RealDRGs， 在这个提案中，我们将结合这种丰富的、可复制的、可靠的人类来源。 “伤害感受器”采用尖端多电极阵列 (MEA) 技术来筛选天然产物组分 在过去的几年中，我们使用带有 MEA 的小鼠 DRG 来证明- 概念 (PoC) 镇痛筛查，包括记录和验证 Z’因子质量测定评估 我们将使用 R61 部分来确定 (1) RealDRG 电镀后的最佳稳定基线。 估计活性电极产量，(2) 外源敏化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 PoC 筛选 在达到我们的定量里程碑后，我们将进行。 R33 阶段的进展：(1) 筛选来自国家癌症研究所的天然产物级分，(2) 最终结果 在 RealDRG 和人类初级 DRG 中测试亚组分。