Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins.

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursor-directed biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide- or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

Hapalindoles from the Cyanobacterium Hapalosiphon sp. Inhibit T Cell Proliferation.

Novel immunomodulating agents are currently sought after for the treatment of autoimmune diseases and cancers. In this context, a screening campaign of a collection of 575 cyanobacteria extracts for immunomodulatory effects has been conducted. The screening resulted in several active extracts. Here we report the results of subsequent studies on an extract from the cyanobacterium Hapalosiphon sp. CBT1235. We identified 5 hapalindoles as the compounds responsible for the observed immunomodulatory effect. These indole alkaloids are produced by several strains of the cyanobacterial family Hapalosiphonaceae. They are known for their anti-infective, cytotoxic, and other bioactivities. Modulation of the activity of human immune cells has not yet been described. The immunomodulatory activity of the hapalindoles was characterized in vitro using flow cytometry-based measurements of T cell proliferation after carboxyfluorescein diacetate succinimidyl ester staining, and apoptosis and necrosis induction after annexin V/propidium iodide staining. The most potent compound, hapalindole A, reduced T cell proliferation with an IC50 of 1.56 µM, while relevant levels of apoptosis were measurable only at 10-fold higher concentrations. Hapalindole A-formamide and hapalindole J-formamide, isolated for the first time from a natural source, had much lower activity than the nonformylated derivatives while, at the same time, being less selective for antiproliferative over apoptotic effects.