Biocatalytic Generation and Bioactivity Investigation of Naphthylisoquinoline Alkaloids

About This Grant

ABSTRACT Global cases of malaria reached 263 million people in 2023, an eight percent increase from 2022, and the disease has led to estimated annual death tolls of half a million people. Over the next two decades, the global health burden of neglected tropical diseases (NTDs) and malaria is projected to increase further based on an analysis of data from the 2021 Global Burdens of Disease Study. The emergence of Plasmodium strains that are resistant to the current suite of registered antimalarial drugs creates significant challenges in combatting the disease and necessitates the development of novel small molecule antimalarial therapies. Looking to nature, root extracts from the Southeast Asian woody liana Ancistrocladus tectorius have historically been used to treat malaria. The plant species A. tectorius showcases a wide breadth of biaryl naphthylisoquinoline alkaloid (NIQ) metabolites. These NIQs have garnered interest due to their complex architectures and pharmacological properties against tropical diseases. Although the bioactivity of some NIQs has been characterized, our inability to readily obtain many of the molecules in this class of natural products hinders broader characterization. Thus, the biaryl NIQs are fascinating complex targets for selective synthesis with potential utility in combatting malaria and other NTDs. Strategies to access these molecules can require pre-functionalization of the coupling partners, forcing conditions, and can suffer from limited yields and selectivity challenges. In contrast, Nature has evolved versatile heme-dependent cytochrome P450 monooxygenases (P450s) capable of a wide breadth of oxidation chemistry. A biocatalytic oxidative coupling approach offers an efficient alternative strategy for catalyst-controlled selective biaryl C–C bond formation under mild conditions with molecular oxygen as the stoichiometric oxidant. Herein, I propose that profiling of a wild-type P450 library coupled with enzyme engineering to improve both enzyme reactivity and selectivity will enable the chemoenzymatic synthesis of naphthylisoquinoline alkaloids (NIQs) and their subsequent bioactivity characterization. A biocatalyst will be optimized via directed enzyme evolution for the target-oriented synthesis of the antimalarial NIQ ancistrotanzanine C. Further P450 library reactivity profiling and enzyme engineering will generate a suite of biocatalysts a broader scope of NIQs with varying site-selectivity and coupling partners. Synthesized NIQs will be characterized for potential inhibitory activity against a cysteine protease target implicated in malaria and crystal formation that protects Plasmodium from a toxic environment during hemolysis. The proposed work will accelerate the development of novel small molecule alternatives for treating malaria and other neglected tropical diseases.