Search

Natural products are a sustainable resource for drug discovery, but their identification in complex mixtures remains a daunting task. We present an automated pipeline that compares, harmonizes and ranks the annotations of LC-HRMS data by different tools. When applied to 7,400 extracts derived from 6,566 strains belonging to 86 actinomycete genera, it yielded 150,000 molecules after processing over 50 million MS features. The web-based Molecules Gateway provides a highly interactive access to experimental and calculated data for these molecules, along with the metadata related to extracts and producer strains. We show how the Molecules Gateway can be used to rapidly identify known hard to find microbial products, unreported analogs of known families and not yet described metabolites. The Molecules Gateway, which complements available repositories, contains annotated MS data, both acquired and computationally processed under an identical workflow, making it suitable for global analyses which reveal a large and untapped chemical diversity afforded by actinomycetes.

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 C 20 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., Competing Interests: The authors declare the following competing financial interest(s): M. I., C. B., S. I. M., S. D. and M. S. are inventors of patent on allopeptimicins owned by NAICONS. M. I., A. G., C. B., A. T., S. I. M., S. D. and M. S. are employees and shareholders of NAICONS., (This journal is © The Royal Society of Chemistry.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Microbial natural products impress by their bioactivity, structural diversity, and ingenious biosynthesis. While screening the less exploited actinobacterial genus Planomonospora, two cyclopeptides were discovered, featuring an unusual Tyr-His biaryl bridging across a tripeptide scaffold, with the sequences N-acetyl-Tyr-Tyr-His and N-acetyl-Tyr-Phe-His. Planomonospora genomes pointed toward a ribosomal synthesis of the cyclopeptide from a pentapeptide precursor encoded by 18-bp bytA, to our knowledge the smallest coding gene ever reported. Closely linked to bytA is bytO, encoding a cytochrome P450 monooxygenase likely responsible for biaryl installment. In Streptomyces, the bytAO segment was sufficient to direct production of the crosslinked N-acetylated Tyr-Tyr-His tripeptide. Bioinformatic analysis of related cytochrome P450 monooxygenases indicated that they constitute a widespread family of enzymes, and the corresponding genes are closely linked to 5-amino acid coding sequences in approximately 200 (actino)bacterial genomes, all with potential for biaryl linkage between amino acids 1 and 3. We propose the named biarylitides this family of RiPPs., Competing Interests: Declaration of interests M.M.Z., M.S., S.I.M., and S.D. have been and/or are employees and/or shareholders of Naicons Srl. M.H.M. is a co-founder of Design Pharmaceuticals and a member of the scientific advisory board of Hexagon Bio. The other authors declare that no competing interests exist., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

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 .

Glycopeptide antibiotics are used to treat severe multidrug resistant infections caused by Gram-positive bacteria. Dalbavancin is a second generation glycopeptide approved for human use, which is obtained from A40926, a lipoglycopeptide produced by Nonomuraea sp. ATCC39727 as a mixture of biologically active congeners mainly differing in the fatty acid chains present on the glucuronic moiety. In this study, we constructed a double mutant of the A40926 producer strain lacking dbv23, and thus defective in mannose acetylation, a feature that increases A40926 production, and lacking the acyltransferases Dbv8, and thus incapable of installing the fatty acid chains. The double mutant afforded the desired deacyl, deacetyl A40926 intermediates, which could be converted by chemical reacylation yielding A40926 analogs with a greatly reduced number of congeners. The newly acylated analogs could then be transformed into dalbavancin analogs possessing the same in vitro properties as the approved drug.

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.

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 13 C- 15 N-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.

In Escherichia coli, an increase in the frequency of chromosome replication is lethal. In order to identify compounds that affect chromosome replication, we screened for molecules capable of restoring the viability of hyper-replicating cells. We made use of two E. coli strains that over-initiate DNA replication by keeping the DnaA initiator protein in its active ATP bound state. While viable under anaerobic growth or when grown on poor media, these strains become inviable when grown in rich media. Extracts from actinomycetes strains were screened, leading to the identification of deferoxamine (DFO) as the active compound in one of them. We show that DFO does not affect chromosomal replication initiation and suggest that it was identified due to its ability to chelate cellular iron. This limits the formation of reactive oxygen species, reduce oxidative DNA damage and promote processivity of DNA replication. We argue that the benzazepine derivate (±)-6-Chloro-PB hydrobromide acts in a similar manner.

In screening for novel antibiotics, an attractive element of novelty can be represented by screening previously underexplored groups of microorganisms. We report the results of screening 200 strains belonging to the actinobacterial genus Actinoallomurus for their production of antibacterial compounds. When grown under just one condition, about half of the strains produced an extract that was able to inhibit growth of Staphylococcus aureus . We report here on the metabolites produced by 37 strains. In addition to previously reported aminocoumarins, lantibiotics and aromatic polyketides, we described two novel and structurally unrelated polyethers, designated α-770 and α-823. While we identified only one producer strain of the former polyether, 10 independent Actinoallomurus isolates were found to produce α-823, with the same molecule as main congener. Remarkably, production of α-823 was associated with a common lineage within Actinoallomurus , which includes A. fulvus and A. amamiensis . All polyether producers were isolated from soil samples collected in tropical parts of the world., Competing Interests: The authors declare no conflict of interest.

Pseudouridimycin (PUM) is a selective nucleoside-analog inhibitor of bacterial RNA polymerase with activity against Gram-positive and Gram-negative bacteria. PUM, produced by Streptomyces sp. ID38640, consists of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 5'-aminopseudouridine. We report the characterization of the PUM gene cluster. Bioinformatic analysis and mutational knockouts of pum genes with analysis of accumulated intermediates, define the PUM biosynthetic pathway. The work provides the first biosynthetic pathway of a C-nucleoside antibiotic and reveals three unexpected features: production of free pseudouridine by the dedicated pseudouridine synthase, PumJ; nucleoside activation by specialized oxidoreductases and aminotransferases; and peptide-bond formation by amide ligases. A central role in the PUM biosynthetic pathway is played by the PumJ, which represents a divergent branch within the TruD family of pseudouridine synthases. PumJ-like sequences are associated with diverse gene clusters likely to govern the biosynthesis of different classes of C-nucleoside antibiotics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 13 C-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.

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides containing thioether rings. In addition to these cross-links, the clinical candidate lantibiotic NAI-107 also possesses a C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) and a unique 5-chloro-l-tryptophan (ClTrp) moiety linked to its potent bioactivity. Bioinformatic and genetic analyses on the NAI-107 biosynthetic gene cluster identified mibH and mibD as genes encoding flavoenzymes responsible for the formation of ClTrp and AviCys, respectively. The biochemical basis for the installation of these modifications on NAI-107 and the substrate specificity of either enzyme is currently unknown. Using a combination of mass spectrometry, liquid chromatography, and bioinformatic analyses, we demonstrate that MibD is an FAD-dependent Cys decarboxylase and that MibH is an FADH 2 -dependent Trp halogenase. Most FADH 2 -dependent Trp halogenases halogenate free Trp, but MibH was only active when Trp was embedded within its cognate peptide substrate deschloro NAI-107. Structural comparison of the 1.88-Å resolution crystal structure of MibH with other flavin-dependent Trp halogenases revealed that subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining the substrate specificity of this unusual peptide halogenase.

A screening program on a limited number of strains belonging to the Actinoallomurus genus yielded a series of new angucyclinones. NMR and MS analyses established that these compounds are characterized by an unusual lactone ring and present up to four halogens per molecule, with one congener representing the first natural product containing a trichloromethyl substitution on an aromatic system. Remarkably, this family of metabolites seems to be produced by phylogenetically distinct Actinoallomurus isolates. Because of the unique structural features and wide distribution among Actinoallomurus, we have designated these angucyclinones as allocyclinones. Allocyclinones possess interesting activity against different Gram-positive bacteria, including antibiotic-resistant strains, with antibacterial potency increasing with the number of chlorine substituents. The tetrachlorinated compound is the most abundant congener in the allocyclinone complex.

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.

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.

Lantibiotics, an abbreviation for "lanthionine-containing antibiotics", interfere with bacterial metabolism by a mechanism not exploited by the antibiotics currently in clinical use. Thus, they have aroused interest as a source for new therapeutic agents because they can overcome existing resistance mechanisms. Starting from fermentation broth extracts preselected from a high-throughput screening program for discovering cell-wall inhibitors, we isolated a series of related class I lantibiotics produced by different genera of actinomycetes. Analytical techniques together with explorative chemistry have been used to establish their structures: the newly described compounds share a common 24 aa sequence with the previously reported lantibiotic planosporicin (aka 97518), differing at positions 4, 6, and 14. All of these compounds maintain an overall -1 charge at physiological pH. While all of these lantibiotics display modest antibacterial activity, their potency can be substantially modulated by progressively eliminating the negative charges, with the most active compounds carrying basic amide derivatives of the two carboxylates originally present in the natural compounds. Interestingly, both natural and chemically modified lantibiotics target the key biosynthetic intermediate lipid II, but the former compounds do not bind as effectively as the latter in vivo. Remarkably, the basic derivatives display an antibacterial potency and a killing effect similar to those of NAI-107, a distantly related actinomycete-produced class I lantibiotic which lacks altogether carboxyl groups and which is a promising clinical candidate for treating Gram-positive infections caused by multi-drug-resistant pathogens.

Among the growing family of ribosomally synthesized, post-translationally modified peptides, particularly intriguing are class III lanthipeptides containing the triamino acid labionin. In the course of a screening program aimed at finding bacterial cell wall inhibitors, we discovered a new lanthipeptide produced by an Actinoplanes sp. The molecule, designated NAI-112, consists of 22 amino acids and contains an N-terminal labionin and a C-terminal methyl-labionin. Unique among lanthipeptides, it carries a 6-deoxyhexose moiety N-linked to a tryptophan residue. Consistently, the corresponding gene cluster encodes, in addition to the LanKC enzyme characteristic of this lanthipeptide class, a glycosyl transferase. Despite possessing weak antibacterial activity, NAI-112 is effective in experimental models of nociceptive pain, reducing pain symptoms in mice in both the formalin and the chronic constriction injury tests. Thus, NAI-112 represents, after the labyrinthopeptins, the second example of a lanthipeptide effective against nociceptive pain.

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.

Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G-dependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected.

Lantibiotics are biologically active peptides produced by Gram-positive bacteria. Starting from fermentation broth extracts preselected from a high-throughput screening program for discovering cell-wall inhibitors, we successfully isolated a new lantibiotic produced by Actinoplanes sp., designated as NAI-802. MS and NMR analysis together with explorative chemistry established that NAI-802 consists of 21 amino acids, 19 of which are identical to those present in the class II lantibiotic actagardine. Interestingly, NAI-802 carries one extra alanine and one extra arginine at the N- and C-termini, respectively. As expected from the overall higher positive charge, NAI-802 was slightly more active than actagardine against staphylococci and streptococci. Further improvement of its antibacterial activity was achieved by adding one additional positive charge through conversion of the C-terminal carboxylate into the corresponding basic amide. NAI-802 thus represents a novel promising candidate for treating Gram-positive infections caused by multidrug-resistant pathogens.

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.

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.

The lantibiotic 97518, produced by a Planomonospora sp., was reported as a 2194 Da polypeptide comprising 24 amino acid residues with five thioether bridges. It was assigned to the mersacidin subgroup of type B lantibiotics by Castiglione et al. (Biochemistry 2007, 46, 5884-5897) and named planosporicin. New analytical, chemical, and genetic data and reinterpretation of the published NMR chemical shifts enable structure revision of 97518. The resulting revision of the 97518 structure involves both a shift of two amino acids and a reorganization of two thioether bridges. With this revision, the lantibiotic 97518 becomes a clear member of the nisin subgroup of compounds.

RNA polymerase (RNAP) currently represents an important target for the development of new antibacterial agents. RNAP is a nucleotidyl transferase enzyme able to generate an RNA copy of a DNA or RNA template chain, controlling initiation and termination of transcription. RNAP is found in nature in all eukaryotes, prokaryotes and archaea, as well as in many viruses. Bacterial RNAP is a large molecule (about 400 kDa) and its core structure is composed of four polypeptide subunits: alpha (alpha) required for assembly of the enzyme, beta (beta) involved in chain initiation and elongation, beta' (beta') which binds to the DNA template, and omega (omega) which constrains the beta' subunit and aids its assembly into RNAP, in the stoichiometry alpha2betabeta'omega. The bacterial enzyme differs both from eukaryotic RNAP, which is composed of different subunits and is present in several variants, and from archaeal or viral RNAP. These differences allow to selectively target the bacterial enzyme with appropriately designed inhibitors, excluding interactions with eukaryotic RNAP, accounting for the deep interest developed around these compounds as selective antibacterial agents. In this review the known natural and synthetic inhibitors of RNAP will be described considering their mechanism of action, biological activity, availability of analogues, Structure Activity Relationship (SAR) information and clinical use when already approved or recently entered into clinical trials.

Ramoplanin is a glycolipodepsipeptide antibiotic active against Gram-positive bacteria including vancomycin-resistant enterococci. Ramoplanin inhibits bacterial cell wall biosynthesis by a mechanism different from that of glycopeptides and hence does not show cross-resistance with these antibiotics. The systemic use of ramoplanin has been so far prevented because of its low local tolerability when injected intravenously. To overcome this problem, the fatty acid side chain of ramoplanin was selectively removed and replaced with a variety of different carboxylic acids. Many of the new ramoplanin derivatives showed antimicrobial activity similar to that of the natural precursor coupled with a significantly improved local tolerability. Among them the derivative in which the 2-methylphenylacetic acid has replaced the di-unsaturated fatty acid side chain (48) was selected as the most interesting compound and submitted to further in vitro and in vivo characterization studies.

[reaction: see text] A new, mild, and reversible method to convert primary amides to nitriles in good yields using PdCl2 in aqueous acetonitrile is described.

GE23077 is a novel RNA polymerase inhibitor that is isolated from the fermentation broth of an Actinomadura sp. It is a cyclic heptapeptide complex made up of four factors, differing in the structure of acyl group connected to the side chain of an alpha,beta-diaminopropanoic acid moiety and in the configuration of the stereocenter of an alpha-amino-malonic acid residue. Although GE23077 shows strong inhibitory activity on both Rifampicin-sensitive and -resistant polymerases, it exhibits poor antimicrobial activity. The most reasonable explanation for this property has been based on the lack of penetration of the molecule across the bacterial membrane, owing to its strong hydrophilic character. To improve penetration, several parts of the molecule were accordingly modified with the aim of altering the physico-chemical properties of GE23077. The current SAR study has identified moieties important for RNA polymerase activity.

New derivatives of the glycopeptide antibiotic A40926 were synthesized and evaluated for antimicrobial activity against VRE. Deacylated A40926 was obtained by microbial transformation of the parent antibiotic with the use of Actinoplanes teichomyceticus ATCC 31121. Regioselective synthesis of alkylated derivatives of Deacyl A40926 was carried out using lipophilic aliphatic and aromatic halides or aldehydes. Further modification of the two carboxylic acids was performed to increase antibiotic activity. Poor antimicrobial activity was observed for the derivatives obtained by lipophilic mono- or dialkylation of the amino groups present on the molecule, while simultaneous condensation of both carboxylic groups, in hydrophobic derivatives, with dibasic amines led to a strong increase in antibiotic activity.