Prodiginines are a family of linear and cyclic oligopyrrole red-pigmented compounds. Herein we describe the in vitro antimalarial activity of 4 natural (IC50 = 1.7-8.0 nM) and 3 sets of synthetic prodiginines against Plasmodium falciparum. Set 1 compounds replaced the terminal non-alkylated pyrrole ring of natural prodiginines and had diminished activity (IC50 >2920 nM). Set 2 and set 3 prodiginines were monosubstituted or disubstituted at either the 3 or 5 position of the right hand terminal pyrrole, respectively. Potent in vitro activity (IC50 = 0.9-16.0 nM) was observed using alkyl or aryl substituents. Metacycloprodiginine and more potent synthetic analogs were evaluated in a P. yoelii murine patent infection using oral administration. Each analog reduced parasitemia by more than 90% after 25 mg/kg/day dosing, and in some cases provided a cure. The most favorable profile was 92% parasite reduction at 5 mg/kg/day, and 100% reduction at 25 mg/kg/day without any evident weight loses or clinical overt toxicity.

The marine Streptomyces sp. CNQ-617 produces two diastereomers, marineosins A and B. These are structurally related to alkyl prodiginines, but with a more complex cyclization and an unusual spiroaminal skeleton. We report the identification of the mar biosynthetic gene cluster and demonstrate production of marineosins through heterologous expression in a S. venezuelae host named JND2. The mar cluster shares the same gene organization and has high homology to the genes of the red cluster (which directs the biosynthesis of undecylprodiginine) but contains an additional gene, named marA. Replacement of marA in the JND2 strain leads to the accumulation of premarineosin, which is identical to marineosin with the exception that the middle pyrrole (Ring B) has not been reduced. The final step of the marineosin pathway is thus a MarA catalyzed reduction of this ring. Replacement of marG (a homologue of redG that directs undecylprodiginine cyclization to give streptorubin B) in the JND2 strain leads to the loss of all spiroaminal products and the accumulation of 23-hydroxyundecylprodiginine and a shunt product, 23-ketoundecylprodiginine. MarG thus catalyzes the penultimate step of the marineosin pathway catalyzing conversion of 23-hydroxyundecylprodiginine to premarineosin. The preceding steps of the biosynthetic marineosin pathway likely mirror that in the red-directed biosynthetic process, with the exception of the introduction of the hydroxyl functionality required for spiroaminal formation. This work presents the first experimentally supported scheme for biosynthesis of marineosin and provides a new biologically active molecule, premarineosin.

Facile and highly efficient synthetic routes for the synthesis of (S)- and (R)-23-hydroxyundecylprodiginines ((23S)-2, and (23R)-2), 23-ketoundecylprodiginine (3), and deuterium-labeled 23-hydroxyundecylprodiginine ([23-d]-2) have been developed. We demonstrated a novel Rieske oxygenase MarG catalyzed stereoselective bicyclization of (23S)-2 to premarineosin A (4), a key step in the tailoring process of the biosynthesis of marineosins, using a marG heterologous expression system. The synthesis of various A–C-ring functionalized prodiginines 32–41 was achieved to investigate the substrate promiscuity of MarG. The two analogues 32 and 33 exhibit antimalarial and cytotoxic activities stronger than those of the marineosin intermediate 2, against Plasmodium falciparum strains (CQS-D6, CQR-Dd2, and 7G8) and hepatocellular HepG2 cancer cell line, respectively. Feeding of 34–36 to Streptomyces venezuelae expressing marG led to production of novel premarineosins, paving a way for the production of marineosin analogues via a combinatorial synthetic/biosynthetic approach. This study presents the first example of oxidative bicyclization mediated by a Rieske oxygenase.

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.

Glycosyltransferases are key enzymes involved in the biosynthesis of valuable natural products providing an excellent drug-tailoring tool. Herein, we report the identification of two cooperative glycosyltransferases from the sqn gene cluster directing the biosynthesis of saquayamycins in Streptomyces sp. KY40-1: SqnG1 and SqnG2. Gene inactivation of sqnG1 leads to 50 folds decrease in saquayamycin production, while inactivation of sqnG2 leads to complete production loss suggesting that SqnG2 acts as dual O- and C-glycosyltransferase. Gene inactivation of a third putative glycosyltransferase-encoding gene, sqnG3, does not majorly affect saquayamycin production suggesting that SqnG3 has no or supportive role in glycosylation. The data indicate that SqnG1 and SqnG2 are solely and possibly cooperatively responsible for the sugar diversity observed in saquayamycins 1–7. This is the first evidence of a proposed glycosyltransferase complex with dual O- and C-glycosyltransferase activity, utilizing NDP-activated d-olivose, l-rhodinose as well as an unusual amino sugar, presumably 3,6-dideoxy-l-idosamine.