Synthesis and in vitro and in vivo screening of fused and tethered heterocyclic peptidomimetics for the discovery of new analgesics with decreased side effects

融合和束缚杂环肽模拟物的合成以及体外和体内筛选，以发现副作用减少的新型镇痛药

• 批准号: 10297832
• 负责人: Jay P. McLaughlin
• 金额: $ 29.71万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-11 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10297832
• 关键词: Absence of pain sensation; Acetic Acids; Address; Affinity; Agonist; Amino Acids; Analgesics; Area; BCL2L11 gene; Behavior assessment; Binding; Binding Proteins; Biological; Biological Assay; Biological Availability; Blood; Blood - brain barrier anatomy; Brain; Clinical; Constipation; Cyclic AMP; Data; Development; Dose; Drug Kinetics; Evaluation; Exhibits; Failure; Generations; Goals; Harvest; Heterocyclic Compounds; Imidazolidines; Imines; In Vitro; Lead; Lead levels; Libraries; Liver; Mass Spectrum Analysis; Measurement; Mediating; Metabolic; Molecular Probes; Morphine; Motor Activity; Mus; Naloxone; Opiate Addiction; Opioid; Opioid Analgesics; Opioid Antagonist; Opioid Receptor; Opioid agonist; Oral; Oral Administration; Outcome; Pain; Parents; Peptides; Peripheral; Permeability; Physiological; Piperazines; Plasma Proteins; Property; Research; Rewards; Sedation procedure; Series; Side; Signal Transduction; Suggestion; System; Tail; Testing; Time; Ventilatory Depression; Vertebral column; Water; Withdrawal; abuse liability; antagonist; aqueous; base; beta-arrestin; computational chemistry; conditioned place preference; design; gastrointestinal epithelium; imidazolone; improved; in vivo; innovation; interest; intravenous administration; kappa opioid receptors; liquid chromatography mass spectrometry; novel; peptidomimetics; radioligand; receptor; recruit; respiratory; scaffold; screening; side effect; small molecule; therapeutic opioid

We hypothesize that the development of peripherally-restricted, mixed-activity opioid receptor agonists will produce robust analgesia while exhibiting reduced side effects, including a lack of respiratory depression or reinforcing properties. In preliminary studies, the in vitro screening of novel bis-imidazolidines and fused heterocyclic lead compounds BIM-22 and FDC-14 identified mixed-opioid receptor agonist activity. After peripheral (i.p.) administration to mice, both compounds demonstrated antinociception equivalent to morphine in the 55oC warm-water tail withdrawal (tail-flick) assay, but FDC-14 was 25 times more potent than morphine in the acetic acid writhing test, with an antinociceptive potency ratio of 72.7, suggestive of peripherally- restricted activity. Confirming this, LC-MS/MS studies detected BIM-22 and FDC-14 in harvested mouse blood, but not brain, after i.v. administration. This proposal addresses two research areas of particular interest for this FOA: i) The development of new and innovative molecular probes for receptors and new strategies for innovative peptidomimetic design, and ii) The identification of structurally diverse, orally active, metabolically stable peripherally-restricted opioid agonists. We propose six interacting aims: In Aim 1, we propose the computationally guided synthesis of heterocyclic peptidomimetics: bis-imidazolidin-2-imines, piperazines, bis- piperazine, bis-imidazolone and fused heterocyclic libraries. In Aim 2, we will screen all compounds in competition radioligand binding assays to determine affinity for μ- (MOR), δ- (DOR), and κ- (KOR) opioid receptors, and in functional assays to identify dual δ/κ or dual δ/µ agonists. In Aim 3, ten selected compounds will be evaluated in vitro for pharmacokinetic properties and screened in vivo for antinociception using the mouse tail-flick assay. Lead compounds identified from this aim will guide 2nd generation SAR studies to enhance peripherally-selective activity. In Aim 4: we will perform full in vivo antinociceptive characterization of lead agonist compounds. The most stable, active 4 agonists not crossing the BBB in Aims 2+3 will be evaluated after i.p. administration with mouse tail-flick and acetic-acid writhing assays for efficacy, duration of action, and opioid receptor selectivity. In Aim 5: we will characterize the bioavailability in mice of the selected 4 agonists following oral administration and confirm their inability to penetrate the blood brain barrier. In Aim 6: The two most potent bioavailable novel agonists identified in Aims 1-5 will be examined for liabilities, specifically antinociceptive tolerance, respiratory and hyperlocomotor effects in the CLAMS physiological and behavioral assessment system, sedation and disruption of coordinated locomotor activity in the rotorod assay, and assessment for rewarding or aversive effects in the conditioned place preference (CPP) assay. Effects on GI transit will also be evaluated to assess effects on constipation. In summary, we expect to generate novel peripherally-restricted, mixed-activity peptidomimetic opioid receptor agonists as both probes and analgesics with reduced side effects, thereby significantly impacting analgesic development.

我们假设外周限制性混合活性阿片受体激动剂的开发将 产生强大的镇痛作用，同时减少副作用，包括缺乏呼吸抑制或 增强性能。在初步研究中，新型双咪唑啉和融合的体外筛选 杂环先导化合物 BIM-22 和 FDC-14 鉴定出混合阿片受体激动剂活性。后 对小鼠进行外周（腹腔注射）给药，两种化合物均表现出与吗啡相当的镇痛作用 在 55oC 温水缩尾（甩尾）测定中，FDC-14 的效力比吗啡强 25 倍 在醋酸扭体试验中，抗伤害效力比为 72.7，提示外周- 限制活动。 LC-MS/MS 研究在采集的小鼠血液中检测到 BIM-22 和 FDC-14，证实了这一点， 但在静脉注射后，大脑就没有了。行政。该提案涉及对此特别感兴趣的两个研究领域 FOA：i) 开发新型创新的受体分子探针和新的受体策略 创新的拟肽设计，以及 ii) 结构多样性、口服活性、代谢活性的鉴定 稳定的外周限制性阿片受体激动剂。我们提出了六个相互作用的目标： 在目标 1 中，我们提出 杂环肽模拟物的计算引导合成：双咪唑烷-2-亚胺、哌嗪、双- 哌嗪、双咪唑酮和稠合杂环文库。在目标 2 中，我们将筛选以下所有化合物 竞争放射性配体结合测定，以确定 μ- (MOR)、δ- (DOR) 和 κ- (KOR) 阿片类药物的亲和力 受体，并在功能测定中识别双 δ/κ 或双 δ/μ 激动剂。在目标 3 中，选择了 10 种化合物 将在体外评估药代动力学特性，并使用以下方法在体内筛选抗伤害作用 小鼠甩尾实验。由此目标确定的先导化合物将指导第二代 SAR 研究 增强外周选择性活性。在目标 4 中：我们将进行完整的体内抗伤害表征 铅激动剂化合物。在目标 2+3 中不穿过 BBB 的最稳定、最活跃的 4 激动剂将是 腹腔注射后评估通过小鼠甩尾和醋酸扭体试验来测定给药效果、持续时间 作用和阿片受体选择性。在目标 5 中：我们将表征所选 4 种药物在小鼠中的生物利用度 口服给药后的激动剂并证实它们无法穿透血脑屏障。目标 6： 将检查目标 1-5 中确定的两种最有效的生物利用度新型激动剂的责任， 特别是蛤蜊生理和运动中的抗伤害耐受性、呼吸和运动亢进作用 行为评估系统、转子试验中的镇静和协调运动活动的破坏， 以及在条件性位置偏好（CPP）测定中评估奖励或厌恶效应。对的影响 还将评估胃肠道运输以评估对便秘的影响。总之，我们期望产生新颖的 外周限制性混合活性肽模拟阿片受体激动剂既作为探针又作为镇痛药 副作用减少，从而显着影响镇痛药的开发。

项目成果

Bis-Cyclic Guanidine Heterocyclic Peptidomimetics as Opioid Ligands with Mixed μ-, κ- and δ-Opioid Receptor Interactions: A Potential Approach to Novel Analgesics.

• DOI: 10.3390/ijms23179623
• 发表时间: 2022-08-25
• 期刊: International journal of molecular sciences
• 影响因子: 5.6