Decoding the Bio-Assembly of Hapalindole-Type Alkaloids from Cyanobacteria for Drug Discovery

Abstract

Biosynthetic exploration of natural products provides a promising opportunity to produce novel bioactive molecules for drug development. Hapalindole-type alkaloids are a large group of secondary metabolites isolated from terrestrial and freshwater Hapalosiphonaceae cyanobacteria. Their diverse pharmacological activity and intriguing polycyclic skeletons have compelled us to pursue a detailed mechanistic understanding of their biosynthesis, hoping to overcome the limitations of chemical synthesis and natural product isolation in developing these metabolites as drug leads. My dissertation research focus on elucidating the molecular basis of hapalindole and fischerindole core ring formation, which are the basic structures to generate more complex ambiguine and welwitindolinone subgroups through late-stage tailoring. All reported hapalindoles and fischerindoles can be categorized into six stereochemical patterns based on the C10, C11, C12, and C15 chiral centers. This stereo- and regiochemically diverse polycyclic ring formation represents a fascinating platform to investigate variations during initial cyclization and subsequent tailoring reactions. In this report, we describe a thorough study of the novel Stig cyclases responsible for catalyzing hapalindole and fischerindole formation from a common intermediate C3-geranylated indole isonitrile, whose coupled Cope rearrangement/6-exo-trig cyclization/electrophilic aromatic substitution together elaborates the four chiral centers and three types of ring systems. We have characterized the enzymatic activity of more than twenty annotated Stig cyclases generating stereo-diverse hapalindoles and fischerindoles. Our crystal structure study, computational modeling and mutational analysis together reveal that the Stig cyclases may function as Ca2+-induced higher-order heteromeric oligomers to collaboratively control the stereochemistry. This work also enable manipulation to diversify these remarkable alkaloids.