Proposed Biosynthesis and Molecular Targets of Manzamine Alkaloids from Acanthostrongylophora ingens (Phylum Porifera: Class Demospongiae).

Manzamine alkaloids are a structurally diverse class of marine-derived alkaloids that have attracted considerable interest because of their complex polycyclic architectures, diverse biological activities, and distinctive biosynthetic features. The Indonesian marine sponge Acanthostrongylophora ingens is a particularly rich source of these alkaloids, providing an opportunity to further explore their chemical diversity, biosynthetic relationships, and biological properties. Detailed analyses of LC-HR-ESI-MS/MS and molecular networking were performed on EtOAc layers obtained from H2O/EtOAc partitioning of methanolic extracts of the marine sponge, Acanthostrongylophora ingens, collected from Raja Ampat (Southwest Papua), the Thousand Islands reef complex (Jakarta Special Region), Spermonde Archipelago (South Sulawesi), and Sangiang Island (Banten). This approach revealed the presence of both known and new manzamine alkaloids, along with their possible precursors and structural modifications, thereby supporting the proposed biosynthetic origin of these alkaloids. In addition, three flexible cytotoxic manzamine alkaloids, namely (+)-manzamine A hydrochloride (1), (+)-manzamine B (2), and (+)-32,33-dihydro-31-hydroxymanzamine A (3), were purified and their relative configurations determined using quantum mechanic NMR-based calculations and validated by X-ray analysis. (+)-Manzamine A hydrochloride (1) displayed the most potent cytotoxic activities against Artemia salina larvae (LC50 value of 0.041 ± 0.012 µM) and HEK293T cells (IC50 value of 0.599 ± 0.057 µM). The potent cytotoxic activity suggested the importance of azocine ring (Z-olefin at C32-C33) in 1. Molecular mechanisms of 1-3 were subsequently revealed through computational approaches identifying GSK-3β as a potential target. Docking results indicated that 3 possessed better binding affinity to GSK-3β, compared with other ligands. All isolated manzamines (1-3) were found to be stable in the binding site. A molecular mechanism is also proposed to provide insights into how manzamines interrupt cancer cells by targeting specifically with GSK-3β.