Bioactive and bioorthogonal molecular tools for studying host-pathogen interactions

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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.