Development of Novel Chemical Probes for the Elucidation of Bacterial and Viral Pathogenesis and Mechanisms of Infection

Abstract

Bioactive small molecules have been a major source of therapeutics for the treatment of human disease. Advances in synthetic and semi-synthetic methods have greatly expanded the repertoire of available small molecules for use in the study of the functional characteristics of biological systems. Chemical proteomic methods, which in some cases harness bioactive small molecules to enhance proteomic techniques, have been developed to study protein targets in physiological conditions. Such approaches allow for maintenance of post-translational modifications, protein-protein interactions, and interactions with endogenous regulators. Functional proteomics, using tandem-labeling strategies that harness bioorthogonal chemistry, have permitted the study of the molecular targets of bioactive small molecules. To this end, herein bio-orthogonal methods and functional proteomics to study the molecular targets of many bioactive small molecules have been developed. In chapter 2, three previously reported bioactive small molecules were screened for activity against HCV replication and 6-hydroxydopamine (6-OHDA) was identified as a potential inhibitor. By generating a novel chemical probe based on 6-OHDA, we determined that 6-OHDA was able to covalently modify a large range of biological targets and initiate cellular oxidative stress, both of which contribute to the antiviral activity of 6-OHDA. In chapter 3, an affinity probe (AfBP) based on the methyltransferase inhibitor sinefungin was developed and used to profile eukaryotic enzyme targets. Using in-gel fluorescence scanning and mass spectrometry analysis, we identified several proteins including methyltransferases and other candidate proteins that may be associated with epigenetic mechanisms. In chapter 4, the characterization of the bacterial and eukaryotic targets of a novel antibacterial small molecule armeniaspirole A is reported. Using a medicinal chemistry strategy, the Cl-ARM-A-yne affinity-based probe was developed and was capable of covalently capturing proteogenic molecular targets. Following mass spectrometry analysis, several lead molecular targets were identified that may be implicated in the antimicrobial effects of armeniaspirole A. Herein, the targets of a novel chemical probe allowed the characterization and elucidation of the previously unknown molecular targets of armeniaspirole A. Finally, related probes were explored as novel photoaffinity ligands of cytosporone B, a small molecule agonist for the nuclear orphan receptor 77, which could be highly effective at capturing and analysing the Nur77 associated co-activators or co-repressors towards exploring the biology of this mysterious receptor.