MISSISSIPPI COBRE: CORE C: IN VITRO PHARMACOLOGY CORE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 体外药理学核心筛选化合物与特定目标相互作用的能力。主要靶点是神经递质和神经调节剂的神经元受体和转运体，这些神经递质和神经调节剂介导神经精神效应。核心还筛选化合物与配体无关的离子通道相互作用的能力。核心也被设计用来评估化合物在它们相互作用的部位的功能，例如，评估它们是否具有激动性或拮抗性。这类检测包括评估GTP-GammaS结合、离子转运，以及cAMP和三磷酸肌醇形成的检测。 到目前为止，核心用来测试提交给核心的样本的主要方法是使用针对感兴趣目标的放射性配基的竞争结合分析，例如针对Mu阿片受体的~3H-DAMGO。今后，该核心还将利用非放射性方法。在可能的情况下，核心使用人类受体的膜制剂导入细胞系。然而，当没有这样的受体时，可以使用从动物来源获得的合适的膜制剂。评估样品与目标的相互作用包括1到3个步骤。最初的步骤是初步筛选，在其中测试单个浓度(例如，化合物为10M和10 mg/ml的提取物或分离部分)竞争与靶点的放射性配体结合的能力。如果一次筛选显示放射性配体的特异性结合抑制超过50%，则进行二次筛选。二次筛以对数单位间隔使用4到6个浓度的样品，范围从10？M到100 PM。通过使用单点竞争方程(棱镜、Graphpad软件)的非线性回归分析来评估竞争曲线以确定IC50值。基于对放射性配基的饱和结合动力学的同时评估，将IC50值转换为KI值。对于更详细的分析，例如对于看起来不具有Hilll斜率=1或可能显示不同亲和力的放射性配体，如上所述运行12点竞争结合分析，并且或者使用希尔系数不设置为1的单位点分析，或者使用多结合位点分析来使用棱镜来拟合数据。 到目前为止，Core已经对Mu、Delta和Kappa阿片受体以及CB1大麻受体进行了分析。正在开发中的是CB2、OFQ受体和钙信号的分析。其他的受体、离子通道和转运体结合分析正在计划阶段。 这些核心职能涉及使用最先进的技术以及经过时间考验的实验室方法。使用多标签阅读器技术和放射性结合技术，建立核心，以快速评估在密西西比大学国家天然产品研究中心的储存库(核心A)中持有的化合物和提取物的特定活性，以及化学核心(核心B)合成的化合物的特定活性。这些研究可以在从动物或培养细胞中获取的组织上进行。对提取物及其化合物作用的机理研究可以使用生化动力学分析以及细胞电生理学和成像技术进行评估，这些技术将在第二年增加。这种探索能力是通过四个核心功能组件实现的：细胞培养设备、功能测试设备、生物化学、受体药理学和组织学设备，以及即将增加的电生理学和成像设备。体外药理学核心今年已经购买了大量的设备，包括带有12个PMT读取器的PerkinElmer Topcount和带有分配单元的PerkinElmer enVision多标签读取器。该中心还为细胞培养设施购买了细胞培养孵化器和层流流罩，并购买了电子多通道吸管，以方便微孔板分析。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The In Vitro Pharmacology Core screens compounds for their ability to interact with specific targets of interest. The primary targets are neuronal receptors and transporters for neurotransmitters and neuromodulators that mediate neuropsychiatric effects. The core also screens compounds for their ability to interact with ligand-independent ion channels. The core is also designed to assess functionality of compounds at the sites where they interact, e.g., to assess whether they have agonistic or antagonistic properties. Such assays include assessment of GTPgammaS binding, ion transport, and assays for cAMP and inositol trisphosphate formation. The primary methodology employed by the core thus far to test samples submitted to the core is by competition binding assays using radioligands specific for targets of interest e.g., 3H-DAMGO for mu opioid receptors. In the future the core will also make use of non-radioactive methodologies. When possible, the core uses membrane preparations of human receptors transfected into cell lines. However, when such receptors are not available suitable membrane preparations obtained from animal sources are used. Evaluation of samples for interactions with targets involves 1 to 3 steps. The initial step is a primary screen in which a single concentration (e.g. 10 ¿M for compounds and 10 mg/ml of extract or isolated fraction) is tested for its ability to compete for radioligand binding to the target site. If the primary screen reveals more than 50% inhibition of specific binding of the radioligand, a secondary screen is carried out. The secondary screen uses 4 to 6 concentrations of the sample ranging from 10 ¿M down to 100 pM at log unit intervals. The competition curve is evaluated to determine an IC50 value by non-linear regression analysis using a one-site competition equation (Prism, Graphpad Software). The IC50 value is converted to a KI value based on simultaneous evaluation of the saturation binding kinetics of the radioligand. For more detailed analyses, e.g., for radioligands that do not appear to have Hilll slopes = 1 or which may show different affinities, 12 point competition binding assays are run as noted above and either one-site with Hill coefficient not set at one, or multiple binding site analyses are used to fit the data using Prism. To date, the Core has assays for mu, delta and kappa opioid receptors, and CB1 cannabinoid receptors. In development are assays for CB2, and OFQ receptors and Ca2+ signaling. Additional receptor, ion channel, and transporter binding assays are in the planning stage. These core functions involve the use of state of the art technologies as well as time-tested laboratory methodologies. Using multi-label reader technologies and radioactive binding techniques, the core is set up to rapidly assess specific activities of compounds and extracts held in the repositories (Core A) at the University of Mississippi National Center for Natural Products Research and of compounds synthesized by the Chemistry Core (Core B). These studies can be performed on tissues harvested from animals or from cultured cells. Mechanistic studies into the actions of extracts and their compounds can be assessed using biochemical kinetic analyses as well as cellular electrophysiology and imaging techniques, which will be added in the 2nd year. This exploratory ability is accomplished through the cores four functional components; cell culture facility, functional testing facility, biochemistry, receptor pharmacology and histology facility, and the soon to be added electrophysiology and imaging facility. The In vitro Pharmacology Core has made significant equipment purchases this year including a PerkinElmer Topcount with 12 PMT readers and a PerkinElmer EnVision multilabel reader with a dispensing unit. The core has also purchased cell culture incubators and laminar flow hoods for the cell culture facility and electronic multichannel pipettors to facilitate microplate assays.