Bioactive and bioorthogonal molecular tools for studying host-pathogen interactions

我们将在这项拟议的NSERC发现拨款计划中进行三个具体的研究主题。首先，我们将开发基于活性的蛋白质图谱工具来研究酶活性的变化。基于活性的蛋白质图谱(ABPP)是一种用来表征整个细胞蛋白质组或活细胞的复杂环境中的酶功能的化学方法。这涉及到使用活性位点定向的化学探针来报告酶的活性。我们将设计、合成和表征新的基于活性的探针，特别关注脂蛋白激酶，以及控制和改变膜微环境的相关酶。我们还将开发新的生物正交化学，以允许修改和跟踪活的病原体，重点是允许分子跟踪的方法。这将通过结合非天然化学基团来实现，这些基团可用于询问特定的致病生物分子。涉及硝酮-炔环加成反应的生物正交反应将进一步发展，以优化生物相关环境中的反应活性，促进有效的生物偶联反应，用于体内应用。这将包括合成和评估新的分子、反应，在某些情况下，还将扩展遗传密码，以便在目标蛋白质中特定地加入生物正交反应性。最后，我们将开发新的方法来理解microRNAs(MiRNA)的作用。MiRNAs是调节信使RNA稳定性和翻译的内源性非编码RNA。然而，识别它们在体内调节的mRNA靶标仍然是该领域的一个挑战。尽管传统的miRNA图谱研究已被证明有助于确定疾病的生物标记物，但这些研究通常没有提供差异表达的miRNA的功能影响和病理相关性的明确指示。在这个研究项目中，我们将重点开发新的方法来鉴定miRNA的功能。我们将使用已知的针对细胞通路的通路特异性小分子来注释与它们相关的miRNAs，识别小分子和非编码RNA之间的功能和潜在的协同作用。这些基础研究将使进一步分析这些途径成为可能，因为它们可能与病原体-宿主相互作用的调节有关。 这项发现赠款计划将继续为在自然科学和健康科学中的应用创造新的化学生物学工具而进行的工作。此外，这笔赠款将使我的实验室继续培养代表下一代加拿大科学家的有才华的多学科研究人员。这些高素质的人才将为加拿大未来在科学、技术和我们知识驱动的加拿大经济方面的竞争力做出贡献。

We will pursue three specific research themes within this proposed NSERC Discovery Grant program. First, we will develop activity-based protein profiling tools to investigate changes in enzyme activity. Activity-based protein profiling (ABPP) is a chemical approach for the characterization of enzymatic function within the complex environment of whole cell proteomes or living cells. This involves the use of active site-directed chemical probes that report on enzyme activity. We will design, synthesize and characterize new activity-based probes, with a special focus on lipid kinases, and related enzymes that control and alter membrane microenvironments. We will also develop new bioorthogonal chemistry to allow the modification and tracking of living pathogens, with an emphasis on methods that allow molecular tracking. This will be accomplished by the incorporation of non-natural chemical groups that can be used to interrogate specific pathogenic biomolecules. Bioorthogonal reactions involving nitrone-alkyne cycloadditions will be further developed to optimize reactivity in biologically relevant environments that promote efficient bioconjugation reactions for in vivo applications. This will involve the synthesis and evaluation of new molecules, reactions, and in some cases expansion of the genetic code for site-specific incorporation of bioorthogonal reactivity within target proteins. Lastly, we will develop new methodologies for understanding the roles of microRNAs (miRNA). miRNAs are endogenous non-coding RNAs that regulate messenger RNA stability and translation. However, identification of the mRNA targets that they regulate in vivo remains a challenge to the field. Although traditional miRNA profiling studies have proven instrumental in identifying biomarkers for disease, these studies typically provide no clear indication of the functional impact and pathological relevance of differentially expressed miRNAs. In this research program we will focus on the development of new methods to identify miRNA function. We will use pathway-specific small molecules known to target cellular pathways in order to annotate miRNAs associated with them, identifying function, and potential synergies between small molecules and non-coding RNAs. These fundamental studies will then enable further analysis of these pathways as they may pertain to the regulation of pathogen-host interactions. This discovery grant program will continue ongoing work in the creation of new chemical biology tools for applications in natural and health sciences. Furthermore, this grant will allow my laboratory to continue its development of talented and multidisciplinary researchers that represent the next generation of Canadian scientists. These highly qualified personnel will contribute to Canada’s future competitiveness in science, technology, and our knowledge-driven Canadian economy.