In Vivo Screening of Mixture-Based Combinatorial Libraries

描述(由申请人提供)：我们的工作假设是，从基于混合物的组合库中直接在体内筛选和鉴定单个化合物将产生更先进的候选治疗方案，同时减少药物发现过程中固有的时间和成本。目前的药物发现筛选策略几乎总是涉及基于靶点的体外生化或基于细胞的分析。这些是鉴定活性化合物的主要手段，下一步在动物研究中评估它们的活性。正是在这个较晚的阶段，大多数化合物由于毒性、缺乏效力和生物利用度较差而失败。 为了支持我们的工作假设，利用小鼠甩尾疼痛模型进行的成功的初步研究表明，可以在活性混合物和非活性混合物之间实现明显的区分。例如，在甩尾试验中发现由125,000个四肽组成的混合物，由三个位置的50个不同的氨基酸组成，只有其N-末端位置被单独定义，具有抗伤害感受活性。这种混合物(含有DMT-DALDA；已知在体内具有活性)的活性比吗啡的作用时间长5-10倍，而每毫克的活性仅比吗啡低3-5倍。此外，在u、Delta或kappa阿片结合试验中选择的其他没有活性的混合物被发现在体内具有明显的甩尾活性。这增加了一种令人兴奋的可能性，即新的受体位置或多个受体相互作用是所发现的活动的原因。 这项建议中的两个目标将作为这一方法的一般概念证明，并将为以后利用一系列现有的杂环混合物库进行研究奠定基础。第一个目标将使用甩尾试验来完成迭代去卷积过程，以确定这种活性混合物的三个位置上最活跃的氨基酸。我们认为，这将使得能够识别相对于DMT-DALDA具有增强活性的活性个体序列，或者至少将识别DMT-DALDA(两者都是概念最终结果的可接受的证据)。第二个目标是在体内筛选组成这个由6,250,000个不同序列(50×503)组成的四肽文库的全部50个混合物。除了识别新的阿片类特定激动剂外，这种方法还可以识别新的抗伤害性化合物，这些化合物对“孤儿”疼痛调制非阿片受体具有活性。如果成功，基于混合物的组合库的直接体内测试不仅将促进疼痛调制，而且将推动生物医学研究和药物发现过程。

DESCRIPTION (provided by applicant): Our working hypothesis is that the direct in vivo screening and identification of individual compounds from mixture-based combinatorial libraries will yield more "advanced" therapeutic candidates, while decreasing the time and costs inherent in the drug discovery process. Current drug discovery screening strategies virtually always involve target based in vitro biochemical or cell-based assays. These serve as the primary means to identify active compounds that are next assessed for their activity in animals studies. It is at this later stage that the majority of compounds fail due to toxicity, lack of efficacy, and poor bioavailability. In support of our working hypothesis, successful preliminary studies utilizing the murine tail flick pain model have demonstrated that clear differentiation can be achieved between active and inactive mixtures. For example, a mixture of 125,000 tetrapeptides, made up of 50 different amino acids at three positions and with only its N-terminal position individually defined, was found to have antinociceptive activity in the tail flick assay. The activity of this mixture (that contained Dmt-DALDA; known to be active in vivo) had a 5 to 10 time longer duration of action than morphine, while being only 3-5 times less active than morphine on a per mg basis. Additionally, other mixtures chosen that had no activity in the mu, delta or kappa opioid binding assays were found that had clear in vivo tail flick activity. This raises the exciting possibility that novel receptor sites or multiple receptor interactions are responsible for the activities found. The two Aims in this proposal will serve as a general proof of concept for this approach and will lay the foundation for later studies with a range of existing heterocyclic mixture-based libraries. The first Aim will use the tail flick assay to complete an iterative deconvolution process to identify the most active amino acids at the three positions of this active mixture. We believe that this will enable the identification of active individual sequences that have enhanced activity relative to Dmt-DALDA or, as a minimum, will identify Dmt-DALDA (both are acceptable proof of concept end results). The second Aim involves the in vivo screening of the entire 50 mixtures making up this tetrapeptide library of 6,250,000 different sequences (50 x 503). In addition to identifying novel opioid specific agonists, this approach may enable the identification of novel antinociceptive compounds that have activity at "orphan" pain modulating non-opioid receptors. If successful, the direct in vivo testing of mixture-based combinatorial libraries will advance not only pain modulation, but biomedical research and the drug discovery process in general.