Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.

Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.

N-Acetyl-Cysteinylated Streptophenazines from Streptomyces.

Here, we describe two N-acetyl-cysteinylated streptophenazines (1 and 2) produced by the soil-derived Streptomyces sp. ID63040 and identified through a metabolomic approach. These metabolites attracted our interest due to their low occurrence frequency in a large library of fermentation broth extracts and their consistent presence in biological replicates of the producer strain. The compounds were found to possess broad-spectrum antibacterial activity while exhibiting low cytotoxicity. The biosynthetic gene cluster from Streptomyces sp. ID63040 was found to be highly similar to the streptophenazine reference cluster in the MIBiG database, which originates from the marine Streptomyces sp. CNB-091. Compounds 1 and 2 were the main streptophenazine products from Streptomyces sp. ID63040 at all cultivation times but were not detected in Streptomyces sp. CNB-091. The lack of obvious candidates for cysteinylation in the Streptomyces sp. ID63040 biosynthetic gene cluster suggests that the N-acetyl-cysteine moiety derives from cellular functions, most likely from mycothiol. Overall, our data represent an interesting example of how to leverage metabolomics for the discovery of new natural products and point out the often-neglected contribution of house-keeping cellular functions to natural product diversification.

Planomonospora: A Metabolomics Perspective on an Underexplored Actinobacteria Genus.

Despite an excellent track record, microbial drug discovery suffers from high rates of rediscovery. Better workflows for the rapid investigation of complex extracts are needed to increase throughput and to allow early prioritization of samples. In addition, systematic characterization of poorly explored strains is seldomly performed. Here, we report a metabolomic study of 72 isolates belonging to the rare actinomycete genus Planomonospora, using a workflow of commonly used open access tools to investigate its secondary metabolites. The results reveal a correlation of chemical diversity and strain phylogeny, with classes of metabolites exclusive to certain phylogroups. We were able to identify previously reported Planomonospora metabolites, including the ureylene-containing oligopeptide antipain, the thiopeptide siomycin including new congeners, and the ribosomally synthesized peptides sphaericin and lantibiotic 97518. In addition, we found that Planomonospora strains can produce the siderophore desferrioxamine or a salinichelin-like peptide. Analysis of the genomes of three newly sequenced strains led to the detection of 59 gene cluster families, of which three were connected to products found by LC-MS/MS profiling. This study demonstrates the value of metabolomic studies to investigate poorly explored taxa and provides a first picture of the biosynthetic capabilities of the genus Planomonospora.

Effective approaches to discover new microbial metabolites in a large strain library.

Natural products have provided many molecules to treat and prevent illnesses in humans, animals and plants. While only a small fraction of the existing microbial diversity has been explored for bioactive metabolites, tens of thousands of molecules have been reported in the literature over the past 80 years. Thus, the main challenge in microbial metabolite screening is to avoid the re-discovery of known metabolites in a cost-effective manner. In this perspective, we report and discuss different approaches used in our laboratory over the past few years, ranging from bioactivity-based screening to looking for metabolic rarity in different datasets to deeply investigating a single Streptomyces strain. Our results show that it is possible to find novel chemistry through a limited screening effort, provided that appropriate selection criteria are in place.

Antibacterial Aromatic Polyketides Incorporating the Unusual Amino Acid Enduracididine.

The increasing incidence of infections caused by drug-resistant pathogens requires new efforts for the discovery of novel antibiotics. By screening microbial extracts in an assay aimed at identifying compounds interfering with cell wall biosynthesis, based on differential activity against a Staphylococcus aureus strain and its isogenic l-form, the potent enduracyclinones (1, 2), containing the uncommon amino acid enduracididine linked to a six-ring aromatic skeleton, were discovered from different Nonomuraea strains. The structures of 1 and 2 were established through a combination of derivatizations, oxidative cleavages, and NMR analyses of natural and 13C-15N-labeled compounds. Analysis of the biosynthetic cluster provides the combination of genes for the synthesis of enduracididine and type II polyketide synthases. Enduracyclinones are active against Gram-positive pathogens (especially Staphylococcus spp.), including multi-drug-resistant strains, with minimal inhibitory concentrations in the range of 0.0005 to 4 µg mL-1 and with limited toxicity toward eukaryotic cells. The combined results from assays and macromolecular syntheses suggest a possible dual mechanism of action in which both peptidoglycan and DNA syntheses are inhibited by these molecules.

Discovery, properties, and biosynthesis of pseudouridimycin, an antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase.

Pseudouridimycin (PUM) is a novel pseudouridine-containing peptidyl-nucleoside antibiotic that inhibits bacterial RNA polymerase (RNAP) through a binding site and mechanism different from those of clinically approved RNAP inhibitors of the rifamycin and lipiarmycin (fidaxomicin) classes. PUM was discovered by screening microbial fermentation extracts for RNAP inhibitors. In this review, we describe the discovery and characterization of PUM. We also describe the RNAP-inhibitory and antibacterial properties of PUM. Finally, we review available information on the gene cluster and pathway for PUM biosynthesis and on the potential for discovering additional novel pseudouridine-containing nucleoside antibiotics by searching bacterial genome and metagenome sequences for sequences similar to pumJ, the pseudouridine-synthase gene of the PUM biosynthesis gene cluster.

Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells.

Objectives: To characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria. Methods: We used MIC determinations and chequerboard experiments to establish the antibacterial activity of the examined compounds against target microorganisms. Time-kill assays were used to evaluate killing of exponential and stationary-phase cells. The effects on biofilms (growth inhibition and biofilm eradication) were evaluated using biofilm-coated pegs. The frequency of spontaneous resistant mutants was evaluated by either direct plating or by continuous sub-culturing at 0.5 × MIC levels, followed by population analysis profiles. Results: The results showed that NAI-107 and its brominated variant are highly active against Neisseria gonorrhoeae and some other fastidious Gram-negative pathogens. Furthermore, all compounds strongly synergized with polymyxin against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and showed bactericidal activity. Surprisingly, NAI-107 alone was bactericidal against non-dividing A. baumannii cells. Against S. aureus, NAI-107 and related lantibiotics showed strong bactericidal activity against dividing and non-dividing cells. Activity was also observed against S. aureus biofilms. As expected for a lipid II binder, no significant resistance to NAI-107 was observed by direct plating or serial passages. Conclusions: Overall, the results of the current work, along with previously published results on the efficacy of NAI-107 in experimental models of infection, indicate that this lantibiotic represents a promising option in addressing the serious threat of antibiotic resistance.

Antibacterial Paramagnetic Quinones from Actinoallomurus.

Four metabolites, designated paramagnetoquinone A, B, C, and D (1-4), were isolated from three strains belonging to the actinomycete genus Actinoallomurus. Compounds 1 and 2 showed potent antibacterial activity with MIC values lower than 0.015 µg/mL against Gram-positive pathogens, including antibiotic-resistant strains. Since compounds 1 and 2 were NMR-silent due to the presence of an oxygen radical, structure elucidation was achieved through a combination of derivatizations, oxidations, and analysis of 13C-labeled compounds. The paramagnetoquinones share the same carbon scaffold as tetracenomycin but carry two quinones and a five-membered lactone fused to the aromatic system. Compounds 2 and 1 are identical except for an unprecedented replacement of a methoxy in 2 by a methylamino group in 1. Related compounds devoid of methyl group(s) and of antibacterial activity were isolated from a different Actinoallomurus strain. The likely pmq biosynthetic gene cluster was identified from strain ID145113. While the cluster encodes many of the expected enzymes involved in the formation of aromatic polyketides, it also encodes a dedicated ketoacid dehydrogenase complex and an unusual acyl carrier protein transacylase, suggesting that an unusual starter unit might prime the polyketide synthase.

Advancing cell wall inhibitors towards clinical applications.

Natural products represent a major source of approved drugs and still play an important role in supplying chemical diversity. Consistently, 2014 has seen new, natural product-derived antibiotics approved for human use by the US Food and Drug Administration. One of the recently approved second-generation glycopeptides is dalbavancin, a semi-synthetic derivative of the natural product A40,926. This compound inhibits bacterial growth by binding to lipid intermediate II (Lipid II), a key intermediate in peptidoglycan biosynthesis. Like other recently approved antibiotics, dalbavancin has a complex history of preclinical and clinical development, with several companies contributing to different steps in different years. While our work on dalbavancin development stopped at the previous company, intriguingly our current pipeline includes two more Lipid II-binding natural products or derivatives thereof. In particular, we will focus on the properties of NAI-107 and related lantibiotics, which originated from recent screening and characterization efforts.

Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727.

UNLABELLED: The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, overexpression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, and dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, and dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation. IMPORTANCE: This report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions.

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

A Derivative of the Thiopeptide GE2270A Highly Selective against Propionibacterium acnes.

A chemical derivative of the thiopeptide GE2270A, designated NAI003, was found to possess a substantially reduced antibacterial spectrum in comparison to the parent compound, being active against just a few Gram-positive bacteria. In particular, NAI003 retained low MICs against all tested isolates of Propionibacterium acnes and, to a lesser extent, against Enterococcus faecalis. Furthermore, NAI003 showed a time- and dose-dependent killing of both a clindamycin-resistant and a clindamycin-sensitive P. acnes isolate. Gel shift experiments indicated that, like the parent compound, NAI003 retained the ability to bind to elongation factors Tu (EF-Tus) derived from Escherichia coli, E. faecalis, or P. acnes, albeit with reduced efficiency. In contrast, EF-Tus derived from the NAI003-insensitive Staphylococcus aureus or Streptococcus pyogenes did not bind this compound. These results were confirmed by in vitro studies using a hybrid translation system, which indicated that NAI003 can inhibit most efficiently protein synthesis driven by the P. acnes EF-Tu. P. acnes mutants resistant to NAI003 were isolated by direct plating. With one exception, all analyzed strains carried mutations in the tuf gene, encoding EF-Tu. Because of its selective effect on P. acnes in comparison to resident skin flora, NAI003 represents a promising candidate for the topical treatment of acne, which has already completed a phase 1 clinical study.

Brominated Variant of the Lantibiotic NAI-107 with Enhanced Antibacterial Potency.

We identified an Actinoallomurus strain producing NAI-107, a chlorinated lantibiotic effective against multidrug-resistant Gram-positive pathogens and previously reported from the distantly related genus Microbispora. Inclusion of KBr in the production medium of either the Actinoallomurus or the Microbispora producer readily afforded brominated variants of NAI-107, which were designated as NAI-108. The other post-translational modifications naturally occurring in this lantibiotic family (i.e., hydroxylation of Pro-14 and C-terminal decarboxylation) were unaffected by the presence of a brominated tryptophan. In addition to being the first example of a bromine-containing lantibiotic, NAI-108 displayed a small but consistent improvement in antibacterial activity against all tested strains. The brominated lantibiotic maintained the same rapid bactericidal activity as NAI-107 but at reduced concentrations, consistent with its increased potency and with the role played by the hydrophobicity of the first lanthionine ring. NAI-108 thus represents an interesting addition to a promising family of potent and effective lantibiotics.

Pharmacological properties of NAI-603, a well-tolerated semisynthetic derivative of ramoplanin.

NAI-603 is a ramoplanin derivative designed to overcome the tolerability issues of the parent drug as a systemic agent. NAI-603 is highly active against aerobic and anaerobic Gram-positive bacteria, with MICs ranging from 0.008 to 8 µg/ml. MICs were not significantly affected by pH (range, 6 to 8), by inoculum up to 10(8) CFU/ml, or by addition of 50% human serum. Against staphylococci and enterococci, NAI-603 was rapidly bactericidal, with minimum bactericidal concentration (MBC)/MIC ratios never exceeding 4. The frequency of spontaneous resistance was low at 2× to 4× MIC (≤1×10(-6) to ≤1×10(-8)) and below the detection limit (about ≤1×10(-9)) at 8×MIC. Serial subcultures at 0.5×MIC yielded at most an 8-fold increase in MICs. In a systemic infection induced by methicillin-resistant Staphylococcus aureus (MRSA), the 50% effective dose (ED50) of intravenous (i.v.) NAI-603 was 0.4 mg/kg, lower than that of oral (p.o.) linezolid (1.4 mg/kg) and subcutaneous (s.c.) teicoplanin (1.4 mg/kg) or vancomycin (0.6 mg/kg). In neutropenic mice infected with vancomycin-resistant enterococci (VRE), the ED50s for NAI-603 were 1.1 to 1.6 mg/kg i.v., compared to 0.5 mg/kg i.v. of ramoplanin. The bactericidal activity was confirmed in vivo in the rat granuloma pouch model induced by MRSA, where NAI-603, at 40 mg/kg i.v., induced about a 2- to 3-log10-reduction in viable bacteria in the exudates, which persisted for more than 72 h. The pharmacokinetic (PK) profiles of NAI-603 and ramoplanin at 20 mg/kg show similar half-lives (3.27 and 3.80 h, respectively) with the maximum concentration (Cmax) markedly higher for NAI-603 (207 µg/ml versus 79 µg/ml). The favorable pharmacological profile of NAI-603, coupled with the absence of local tolerability issues, supports further investigation.

Characterization of the congeners in the lantibiotic NAI-107 complex.

NAI-107, a lantibiotic produced by Microbispora sp. 107891, shows potent activity against multi-drug-resistant bacterial pathogens. It is produced as a complex of related molecules, which is unusual for ribosomally synthesized peptides. Here we describe the identification, characterization, and antibacterial activity of the congeners produced by Microbispora sp. 107891 and by the related Microbispora corallina NRRL 30420. These molecules differ by the presence of two, one, or zero hydroxyl groups at Pro-14, by the presence of a chlorine at Trp-4, and/or by the presence of a sulfoxide on the thioether of the first lanthionine.

Solution structure by nuclear magnetic resonance of the two lantibiotics 97518 and NAI-107.

Lantibiotics 97518 and NAI-107, produced by the related genera Planomonospora and Microbispora respectively, are members of a family of nisin-related compounds. They represent promising compounds to treat infections caused by multiresistant Gram-positive pathogens. Despite their similar structure and a similar antibacterial spectrum, the two lantibiotics exhibit significant differences in their potency. To gain an insight into the structure-activity relationships, their conformational properties in solution are determined by NMR. After carrying out an NOE analysis of 2D (1)H NMR spectra, high-resolution 3D structures are determined using molecular dynamics simulations.

Chrolactomycins from the actinomycete actinospica.

Examination of the metabolites produced by an Actinospica strain led to the identification of 6-hydroxychrolactomycin (compound 1), which is produced along with minor amounts of chrolactomycin (compound 2). The structure of 1 was established on the basis of extensive spectroscopic analysis, including one- and two-dimensional NMR. Compound 1 showed antimicrobial activity against Gram-positive bacteria, although it was generally less active than 2.

Halogenated spirotetronates from Actinoallomurus.

Two new members of the spirotetronate class, nai414-A and nai414-B, were discovered and isolated from an Actinoallomurus sp. Their structures were established by 1D and 2D NMR, UV, and MS analyses and by chemical degradation. They showed antimicrobial and antitumor activity against Gram-positive bacteria and against human microvascular endothelial cells, respectively. Substituting bromide for chloride ions in the growth medium afforded mono- and dibrominated derivatives.

Allopeptimicins: unique antibacterial metabolites generated by hybrid PKS-NRPS, with original self-defense mechanism in Actinoallomurus.

In the search for structurally novel metabolites with antibacterial activity, innovative approaches must be implemented to increase the probability of discovering novel chemistry from microbial sources. Here we report on the application of metabolomic tools to the genus Actinoallomurus, a poorly explored member of the Actinobacteria. From examining extracts derived from 88 isolates belonging to this genus, we identified a family of cyclodepsipeptides acylated with a C20 polyketide chain, which we named allopeptimicins. These molecules possess unusual structural features, including several double bonds in the amino-polyketide chain and four non-proteinogenic amino acids in the octapeptide. Remarkably, allopeptimicins are produced as a complex of active and inactive congeners, the latter carrying a sulfate group on the polyketide amine. This modification is also a mechanism of self-protection in the producer strain. The structural uniqueness of allopeptimicins is reflected in a biosynthetic gene cluster showing a mosaic structure, with dedicated gene cassettes devoted to formation of specialized precursors and modular assembly lines related to those from different pathways.

Insights into an unusual nonribosomal peptide synthetase biosynthesis: identification and characterization of the GE81112 biosynthetic gene cluster.

The GE81112 tetrapeptides (1-3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.