Computation-assisted discovery of bioactive minor cannabinoids from hemp

该项目旨在对大麻中微量大麻素的潜在治疗益处进行机理研究。我们建议将人工智能集成到我们的工作流程中，以加速生物活性次要大麻素的发现。我们专注于慢性疼痛作为我们的非精神活性大麻素的治疗益处。大量文献表明，大麻中的萜酚类化合物可能具有治疗疼痛和其他由低级慢性炎症引起的疾病的潜力。传统的，经典的方法，以确定潜在的治疗来源于天然产物（NP）是费力和耗时的生物测定指导分馏。正如我们的可行性研究所显示的那样，我们用于发现生物活性纳米颗粒的人工智能辅助方法加速了发现管道。我们希望我们能成功地鉴定出以前没有在临床相关生物测定中研究过的生物活性大麻素。我们的具体目标是： 具体目标1：预测和验证CBD耗尽大麻提取物中单个大麻素在与伤害感受相关的细胞培养模型中的作用。哥伦比亚盆地生物科学公司经营着一家工业规模的CBD制造工厂，该工厂利用CBD从富含大麻素的大麻提取物中重结晶。我们将使用各种重结晶阶段的上清液（“母液”）作为我们在计算辅助发现生物活性物质中的次要大麻素的来源。我们将使用稳定过表达重组人TRPV 1的HEK-293细胞作为与伤害感受相关的细胞培养模型进行钙通量测定。我们还将测试FAAH/考克斯-2抑制活性，因为靶向内源性大麻素和内源性香草素系统的双重作用化合物正在成为慢性疼痛管理的新治疗选择。我们将通过在相同的试验中测试单个纯大麻素来验证最有效的活性预测因子。我们还将从我们的大麻素标准库中确定17种次要大麻素的生物活性，这些标准库可能包括也可能不包括我们发现管道中的生物活性预测因子。 具体目标2：预测和验证生物活性大麻素之间的协同/相互作用。我们的人工智能发现管道能够基于对含有生物活性大麻素的粗馏分中NP绝对浓度的测量来检测协同、相加或拮抗作用，我们将建立浓度-效应曲线。我们将量化真实大麻素的一个子集之间的药理学相互作用，包括来自我们的标准品库的17种次要大麻素，这些标准品在我们的试验中显示出生物活性，或者据报道可以减弱TRPV 1相关的疼痛信号。我们预计，所提出的人工智能辅助发现方法将产生减弱伤害性TRPV 1-FAAH/考克斯-1信号通路的大麻素候选物，用于疼痛感觉的临床前模型中的进一步机制测试。从CBD工业的废物中开发微量大麻素作为疼痛缓解剂，不仅有利于慢性疼痛患者，也有利于可持续的农业工业。

This project aims to conduct mechanistic research of potential therapeutic benefits of minor cannabinoids in hemp. We propose to integrate Artificial Intelligence in our workflow to accelerate the discovery of bioactive minor cannabinoids. We focus on chronic pain as our therapeutic benefit of non-psychoactive cannabinoids. A large body of literature suggests that terpenophenolics from hemp may have potential in the treatment of pain and other conditions that are driven by low-grade chronic inflammation. The conventional, classic approach to identify potential therapeutics derived from natural products (NPs) is laborious and time-consuming bioassay-guided fractionation. Our AI-assisted approach for the discovery of bioactive NPs accelerates the discovery pipeline, as our feasibility studies show. We expect that we will succeed in identifying bioactive cannabinoids that have previously not been studied in clinically relevant bioassays. Our Specific Aims are: Specific Aim 1: To predict and validate the effects of individual cannabinoids in CBD-depleted hemp extracts in cell culture models relevant to nociception. Columbia Basin Bioscience operates an industrial scale CBD manufacturing facility which makes use of recrystallization of CBD from cannabinoid-enriched hemp extracts. We will use the supernatants of various recrystallization stages (“mother liquors”) as our source of minor cannabinoids in our computation-assisted discovery of bioactives. We will perform calcium flux assays using HEK-293 cells stably over-expressing recombinant human TRPV1 as a cell culture model relevant to nociception. We will also test for FAAH/COX-2 inhibitory activity as dual acting compounds targeting the endocannabinoid and endovanilloid systems are emerging as a novel treatment option for chronic pain management. We will validate the most potent predictors of activity by testing individual, pure cannabinoids in the same assays. We will also determine the bioactivity of 17 minor cannabinoids from our repository of cannabinoid standards, which may or may not include predictors of bioactivity from our discovery pipeline. Specific Aim 2: To predict and validate synergistic/interactive effects between bioactive cannabinoids. Our AI-discovery pipeline has the ability to detect synergistic, additive, or antagonistic effects based on measurements of absolute concentrations of NPs in crude fractions containing bioactive cannabinoids for which we will establish concentration-effect curves. We will quantify the pharmacological interactions among a subset of authentic cannabinoids including 17 minor cannabinoids from our repository of standards which show bioactivity in our assays or which have been reported to attenuate TRPV1-related pain signaling. We expect that the proposed AI-assisted discovery approach will generate cannabinoid candidates that attenuate nociceptive TRPV1-FAAH/COX-1 signaling pathways for further mechanistic testing in preclinical models of pain sensation. The development of minor cannabinoids as pain mitigators from waste products of the CBD-industry will benefit not only chronic pain patients but also a sustainable agro-industry.