Nucleoside Antibiotics Research Articles

Biosynthesis and combinatorial biosynthesis of antifungal nucleoside antibiotics.

There is an urgent need for new antifungal agents to treat or combat fungal infection in humans and plants. Antifungal nucleoside antibiotics are an important family of natural products with distinctive structural features. Understanding their biosynthetic machinery is of great importance for the improvement of antibiotics titers. More importantly, it is a requisite for combinatorial biosynthesis to create hybrid nucleoside antibiotics. We herein focus on findings on the natural and designed biosynthesis of this important family of nucleoside antibiotics.

ANTIDERMATOPHYTIC ACTIVITY OF STREPTOMYCES CACAOI SUBSP. CACAOI. M20

Objective: This study is an approach to find the alternative drug for the dermatophytosis because some of the existing antibiotics cause toxicity and also found expensive. Methods: There were 25 actinomycetes isolated from the soil sample of the mangrove, Avicennia marina from Ariyankuppam backwater area, Puducherry. Among the 25 isolates, the most active isolate, M20 was selected initially based on its antifungal activity, and it was checked for antidermatophytic activity against three dermatophytes, Microsporum gypseum, Trichophyton mentagrophytes, and Epidermophyton floccosum by agar plug method, well diffusion method followed by food poisoning technique. Release of volatile compounds from the isolate M20 has also been tested. Ultraviolet (UV)-vis spectral analysis of partially purified yellow compound fraction was done. Results: The isolate M20 actively controlled the growth of dermatophytes by agar plug method in the primary screening, well diffusion method in the secondary screening. The isolate was identified as Streptomyces cacaoi sub sp. cacaoi by 16sRNA sequencing analysis. The 10% (10 ml) culture filtrate of the isolate M20 was found to control the radial growth of these three dermatophytic fungi by food poisoning technique. Volatile compounds of the isolate M20 affected the mycelial growth of M. gypseum tremendously. Complete arrest of growth of M. gypseum was noticed with the combination of both volatile and non-volatile antibiotic compound of the isolate M20. The partially purified compound fraction (yellow) (0.5 mg/6 mm disc) inhibited the growth of M. gypseum (12 mm), and it was compared with the standard antibiotic clotrimazole (0.5 mg/6 mm disc), whose inhibition on M. gypseum was only 6 mm. UV-vis spectral analysis revealed the compound belonged to nucleoside antibiotics. Conclusion: It is evident that the mangrove actinomycetes are potential for preparing biomedicines for human welfare.

A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter.

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

Identification and characterization of lbpA, an indigoidine biosynthetic gene in the γ-butyrolactone signaling system of Streptomyces lavendulae FRI-5

Streptomyces lavendulae FRI-5 produces the blue pigment indigoidine and other secondary metabolites (d-cycloserine and nucleoside antibiotics). The production of these useful compounds is controlled by a signaling cascade mediated by the γ-butyrolactone autoregulator IM-2. Previously we revealed that the far regulatory island includes the IM-2 receptor, the IM-2 biosynthetic enzyme, and several transcriptional regulators, and that it contributes to the regulation of indigoidine production in response to the signaling molecule. Here, we found that the vicinity of the far regulatory island includes the putative gene cluster for the biosynthesis of indigoidine and unidentified compounds, and demonstrated that the expression of the gene cluster is under the control of the IM-2 regulatory system. Heterologous expression of lbpA, encoding a plausible nonribosomal peptide synthetase, in the versatile model host Streptomyces avermitilis SUKA22 led to indigoidine production, which was enhanced dramatically by feeding of the indigoidine precursor l-glutamine. These results confirmed that LbpA is an indigoidine biosynthetic enzyme in the IM-2 signaling cascade.

Biosynthesis of 2′-Chloropentostatin and 2′-Amino-2′-Deoxyadenosine Highlights a Single Gene Cluster Responsible for Two Independent Pathways in Actinomadura sp. Strain ATCC 39365

2'-Chloropentostatin (2'-Cl PTN, 2'-chloro-2'-deoxycoformycin) and 2'-amino-2'-deoxyadenosine (2'-amino dA) are two adenosine-derived nucleoside antibiotics coproduced by Actinomadura sp. strain ATCC 39365. 2'-Cl PTN is a potent adenosine deaminase (ADA) inhibitor featuring an intriguing 1,3-diazepine ring, as well as a chlorination at C-2' of ribose, and 2'-amino dA is an adenosine analog showing bioactivity against RNA-type virus infection. However, the biosynthetic logic of them has remained poorly understood. Here, we report the identification of a single gene cluster (ada) essential for the biosynthesis of 2'-Cl PTN and 2'-amino dA. Further systematic genetic investigations suggest that 2'-Cl PTN and 2'-amino dA are biosynthesized by independent pathways. Moreover, we provide evidence that a predicted cation/H+ antiporter, AdaE, is involved in the chlorination step during 2'-Cl PTN biosynthesis. Notably, we demonstrate that 2'-amino dA biosynthesis is initiated by a Nudix hydrolase, AdaJ, catalyzing the hydrolysis of ATP. Finally, we reveal that the host ADA (designated ADA1), capable of converting adenosine/2'-amino dA to inosine/2'-amino dI, is not very sensitive to the powerful ADA inhibitor pentostatin. These findings provide a basis for the further rational pathway engineering of 2'-Cl PTN and 2'-amino dA production.IMPORTANCE 2'-Cl PTN/PTN and 2'-amino dA have captivated the great interests of scientists, owing to their unusual chemical structures and remarkable bioactivities. However, the precise logic for their biosynthesis has been elusive for decades. Actually, the identification and elucidation of their biosynthetic pathways not only enrich the biochemical repertoire of novel enzymatic reactions but may also lay solid foundations for the pathway engineering and combinatorial biosynthesis of this family of purine nucleoside antibiotics to generate novel hybrid analogs with improved features.

Discovery of the Showdomycin Gene Cluster from Streptomyces showdoensis ATCC 15227 Yields Insight into the Biosynthetic Logic of C-Nucleoside Antibiotics.

Nucleoside antibiotics are a large class of pharmaceutically relevant chemical entities, which exhibit a broad spectrum of biological activities. Most nucleosides belong to the canonical N-nucleoside family, where the heterocyclic unit is connected to the carbohydrate through a carbon-nitrogen bond. However, atypical C-nucleosides were isolated from Streptomyces bacteria over 50 years ago, but the molecular basis for formation of these metabolites has been unknown. Here, we have sequenced the genome of S. showdoensis ATCC 15227 and identified the gene cluster responsible for showdomycin production. Key to the detection was the presence of sdmA, encoding an enzyme of the pseudouridine monophosphate glycosidase family, which could catalyze formation of the C-glycosidic bond. Sequence analysis revealed an unusual combination of biosynthetic genes, while inactivation and subsequent complementation of sdmA confirmed the involvement of the locus in showdomycin formation. The study provides the first steps toward generation of novel C-nucleosides by pathway engineering.

Complete genome sequence of Streptomyces griseochromogenes ATCC 14511T, a producer of nucleoside compounds and diverse secondary metabolites

ATCC 14511T (=DSM 40499, =NBRC 13413) is a type strain of Streptomyces griseochromogenes. It is known as a producer of nucleoside antibiotic, blasticidin S. In this report we present the complete genome sequence of S. griseochromogenes ATCC 14511T, which consists of 10,764,674bp with a linear chromosome, 9822 protein-coding genes, 6 rRNA operons, 74 tRNA and 3 sRNA. The genomic analysis revealed that 52 putative gene clusters are involved in the biosynthesis of secondary metabolites, including four gene clusters of nucleoside antibiotics. These gene clusters provide a beneficial source for production of bioactive natural compounds.

Nature's combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in Streptomyces.

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.

An Unusual Protector-Protégé Strategy for the Biosynthesis of Purine Nucleoside Antibiotics

Pentostatin (PTN, deoxycoformycin) and arabinofuranosyladenine (Ara-A, vidarabine) are purine nucleoside antibiotics used clinically to treat hematological cancers and human DNA virus infections, respectively. PTN has a 1,3-diazepine ring, and Ara-A is an adenosine analog with an intriguing epimerization at the C-2' hydroxyl group. However, the logic underlying the biosynthesis of these interesting molecules has long remained elusive. Here, we report that thebiosynthesis of PTN and Ara-A employs an unusual protector-protégé strategy. To our surprise, we determined that a single gene cluster governs PTN and Ara-A biosynthesis via two independent pathways. Moreover, we verified that PenB functions asa reversible oxidoreductase for the final step ofPTN. Remarkably, we provided the first direct biochemical evidence that PTN can protect Ara-A from deamination by selective inhibition of the hostadenosine deaminase. These findings expand our knowledge of natural product biosynthesis and open the way for target-directed genome mining of Ara-A/PTN-related antibiotics.

Construction of an octosyl acid backbone catalyzed by a radical S-adenosylmethionine enzyme and a phosphatase in the biosynthesis of high-carbon sugar nucleoside antibiotics.

Unique bicyclic octosyl uronic acid nucleosides include ezomycin, malayamycin, and octosyl acid (OA). They are structurally characterized by OA, an unusual 8-carbon furanosyl nucleoside core proposed to be the precursor to polyoxin and nikkomycin. Despite the well-known bioactivity of these nucleoside antibiotics, the biosynthesis of OA has not been elucidated yet. Here we report the two pivotal enzymatic steps in the polyoxin biosynthetic pathway leading to the identification of OA as a key intermediate. Our data suggest that this intermediate is formed via a free radical reaction catalyzed by the radical S-adenosylmethionine (SAM) enzyme, PolH, and using 3'-enolpyruvyl uridine 5'-monophosphate (3'-EUMP) as a substrate. Subsequent dephosphorylation catalyzed by phosphatase PolJ converts the resulting octosyl acid 5'-phosphate (OAP) to OA. These results provide, for the first time, significant in vitro evidence for the biosynthetic origins of the C8 backbone of OA.

Trans-glycosylation capacity of a highly glycosylated multi-specific β-glucosidase from Fusarium solani.

An extracellular β-glucosidase from Fusaruim solani cultivated on wheat bran was purified by only two chromatographic steps. The purified enzyme exhibited optimal temperature and pH at 60°C and pH 5, respectively. The purified β-glucosidase behaves as a very large protein due to its high degree of glycosylation. More interestingly, the endoglycosidase H (Endo H) treatment led to 97.55% loss of its initial activity after 24h of treatment. Besides, the addition of Tunicamycin (nucleoside antibiotic blocking the N-glycosylation first step) during the culture of the fungus affected seriously the glycosylation of the enzyme. Both treatments (endo H and Tunicamycin) strengthened the idea that the hyperglycosylation is involved in the β-glucosidase activity and thermostability. This enzyme was also shown to belong to class III of β-glucosidases (multi-specific) since it was able to act on either cellobiose, gentiobiose or sophorose which are disaccharide composed of two units of D-glucose connected by β1-4, β1-6 and β1-2 linkage, respectively. The β-glucosidase activity was strongly enhanced by ferrous ion (Fe2+) and high ionic strength (1M KCl). The purified enzyme exhibited an efficient transglycosylation capacity allowing the synthesis of cellotriose and cellotetraose using cellobiose as donor.

Critical role of the proton-dependent oligopeptide transporter (POT) in the cellular uptake of the peptidyl nucleoside antibiotic, blasticidin S

Blasticidin S (BlaS) interferes in the cell growth of both eukaryotes and prokaryotes. Its mode of action as a protein synthesis inhibitor has been investigated extensively. However, the mechanism of BlaS transport into the target cells is not understood well. Here, we show that Ptr2, a member of the proton-dependent oligopeptide transporter (POT) family, is responsible for the uptake of BlaS in yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Notably, some mutants of Ptr2 that are dysfunctional in dipeptide uptake were still competent to transport BlaS. Mouse-derived oligopeptide transporter PepT1 conferred BlaS sensitivity in the S. cerevisiae ptr2∆ mutant. Furthermore, bacterial POT family proteins also potentiated the BlaS sensitivity of E. coli. The role of the POT family oligopeptide transporters in the uptake of BlaS is conserved across species from bacteria to mammals.

A simple extraction method for the detection and quantification of polyoxin D, a nucleoside antibiotic, in butterbur using UPLC-MS/MS.

An effective analytical method was developed for the detection and quantification of polyoxin D in butterbur using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were extracted using acidified deionised water and purified via solid-phase extraction (SPE) using an HLB cartridge. An external matrix-matched standard calibration curve was prepared, which provided an excellent linearity with a coefficient of determination (R2) ⩾0.999. Limits of detection (LOD) and quantification (LOQ) were 0.015 and 0.0.05μg/g, respectively. The developed method was validated in terms of recovery performance using two fortification levels in triplicate. The storage stabilities of the various field samples were also determined. Our method provided consistent recovery (86.26-87.37%) with a relative standard deviation (RSD) of <5%, and was successfully applied to field-treated butterbur grown under greenhouse conditions and collected at various times following commercial fungicide application. As expected, a gradual degradation of polyoxin D was observed, with a half-life (t1/2) of 2.11d being recorded. Finally, we propose that the developed method can be extrapolated to other crops for routine analysis and can be used to determine the pre-harvest intervals (PHIs), thus preventing the development of antibiotic resistance genes in humans and in the environment.

Tunicamycin aggravates endoplasmic reticulum stress and airway inflammation via PERK-ATF4-CHOP signaling in a murine model of neutrophilic asthma

Introduction: Endoplasmic reticulum (ER) stress has been considered to be an important regulator of airway inflammation in the pathogenesis of bronchial asthma, but the mechanism of ER stress involved in neutrophilic asthma remain not fully understood. Methods: Tunicamycin is a mixture of homologous nucleoside antibiotics, which is used to induce ER stress. In the present study, Tunicamycin was administered to mouse bronchial epithelial cells and a neutrophilic asthma model (OVALPS-OVA mice), and ER stress indicators and inflammatory cytokines were measured by Western blotting and Elisa. Results: Tunicamycin not only induced ER stress in mouse bronchial epithelial cells, but also increased expression of inflammation indicators such as IL-6, IL-8, and TNF-α via PERK-ATF4-CHOP signaling. Additionally, the phosphorylation of PERK and the expression levels of ATF4 and CHOP proteins and inflammatory cytokines (IL-6, IL-8 and TNF-α) were elevated in the lung tissue of OVALPS-OVA mice. Administering tunicamycin further increased protein expression levels of ER stress indicators and inflammatory cytokines, and resulted in more severe asthma phenotypes in OVALPS-OVA mice, suggesting that PERK-ATF4-CHOP signaling is associated with airway inflammation in neutrophil-dominant asthma. Conclusions: These data support the emerging notion that regulation of ER stress could be strongly associated with the development of neutrophilic asthma.

Five-Membered Cyclitol Phosphate Formation by a myo-Inositol Phosphate Synthase Orthologue in the Biosynthesis of the Carbocyclic Nucleoside Antibiotic Aristeromycin.

Aristeromycin is a unique carbocyclic nucleoside antibiotic produced by Streptomyces citricolor. In order to elucidate its intriguing carbocyclic formation, we used a genome-mining approach to identify the responsible enzyme. In silico screening with known cyclitol synthases involved in primary metabolism, such as myo-inositol-1-phosphate synthase (MIPS) and dehydroqunate synthase (DHQS), identified a unique MIPS orthologue (Ari2) encoded in the genome of S. citricolor. Heterologous expression of the gene cluster containing ari2 with a cosmid vector in Streptomyces albus resulted in the production of aristeromycin, thus indicating that the cloned DNA region (37.5 kb) with 33 open reading frames contains its biosynthetic gene cluster. We verified that Ari2 catalyzes the formation of a novel five-membered cyclitol phosphate from d-fructose 6-phosphate (F6P) with NAD+ as a cofactor. This provides insight into cyclitol phosphate synthase as a member of the MIPS family of enzymes. A biosynthetic pathway to aristeromycin is proposed based on bioinformatics analysis of the gene cluster.

An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis.

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117–131), Loop 2 (residues 192–201) and the substrate recognition peptide (residues 94–102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0289-y) contains supplementary material, which is available to authorized users.

Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes 1628.

Modification of enzymes involved in transcription- or translation-processes is an interesting way to increase secondary metabolite production in Streptomycetes. However, application of such methods has not been widely described for strains which produce nucleoside antibiotics. The nucleoside antibiotic toyocamycin (TM) is produced by Streptomyces diastatochromogenes 1628. For improving TM production in S. diastatochromogenes 1628, the strain was spread on rifamycin-resistant (Rif(r)) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase β-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5% among the total Rif(r) mutants. Mutant 1628-T15 harboring amino acid substitution His437Arg was the best TM producer with a 4.5-fold increase in comparison to that of the wild-type strain. The worst producer was mutant 1628-T62 which also showed a poor sporulation behavior. RT-PCR was performed to study the transcription levels of the TM biosynthetic gene toyG in the parental strain as well as in mutants 1628-T15 and 1628-T62. The transcriptional level of toyG was higher in mutant 1628-T15 than that in parental strain 1628, while much lower in mutant 1628-T62. In mutant strain 1628-T62 the expression of adpA sd gene, which is required for morphological differentiation, was also much lower. Our studies also indicate that the introduction of mutations into rpoB is an effective strategy to improve the production of TM which is an important nucleoside antibiotic.

Natural and engineered biosynthesis of nucleoside antibiotics in Actinomycetes.

Nucleoside antibiotics constitute an important family of microbial natural products bearing diverse bioactivities and unusual structural features. Their biosynthetic logics are unique with involvement of complex multi-enzymatic reactions leading to the intricate molecules from simple building blocks. Understanding how nature builds this family of antibiotics in post-genomic era sets the stage for rational enhancement of their production, and also paves the wayfor targeted persuasion of the cell factories to make artificial designer nucleoside drugs and leads via synthetic biology approaches. In this review, we discuss the recent progress and perspectives on the natural and engineered biosynthesis of nucleoside antibiotics.

Synthesis and pharmacological evaluation of nucleoside prodrugs designed to target siderophore biosynthesis in Mycobacterium tuberculosis

The nucleoside antibiotic, 5′-O-[N-(salicyl)sulfamoyl]adenosine (1), possesses potent whole-cell activity against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). This compound is also active in vivo, but suffers from poor drug disposition properties that result in poor bioavailability and rapid clearance. The synthesis and evaluation of a systematic series of lipophilic ester prodrugs containing linear and α-branched alkanoyl groups from two to twelve carbons at the 3′-position of a 2′-fluorinated analog of 1 is reported with the goal to improve oral bioavailability. The prodrugs were stable in simulated gastric fluid (pH 1.2) and under physiological conditions (pH 7.4). The prodrugs were also remarkably stable in mouse, rat, and human serum (relative serum stability: human∼rat≫mouse) displaying a parabolic trend in the SAR with hydrolysis rates increasing with chain length up to eight carbons (t1/2=1.6h for octanoyl prodrug 7 in mouse serum) and then decreasing again with higher chain lengths. The permeability of the prodrugs was also assessed in a Caco-2 cell transwell model. All of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction and enhanced permeation in the basolateral-to-apical direction relative to the parent compound 2, resulting in efflux ratios 5–28 times greater than 2. Additionally, Caco-2 cells were found to hydrolyze the prodrugs with SAR mirroring the serum stability results and a preference for hydrolysis on the apical side. Taken together, these results suggest that the described prodrug strategy will lead to lower than expected oral bioavailability of 2 and highlight the contribution of intestinal esterases for prodrug hydrolysis.

A Modular Approach to Phosphoglycosyltransferase Inhibitors Inspired by Nucleoside Antibiotics.

Phosphoglycosyltransferases (PGTs) represent "gatekeeper" enzymes in complex glycan assembly pathways by catalyzing transfer of a phosphosugar from an activated nucleotide diphosphosugar to a membrane-resident polyprenol phosphate. The unique structures of selected nucleoside antibiotics, such as tunicamycin and mureidomycin A, which are known to inhibit comparable biochemical transformations, are exploited as the foundation for the development of modular synthetic inhibitors of PGTs. Herein we present the design, synthesis, and biochemical evaluation of two readily manipulatable modular scaffolds as inhibitors of monotopic bacterial PGTs. Selected compounds show IC50 values down to the 40 μm range, thereby serving as lead compounds for future development of selective and effective inhibitors of diverse PGTs of biological and medicinal interest.