Assessing the polypharmacology of kinase inhibitors by transcription factor activity profiling

摘要 评价蛋白激酶抑制剂（PKI）的生物活性是目前研究中最具挑战性的问题之一。 药物开发在这里，我们提出了一种新的评估方法（ATTAKIN™），我们推断PKI 来自细胞信号应答的生物活性。作为读数，我们评估转录因子的活性， 将信号通路与受调控基因连接起来。在初步研究中，我们发现特定的TF活性 AKT、mTOR、MEK、ERK、CDK和Aurora激酶抑制剂的TFAP谱。值得注意的是，在每个 在这种情况下，给定激酶的大多数抑制剂产生共同的TFAP签名，而不管它们的结构如何。 差异和MOA。因此，这些不变的PKI TFAP签名可以表示用于认证的特定标记。 目标PKI活动。单个PKI在特定浓度范围内显示出靶向TFAP (the“特异性窗口”）;除此之外，我们观察到明显不同的签名，反映了脱靶 方面的影响.对照已知的化学品的参考签名查询脱靶PKI签名 生物活性提供了对非预期激酶和非靶向效应的直接鉴定。 激酶效应物。因此，PKI TFAP为评估PKI提供了明确的量化指标 多元药理学根据这项建议，我们会为一组集中的 40至50种治疗相关激酶（特定目标1）。利用这些特征，我们将评估 窗口和所评价的PKI的脱靶效应（具体目标2）。在具体目标3下，我们将测试 ATTAKIN™方法用于发现新型抑制剂的实用性。为此，我们将查询我们的数据库， 包含超过30，000个TFAP药物和环境化学品条目，并将验证检索到的 通过基于蛋白质组学的激酶谱分析技术。该项目将（1）验证新的，无偏见的 PKI多药药理学评价方法;（2）产生靶向活性的特异性标志物， 许多治疗相关的激酶;和（3）评估大量PKIs的多药理学。在 总之，该项目将坚定地验证ATTAKIN™方法作为激酶生物学评价的工具 抑制剂，这是正交和补充现有的方法。这项研究将建立 新的，签名引导的PKI评估方法作为主流药物评估技术。的 ATTAKIN™将补充和扩展Attagene现有的药物评估服务。

ABSTRACT Evaluating the biological activity of protein kinase inhibitors (PKI) is one of the most challenging problems of drug development. Here, we propose a new evaluation approach (the ATTAKIN™) in which we infer PKI bioactivities from responses of cellular signaling. As a readout, we assess the activity of transcription factors that connect the signaling pathways to regulated genes. In preliminary studies, we found specific TF activity profiles (TFAPs) for inhibitors of AKT, mTOR, MEK, ERK, CDK, and Aurora kinases. Remarkably, in each case, most inhibitors of a given kinase produced a common TFAP signature, regardless of their structural dissimilarities and MOAs. Therefore, these invariant PKI TFAP signatures may represent specific markers for the on-target PKI activity. Individual PKIs exhibited the on-target TFAPs within certain concentration ranges (the “specificity windows”); beyond those, we observed distinctly different signatures, reflecting off-target effects. Querying the off-target PKI signatures against reference signatures of chemicals with known bioactivities afforded straightforward identification of the off-target effects on unintended kinases and non- kinase effectors. Therefore, PKI TFAPs provide clear quantitative metrics for the evaluation of PKI polypharmacology. Under this proposal, we will identify the on-target PKI TFAP signatures for a focused set of 40 to 50 therapeutically relevant kinases (Specific Aim 1). Using these signatures, we will assess the specificity windows and the off-target effects of the evaluated PKIs (Specific Aim 2). Under Specific Aim 3, we will test the utility of the ATTAKIN™ approach for discovery of novel inhibitors. For that, we will query our database, containing over 30,000 TFAP entries for drugs and environmental chemicals, and will validate the retrieved chemicals by a proteomics-based kinase profiling technique. This project will (1) validate the new, unbiased approach to evaluation of PKI polypharmacology; (2) produce specific markers of the on-target activity for many therapeutically relevant kinases; and (3) assess the polypharmacology of a large number of PKIs. In summary, this project will firmly validate the ATTAKIN™ approach as a tool for biological evaluation of kinase inhibitors, which is orthogonal and complementary to existing methodologies. This study will establish the new, signature-guided PKI assessment approach as a mainstream drug evaluation technology. The ATTAKIN™ will complement and expand the existing Attagene line of drug evaluation services.