Biosynthesis of C-nucleoside antibiotics in actinobacteria: recent advances and future developments

Epidemic diseases and antibiotic resistance are urgent threats to global health, and human is confronted with an unprecedented dilemma to conquer them by expediting development of new natural product related drugs. C-nucleoside antibiotics, a remarkable group of microbial natural products with diverse biological activities, feature a heterocycle base linked with a ribosyl moiety via an unusual C-glycosidic bond, and have played significant roles in healthcare and for plant protection. Elucidating how nature biosynthesizes such a group of antibiotics has provided the basis for engineered biosynthesis as well as targeted genome mining of more C-nucleoside antibiotics towards improved properties. In this review, we mainly summarize the recent advances on the biosynthesis of C-nucleoside antibiotics, and we also tentatively discuss the future developments on rationally accessing C-nucleoside diversities in a more efficient and economical way via synthetic biology strategies.

Nucleoside antibiochemotherapy repressed the growth, chemoresistance, survival, and metastatic potentials of castration-resistant prostate cancer cells

ABSTRACTThere is currently no definitive cure for metastatic castration-resistant prostate cancer (mCRPC), therefore justifying the incessant need for more investigative studies to either repurpose old drugs or identify novel and effective therapeutics. In this study, we investigated the possible anticancer effects of two nucleoside antibiotics: puromycin and blasticidin. We hypothesized that the two antibiotics alone or combined with other drugs will inhibit prostate cancer (PCa) cell proliferation and metastasis and induce cell death via apoptosis. mCRPC cell lines (PC3 and DU145) with different p53-gene statuses were cultured and seeded in 96 well-plates, and thereafter treated with varying concentrations of puromycin and blasticidin (1 ng/mL - 100 μg/mL) for 24 - 48 hours. Resazurin reduction and/or MTT assays were done to evaluate the treatment-induced effects on mCRPC cell viability and proliferation. The colony-forming assay measured the cell survival rate following treatment nucleoside antibiotics while scratch migration assay and dual-fluorescent microscopy assessed the effects on metastatic potential and cell death, respectively. The two antibiotics were combined with either paclitaxel, docetaxel, or cabazitaxel to check for synergism. Our results indicate that both antibiotics exhibit dose- and time-dependent anticancer effects on growth, survival, and metastasis of mCRPCs. PC3 cells were significantly more susceptible to both antibiotics compared to DU145 cells. Both cell lines were more susceptible to puromycin compared to blasticidin. Synergism was observed when each antibiotic compound was combined with any of the three taxanes. In conclusion, we have demonstrated that both puromycin and blasticidin could be explored for the treatment of mCRPC.GRAPHICAL ABSTRACT

Identification and characterization of enzymes involved in the biosynthesis of pyrimidine nucleoside antibiotics

This review highlights the functional assignment and partial characterization of multiple proteins involved in the biosynthesis of structurally complex pyrimidine-derived nucleoside antibiotics.

Muraymycin Nucleoside Antibiotics: Structure-Activity Relationship for Variations in the Nucleoside Unit

Muraymycins are a subclass of naturally occurring nucleoside antibiotics with promising antibacterial activity. They inhibit the bacterial enzyme translocase I (MraY), a clinically yet unexploited target mediating an essential intracellular step of bacterial peptidoglycan biosynthesis. Several structurally simplified muraymycin analogues have already been synthesized for structure–activity relationship (SAR) studies. We now report on novel derivatives with unprecedented variations in the nucleoside unit. For the synthesis of these new muraymycin analogues, we employed a bipartite approach facilitating the introduction of different nucleosyl amino acid motifs. This also included thymidine- and 5-fluorouridine-derived nucleoside core structures. Using an in vitro assay for MraY activity, it was found that the introduction of substituents in the 5-position of the pyrimidine nucleobase led to a significant loss of inhibitory activity towards MraY. The loss of nucleobase aromaticity (by reduction of the uracil C5-C6 double bond) resulted in a ca. tenfold decrease in inhibitory potency. In contrast, removal of the 2′-hydroxy group furnished retained activity, thus demonstrating that modifications of the ribose moiety might be well-tolerated. Overall, these new SAR insights will guide the future design of novel muraymycin analogues for their potential development towards antibacterial drug candidates.

Comparative Investigation into Formycin A and Pyrazofurin A Biosynthesis Reveals Branch Pathways for the Construction of C-Nucleoside Scaffolds

ABSTRACT Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond. However, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria and propose that their biosynthesis is likely initiated by a lysine N6-monooxygenase. Moreover, we show that forT and prfT (involved in FOR-A and PRF-A biosynthesis, respectively) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates 4-amino-3,5-dicarboxypyrazole and 3,5-dicarboxy-4-oxo-4,5-dihydropyrazole. We also decipher the enzymatic mechanism of ForT/PrfT for C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfT represents an example of β-RFA-P (β-ribofuranosyl-aminobenzene 5ʹ-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics and also open the way for target-directed genome mining of PRF-A/FOR-A-related antibiotics. IMPORTANCE FOR-A and PRF-A are C-nucleoside antibiotics known for their unusual chemical structures and remarkable biological activities. Deciphering the enzymatic mechanism for the construction of a C-nucleoside scaffold during FOR-A/PRF-A biosynthesis will not only expand the biochemical repertoire for novel enzymatic reactions but also permit target-oriented genome mining of FOR-A/PRF-A-related C-nucleoside antibiotics. Moreover, the availability of FOR-A/PRF-A biosynthetic gene clusters will pave the way for the rational generation of designer FOR-A/PRF-A derivatives with enhanced/selective bioactivity via synthetic biology strategies.