Synthesis, activity and molecular target identification of clionamine B analogues : autophagy stimulating and Mtb clearing amino steroids

Natural products are a rich resource for the discovery and development of chemical tools which can be used to study biological processes. The marine environment contains unique niches where organisms have the ability to adapt in producing chemical compounds that are shown to have various biological utilities. In the following chapters, the synthesis of various analogues of Clionamine B, a biologically useful aminosteroid isolated from the marine sponge Cliona celata and their use as chemical biology tools are outlined. Clionamine B was previously synthesized in the Andersen Lab and was found to potently stimulate autophagy and clear latent Mycobacterium tuberculosis (Mtb) from human macrophages. In chapter 2, a more efficient semi-synthesis of analogues of clionamine B is presented, using environmentally benign reagents, in order to perform structure-activity relationship (SAR) studies. Using this methodology, ten analogues of clionamine B were synthesized, all of which were found to stimulate autophagy and clear Mtb to varying degrees. One of the analogues synthesized, an N-benzyl derivative of clionamine B was found to stimulate autophagy very potently compared to the natural product. In addition, the molecular target of the clionamines was identified as PIK1, a phosphatidylinositol 4-kinase which is known to regulate trafficking to and from the Golgi; and play an important role in the mechanism of autophagy and affect Atg9 trafficking. Chapters 3 and 4 highlight efforts towards the synthesis of analogues with reduced lipophilicity and their effect on autophagy stimulation and Mtb inhibition. This was done systematically by introducing heteroatoms in the lactone side chain. However, the effects of reducing the lipophilicity on the autophagy stimulating and Mtb clearing activities of analogues of clionamine B were not conclusively determined. Chapter 5 highlights efforts towards the synthesis of various molecular probes of the analogue of clionamine B displaying the most desirable combination of autophagy stimulating and Mtb clearing activities observed so far. The synthesis of a biotinylated molecular probe was achieved, and was shown to be effective in corroborating PIK1 as the molecular target of the clionamines using yeast cell lysate.Science, Faculty ofChemistry, Department ofGraduat