Molecular Basis for Selective Late-Stage Transformations in Fungal Indole Alkaloid Biosynthesis

Abstract

The class of fungal indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring is comprised of diverse molecules that display a range of biological activities. While much interest has been garnered due to their therapeutic potential, this class of molecules also displays unique chemical functionality, making them intriguing synthetic targets. Many elegant and intricate total syntheses have been developed to generate these alkaloids, but selectivity and yield have always presented barriers to efficient synthesis. Alternatively, if we can understand the molecular mechanisms behind how the fungi produce these complex molecules, we can leverage the power of nature to perform these chemical transformations. This work has delved into the enzymatic machinery responsible for key biosynthetic transformations in the production of the fungal indole alkaloids. With full mechanistic characterization, these enzymes are now available as biocatalytic tools for generating pre-existing and novel indole alkaloids in the search for the next line of therapeutic molecules. The studies presented in this thesis start with the discovery of the Diels-Alderase enzymes responsible for forming the characteristic bicyclo[2.2.2]diazaoctane ring. This core component is the signature of this class of molecules and we have found that the enzymes responsible for its formation perform multiple roles in the biosynthesis, including reduction and cyclization. Halogen atoms are found within a small number of the fungal indole alkaloids, but the halogen moieties on these molecules significantly contribute to the biological activity. This is particularly true in the case of malbrancheamide, for which the dichlorination is required for its biological activity. This work has thoroughly characterized the iterative late-stage halogenase (MalA) which performs dichlorination as the last step in malbrancheamide biosynthesis. Cocrystal structures and computational studies have led to the generation of site-selective variants produced through structure-based engineering of the halogenase. A collaboration with Novartis Institutes for BioMedical Research led to the discovery that this halogenase has a broad substrate scope, and thus great utility as a biocatalyst for late-stage halogenation. A common functionality of the fungal indole alkaloids is the spirooxindole center. This is another core component of many molecules within this class, and it provides dimension to an otherwise relatively planar structure. This thesis presents the discovery of a flavin monooxygenase that is responsible for the selective spirocyclization of the potent antihelmintic paraherquamides. The natural substrates of this enzyme were identified and cocrystal structures demonstrated that the enzyme binds the molecules in a manner conducive for stereocontrol. The broad substrate scope demonstrated with PhqK provides further evidence that it could be utilized to develop new therapeutic molecules. As mentioned, this class of fungal indole alkaloids has displayed a wide range of biological activities. There has been significant interest in the malbrancheamides because they have displayed a potent vasorelaxant effect, and a complete reversal of the swelling of cardiac tissue. With this in mind, the culmination of my thesis work demonstrated the utility of malbrancheamide as a probe for protein-protein interactions that have been implicated in cardiovascular disease. Malbrancheamide was characterized as a selective inhibitor of calmodulin, and its unique binding mode relieved the effects of Ca2+-calmodulin-induced cardiac hypertropy. We used the in vitro data with malbrancheamide and the Ca2+-CaM·GRK5 complex to validate a much-debated model for this protein-protein interaction, providing valuable insight for the field of Ca2+-calmodulin-dependent kinase signaling.