Structural diversification of hapalindole and fischerindole natural products via cascade biocatalysis.

Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole alkaloid natural products. They are typically isolated from the Stigonemataceae order of cyanobacteria and possess a broad scope of biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate the core ring system, L-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process including a Cope rearrangement, 6-exo-trig cyclization and electrophilic aromatic substitution to create a polycyclic core. Formation of the initial alkaloid is followed by diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to the wide array of structural variations observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases toward core structural diversification of this family of indole alkaloids. Through synthesis of cis-indole isonitrile subunit derivatives, and aided by protein engineering and computational analysis, we have employed cascade biocatalysis to generate a range of derivatives, and gained insights into the basis for substrate flexibility in this system.

Multicomponent Microscale Biosynthesis of Unnatural Cyanobacterial Indole Alkaloids.

Genome sequencing and bioinformatics tools have facilitated the identification and expression of an increasing number of cryptic biosynthetic gene clusters (BGCs). However, functional analysis of all components of a metabolic pathway to precisely determine biocatalytic properties remains time-consuming and labor intensive. One way to speed this process involves microscale cell-free protein synthesis (CFPS) for direct gene to biochemical function analysis, which has rarely been applied to study multicomponent enzymatic systems in specialized metabolism. We sought to establish an in vitro transcription/translation (TT)-assay to assess assembly of cyanobacterial-derived hapalindole-type natural products (cNPs) because of their diverse bioactivity profiles and complex structural diversity. Using a CFPS system including a plasmid bearing famD2 prenyltransferase from Fischerella ambigua UTEX 1903, we showed production of the central prenylated intermediate (3GC) in the presence of exogenous geranyl-pyrophosphate (GPP) and cis-indole isonitrile. Further addition of a plasmid bearing the famC1 Stig cyclase resulted in synthesis of both FamD2 and FamC1 enzymes, which was confirmed by proteomics analysis, and catalyzed assembly of 12-epi-hapalindole U. Further combinations of Stig cyclases (FamC1-C4) produced hapalindole U and hapalindole H, while FisC identified from Fischerella sp. SAG46.79 generated 12-epi-fischerindole U. The CFPS system was further employed to screen six unnatural halogenated cis-indole isonitrile substrates using FamC1 and FisC, and the reactions were scaled-up using chemoenzymatic synthesis and identified as 5- and 6-fluoro-12-epi-hapalindole U, and 5- and 6-fluoro-12-epi-fischerindole U, respectively. This approach represents an effective, high throughput strategy to determine the functional role of biosynthetic enzymes from diverse natural product BGCs.

Molecular basis for the plasticity of aromatic prenyltransferases in hapalindole biosynthesis.

Aromatic prenyltransferases (PTases) are enzymes that catalyze Friedel-Crafts reactions between aromatic compounds and isoprenoid diphosphates. In hapalindole biosynthesis, the aromatic PTases AmbP1 and AmbP3 exhibit surprisingly plastic selectivities. AmbP1 not only transfers the geranyl group on the C-3 of cis-indolylvinyl isonitrile, but also on the C-2, which is supressed in the presence of Mg2+ ions. AmbP3 transfers the dimethylallyl group on C-2 of hapalindole U in the reverse manner, but on C-2 of its C-10 stereoisomer in the normal manner. This review highlights the molecular bases of the AmbP1 and AmbP3 functions, elucidated through their X-ray crystal structures. The knowledge presented here will contribute to the understanding of aromatic PTase reactions and will enhance their uses as biocatalysts.

Hapalindole H Induces Apoptosis as an Inhibitor of NF-ĸB and Affects the Intrinsic Mitochondrial Pathway in PC-3 Androgen-insensitive Prostate Cancer Cells.

BACKGROUND: Prostate cancer presents the highest incidence rates among all cancers in men. Hapalindole H (Hap H), isolated from Fischerella muscicola (UTEX strain number LB1829) as part of our natural product anticancer drug discovery program, was found to be significantly active against prostate cancer cells. MATERIALS AND METHODS: In this study, Hap H was tested for nuclear factor-kappa B (NF-ĸB) inhibition and selective cytotoxic activity against different cancer cell lines. The apoptotic effect was assessed on PC-3 prostate cancer cells by fluorescence-activated cell sorting analysis. The underlying mechanism that induced apoptosis was studied and the effect of Hap H on mitochondria was evaluated and characterized using western blot and flow cytometric analysis. RESULTS: Hap H was identified as a potent NF-ĸB inhibitor (0.76 µM) with selective cytotoxicity against the PC-3 prostate cancer cell line (0.02 µM). The apoptotic effect was studied on PC-3 cells. The results showed that treatment of PC-3 cells with Hap H reduced the formation of NAD(P)H, suggesting that the function of the outer mitochondrial membrane was negatively affected. Thus, the mitochondrial transmembrane potential was assessed in Hap H treated cells. The results showed that the outer mitochondrial membrane was disrupted as an increased amount of JC-1 monomers were detected in treated cells (78.3%) when compared to untreated cells (10.1%), also suggesting that a large number of treated cells went into an apoptotic state. CONCLUSION: Hap H was found to have potent NF-ĸB p65-inhibitory activity and induced apoptosis through the intrinsic mitochondrial pathway in hormone-independent PC-3 prostate cancer cells.

Indole Alkaloids of the Stigonematales (Cyanophyta): Chemical Diversity, Biosynthesis and Biological Activity.

The cyanobacteria are well recognized as producers of a wide array of bioactive metabolites including toxins, and potential drug candidates. However, a limited number of taxa are generally considered with respect to both of these aspects. That said, the order Stigonematales, although largely overlooked in this regard, has become increasingly recognized as a source of bioactive metabolites relevant to both human and environmental health. In particular, the hapalindoles and related indole alkaloids (i.e., ambiguines, fischerindoles, welwitindolinones) from the order, represent a diverse, and phylogenetically characteristic, class of secondary metabolites with biological activity suggestive of potential as both environmental toxins, and promising drug discovery leads. The present review gives an overview of the chemical diversity of biologically active metabolites from the Stigonematales-and particularly the so-called hapalindole-type alkaloids-including their biosynthetic origins, and their pharmacologically and toxicologically relevant bioactivities. Taken together, the current evidence suggests that these alkaloids, and the associated cyanobacterial taxa from the order, warrant future consideration as both potentially harmful (i.e., "toxic") algae, and as promising leads for drug discovery.

Total synthesis of hapalindole-type natural products.

A unified and bioinspired oxidative cyclization strategy was used in the first total syntheses of naturally occurring 12-epi-hapalindole Q isonitrile, hapalonamide H, deschloro 12-epi-fischerindole I nitrile, and deschloro 12-epi-fischerindole W nitrile, as well as the structural revision of the latter. Hapalindoles H and Q were also synthesized.