Metal-binding Isosteres for Influenza Endonuclease Inhibitors and Beyond

项目总结/摘要 本研究计划的重点是开发新的策略，发现金属酶 抑制剂的金属酶是许多生物过程所必需的，并且与治疗有关。 疾病，包括癌症、细菌/病毒感染、高血压等。尽管流行的 金属酶（>40%的所有酶是金属酶）及其在疾病增殖中的关键作用， 新的金属酶抑制剂的开发还非常不充分。PI（科恩） 开发了一个研究计划，结合生物无机与药物化学的原则， 被广泛认为是关注金属酶抑制挑战的少数努力之一。 抑制金属酶的小分子利用金属结合药效团（MBP）功能 基团以结合到靶中的活性位点金属离子。在上一个项目期间，一个聚焦的MBP片段 组装了用于针对金属酶的基于片段的药物发现（FBDD）的文库。在这 更新应用，这些MBP片段的药物样特征将通过应用 电子等排体置换这有望为鉴定金属酶提供新的化学物质 抑制剂，同时获得更广泛的物理化学性质（例如，酸度、亲油性）， 脚手架这些金属结合电子等排体（MBIs）将用于改善一类高活性的 在上一个项目期间开发的抑制剂针对流感病毒N-末端核酸内切酶结构域， 聚合酶酸性蛋白（PAN）。虽然对PAN内切酶有活性，但其吸收特性差， 这些抑制剂导致了细胞中抗病毒的次优活性。MBIs将用于改善 物理化学性质，同时保留基于酶的活性，以产生高活性抑制剂 对抗活细胞中的病毒最后，考察金属酶的靶向活性和选择性 抑制剂，我们的MBP，MBI和PAN抑制剂将通过细胞热位移试验（CETSA）进行检查 和亲和层析。这些实验将验证目标参与并评估选择性如何 随着MBP发展成全长PAN核酸内切酶抑制剂，详细的细胞目标 使用这些方法的金属酶抑制剂的参与数据很少;因此，这些研究将 对于阐明这些治疗化合物的选择性以及因此的临床前景是有价值的。 上一个项目期间产生了许多合作，专利披露，会议 论文集及13篇出版物。此外，还为生物技术劳动力提供了熟练的受训人员， 指导，并实现了我们的成果转化为创业公司。我们将继续培养 合作，以发现最好的和一流的金属酶抑制剂，有可能改善 人体健康总的来说，这项研究计划将继续发挥主导作用，确定小 分子抑制剂，用于具有挑战性的目标类别，对改善人类健康具有巨大价值。

PROJECT SUMMARY/ABSTRACT This research program is focused on developing new strategies for the discovery of metalloenzyme inhibitors. Metalloenzymes are essential to numerous biological processes and are relevant to treating diseases, including cancer, bacterial/viral infections, hypertension, and others. Despite the prevalence of metalloenzymes (>40% of all enzymes are metalloenzymes) and their critical role in disease proliferation, the development of new metalloenzyme inhibitors is extremely underexplored. The PI (Cohen) has developed a research program that combines the principles of bioinorganic with medicinal chemistry and is widely recognized as one of the few efforts focused on the challenges of metalloenzyme inhibition. Small molecules that inhibit metalloenzymes utilize a metal-binding pharmacophore (MBP) functional group to bind to the active site metal ion(s) in the target. In the last project period, a focused MBP fragment library for use in fragment-based drug discovery (FBDD) against metalloenzymes was assembled. In this renewal application, the drug-like features of these MBP fragments will be improved by the application of isostere replacement. This is expected to yield new chemical matter for identifying metalloenzyme inhibitors, while accessing a wider range of physicochemical properties (e.g., acidity, lipophilicity) in these scaffolds. These metal-binding isosteres (MBIs) will then be used to improve a class of highly active inhibitors developed during the last project period against the influenza N-terminal endonuclease domain of the polymerase acidic protein (PAN). Although active against PAN endonuclease, the poor uptake properties of these inhibitors have led to suboptimal activity against the virus in cells. MBIs will be used to improve physicochemical properties, while retaining enzyme-based activity, to produce highly active inhibitors against the virus in live cells. Finally, to examine the on-target activity and selectivity of metalloenzyme inhibitors, our MBPs, MBIs, and PAN inhibitors will be examined by Cellular Thermal Shift Assay (CETSA) and affinity chromatography. These experiments will verify target engagement and evaluate how selectivity improves as the MBP is developed into a full-length PAN endonuclease inhibitor. Detailed cellular target engagement data using these methods for metalloenzyme inhibitors is scarce; therefore, these studies will be valuable for clarifying the selectivity, and hence the clinical prospects, of these therapeutic compounds. The previous project period generated many collaborations, patent disclosures, conference proceedings, and ~13 publications. In addition, skilled trainees for the biotechnology workforce were mentored, and translation of our results into startup companies was achieved. We will continue to nurture collaborations to discover best- and first-in-class metalloenzyme inhibitors that have the potential to improve human health. Overall, this research program will continue to play a leading role in identifying small molecule inhibitors for a challenging target class that has great merit for improving human health.