In Vivo Screening of Mixture-Based Combinatorial Libraries

基于混合物的组合文库的体内筛选

基本信息

批准号：
7477311
负责人：
Richard Allen Houghten
金额：
$ 26.75万
依托单位：
TORREY PINES INST FOR MOLECULAR STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2010-07-31
项目状态：
已结题

项目摘要

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.

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体内筛选和鉴定单个化合物将产生更“先进”的候选治疗药物，同时减少药物发现过程中固有的时间和成本。当前的药物发现筛选策略实际上总是涉及基于靶标的体外生化或基于细胞的测定。这些是鉴定活性化合物的主要手段，接下来在动物研究中评估其活性。正是在这个后期阶段，大多数化合物因毒性、缺乏功效和生物利用度差而失败。为了支持我们的工作假设，利用小鼠甩尾疼痛模型的成功初步研究表明，可以在活性和非活性混合物之间实现清晰的区分。例如，125,000 个四肽的混合物由三个位置的 50 个不同氨基酸组成，并且仅单独定义其 N 端位置，在甩尾测定中发现具有抗伤害活性。该混合物（含有 Dmt-DALDA；已知在体内具有活性）的活性比吗啡长 5 至 10 倍，而每毫克的活性仅比吗啡低 3-5 倍。此外，发现在 mu、delta 或 kappa 阿片类药物结合测定中没有活性的其他所选混合物具有明显的体内甩尾活性。这提出了令人兴奋的可能性，即新的受体位点或多种受体相互作用是所发现的活性的原因。该提案中的两个目标将作为该方法的一般概念证明，并将为后续研究一系列现有的基于杂环混合物的库奠定基础。第一个目标将使用甩尾测定来完成迭代解卷积过程，以识别该活性混合物的三个位置处最活跃的氨基酸。我们相信，这将能够识别相对于 Dmt-DALDA 具有增强活性的活性个体序列，或者至少能够识别 Dmt-DALDA（两者都是可接受的概念验证最终结果）。第二个目标涉及对组成这个包含 6,250,000 个不同序列 (50 x 503) 的四肽文库的全部 50 种混合物进行体内筛选。除了鉴定新型阿片类特异性激动剂之外，该方法还可以鉴定具有“孤儿”疼痛调节非阿片受体活性的新型抗伤害化合物。如果成功，基于混合物的组合文库的直接体内测试不仅将推进疼痛调节，而且还将推进生物医学研究和总体药物发现过程。