Inhibitors Of Bacterial RNA Polymerase Research Articles

Erratum for Balthazar et al., "A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase".

Erratum for Balthazar et al., "A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase".

A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase.

The continued emergence of multi-antibiotic-resistant strains of Neisseria gonorrhoeae necessitates the development of new antibiotics that are effective against this human pathogen. We previously described that the RNA polymerase-targeting antibiotic corallopyronin A (CorA) has potent activity against a large collection of clinical strains that express different antibiotic resistance phenotypes including when such gonococci are in a biofilm state. Herein, we tested whether a CorA-sensitive gonococcal strain could develop spontaneous resistance. Our finding that CorA resistance could only be achieved by a multi-step process involving over-expression of the MtrCDE efflux pump and single amino acid changes in RpoB and RpoC suggests that such resistance may be difficult for gonococci to evolve if this antibiotic is used in the future to treat gonorrheal infections that are refractory to cure by other antibiotics.

Beyond the approved: target sites and inhibitors of bacterial RNA polymerase from bacteria and fungi.

Covering: 2016 to 2022RNA polymerase (RNAP) is the central enzyme in bacterial gene expression representing an attractive and validated target for antibiotics. Two well-known and clinically approved classes of natural product RNAP inhibitors are the rifamycins and the fidaxomycins. Rifampicin (Rif), a semi-synthetic derivative of rifamycin, plays a crucial role as a first line antibiotic in the treatment of tuberculosis and a broad range of bacterial infections. However, more and more pathogens such as Mycobacterium tuberculosis develop resistance, not only against Rif and other RNAP inhibitors. To overcome this problem, novel RNAP inhibitors exhibiting different target sites are urgently needed. This review includes recent developments published between 2016 and today. Particular focus is placed on novel findings concerning already known bacterial RNAP inhibitors, the characterization and development of new compounds isolated from bacteria and fungi, and providing brief insights into promising new synthetic compounds.

Total synthesis and chemical stability of pseudouridimycin.

We report the chemical synthesis of pseudouridimycin (1), an antimicrobial natural product that potently and selectively inhibits bacterial RNA polymerase. Chemical stability studies revealed intramolecular hydroxamate bond scission to be a major decomposition pathway for 1 in aqueous buffer. Replacement of the hydroxamate bond with a tertiary amide, as in 16, afforded a conformational isostere resistant to degradation. These studies pave the way for the design and synthesis of analogues with improved chemical stability and biological activity.

Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin.

Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete, thus permitting the reinitiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase, and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.

Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain

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.

Description of the bacterial RNA polymerase inhibitor GE23077-producer Actinomadura sp. NRRL B-65521T as Actinomadura lepetitiana sp. nov.

The filamentous actinomycete that produces the antibiotic GE23077 was isolated by the Lepetit Research Group from a soil sample collected in Thailand, and it was classified as a member of the genus Actinomadura on the basis of its morphology and cell-wall composition. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain formed a distinct monophyletic line within the genus Actinomadura, and it was most closely related to Actinomadura bangladeshensis DSM 45347T (99.31 % similarity) and Actinomadura mexicana DSM 44485T (98.94 %). The GE23077-producing strain formed an extensively branched, non-fragmented vegetative mycelium; no pseudosporangia were formed and the arthrospores were organized in slightly twisted chains. The cell wall contained meso-2,6-diaminopimelic acid and the diagnostic sugar was madurose. The predominant menaquinone was MK-9(H6), with minor amounts of MK-9(H8) and MK-9(H4). The diagnostic phospholipids were phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were C16 : 0 and tuberculostearic acid (10-methyloctadecanoic acid), followed by minor amounts of C18:1ω9c, C16:1ω7c and 10-methylheptadecanoic acid. The genomic DNA G+C content was 71.77 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, and the low DNA-DNA relatedness between the GE23077-producing strain and closely related type strains clearly demonstrate that it represents a novel species of the genus Actinomadura, for which the name Actinomadura lepetitiana sp. nov. is proposed. The type strain is NRRL B-65521T(=LMG 31258T=DSM 109019T).

Discovery of novel N-aryl pyrrothine derivatives as bacterial RNA polymerase inhibitors.

Bacterial RNA polymerase (RNAP) is a validated drug target for broad-spectrum antibiotics, and its "switch region" is considered as the promising binding site for novel antibiotics. Based on the core scaffold of dithiolopyrrolone, a series of N-aryl pyrrothine derivatives was designed, synthesized, and evaluated for their antibacterial activity. Compounds generally displayed more active against Gram-positive bacteria, but less against Gram-negative bacteria. Among them, compound 6e exhibited moderate antibacterial activity against clinical isolates of rifampin-resistant Staphylococcus aureus with minimum inhibition concentration value of 1-2μg/ml and inhibited Escherichia coli RNAP with IC50 value of 12.0±0.9μM. In addition, compound 6e showed certain degree of cytotoxicity against HepG2 and LO2 cells. Furthermore, molecular docking studies suggested that compound 6e might interact with the switch region of bacterial RNAP in a similar conformation to myxopyronin A. Together, the N-aryl pyrrothine scaffold is a promising lead for discovery of antibacterial drugs acting against bacterial RNAP.

Synthesis and biological evaluation of novel N-2,4-dimethoxyphenyl dithiolopyrrolone derivatives as bacterial RNA polymerase inhibitors

Eighteen novel N-2,4-dimethoxyphenyl dithiolopyrrolone derivatives inhibiting bacterial RNA polymerase (RNAP) were synthesized based on dithiolopyrrolone scaffold. Some compounds displayed potent antimicrobial activity against Gram-positive bacteria of Staphylococcus aureus and Streptococcus pneumoniae, but not the Gram-negative bacteria of Escherichia coli and Pseudomonas aeruginosa. Moreover, the most promising compound 7b showed potent antibacterial activity against clinical isolates of MRSA, VRSA, RRSA, and MPRSP with MIC values in the range of 0.125–2 μg/mL, and potent inhibitory activity against Escherichia coli RNAP with IC50 value of 19.4 ± 1.3 μM. In addition, compound 7b showed cytotoxicity against LO2 cells with IC50 value of 18.5 ± 1.89 μM. Molecular docking studies revealed that compound 7b interacted with the switch region of the bacterial RNAP. Taken together, compound 7b might serve as a lead structure for developing potent bacterial RNAP inhibitors.

Design, synthesis and biological evaluation of novel hybrids of N-aryl pyrrothine-base α-pyrone as bacterial RNA polymerase inhibitors

Antibiotic resistance in bacteria has been an emerging public health problem, thus discovery of novel and effective antibiotics is urgent. A series of novel hybrids of N-aryl pyrrothine-base α-pyrone hybrids was designed, synthesized and evaluated as bacterial RNA polymerase (RNAP) inhibitors. Among them, compound 13c exhibited potent antibacterial activity against antibiotic-resistant S. aureus with the minimum inhibitory concentration (MIC) in the range of 1–4 μg/mL. Moreover, compound 13c exhibited strong inhibitory activity against E.coli RNAP with IC50 value of 16.06 μM, and cytotoxicity in HepG2 cells with IC50 value of 7.04 μM. The molecular docking study further suggested that compound 13c binds to the switch region of bacterial RNAP. In summary, compound 13c is a novel bacterial RNAP inhibitor, and a promising lead compound for further optimization.

Characterization of C-nucleoside Antimicrobials from Streptomyces albus DSM 40763: Strepturidin is Pseudouridimycin

Pseudouridimycin (PUM), a selective inhibitor of bacterial RNA polymerase has been previously detected in microbial-extracts of two strains of Streptomyces species (strain ID38640 and ID38673). Here, we isolated PUM and its deoxygenated analogue desoxy-pseudouridimycin (dPUM) from Streptomyces albus DSM 40763, previously reported to produce the metabolite strepturidin (STU). The isolated compounds were characterized by HRMS and spectroscopic techniques and they selectively inhibited transcription by bacterial RNA polymerase as previously reported for PUM. In contrast, STU could not be detected in the cultures of S. albus DSM 40763. As the reported characteristics reported for STU are almost identical with that of PUM, the existence of STU was questioned. We further sequenced the genome of S. albus DSM 40763 and identified a gene cluster that contains orthologs of all PUM biosynthesis enzymes but lacks the enzymes that would conceivably allow biosynthesis of STU as an additional product.

New inhibitors targeting bacterial RNA polymerase

Rifamycins, a group of bacterial RNA polymerase inhibitors, are the firstline antimicrobial drugs to treat tuberculosis. In light of the emergence of rifamycinresistant bacteria, development of new RNA polymerase inhibitors that kill rifamycinresistant bacteria with high bioavailability is urgent. Structural analysis of bacterial RNA polymerase in complex with inhibitors by crystallography and cryo-EM indicates that RNA polymerase inhibitors function through five distinct molecular mechanisms:inhibition of the extension of short RNA; competition with substrates; inhibition of the conformational change of the'bridge helix'; inhibition of clamp opening;inhibition of clamp closure. This article reviews the research progress of these five groups of RNA polymerase inhibitors to provide references for the modification of existing RNA polymerase inhibitors and the discovery of new RNA polymerase inhibitors.

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.

Analysis of the Pseudouridimycin Biosynthetic Pathway Provides Insights into the Formation of C-nucleoside Antibiotics

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 togovern the biosynthesis of different classes of C-nucleoside antibiotics.

Total Synthesis of Ripostatin B and Structure-Activity Relationship Studies on Ripostatin Analogs.

Described is the total synthesis of the myxobacterial natural product ripostatin B and of a small number of analogs. Ripostatin B is a polyketide-derived 14-membered macrolide that acts as an inhibitor of bacterial RNA-polymerase, but is mechanistically distinct from rifamycin-derived RNA-polymerase inhibitors that are in use for tuberculosis treatment. The macrolactone ring of ripostatin B features two stereocenters and a synthetically challenging doubly skipped triene motif, with one of the double bonds being in conjugation with the ester carbonyl. Appended to the macrolactone core are an extended hydroxy-bearing phenylalkyl side chain at C13 and a carboxymethyl group at C3. The triene motif was established with high efficiency by ring-closing olefin metathesis, which proceeded in almost 80% yield. The side chain-bearing stereocenter α to the ester oxygen was formed in a Paterson aldol reaction between a methyl ketone and a β-chiral β-hydroxy aldehyde with excellent syn selectivity (dr >10:1). The total synthesis provided a blueprint for the synthesis of analogs with modifications in the C3 and C13 side chains. The C3-modified analogs showed good antibacterial activity against efflux-deficient Escherichia coli but, as ripostatin B, were inactive against Mycobacterium tuberculosis, in spite of significant in vitro inhibition of M. tuberculosis RNA-polymerase.

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 againstdrug-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 invitro, 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 ofthe 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.

Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to RNAP, inhibits RNAP, and exhibits antibacterial activity. MRL-436 binds to RNAP through a binding site that differs from the rifampin binding site, inhibits rifampin-resistant RNAP derivatives, and exhibits antibacterial activity against rifampin-resistant strains. Isolation of mutants resistant to the antibacterial activity of MRL-436 yields a missense mutation in codon 622 of the rpoC gene encoding the RNAP β' subunit or a null mutation in the rpoZ gene encoding the RNAP ω subunit, confirming that RNAP is the functional cellular target for the antibacterial activity of MRL-436, and indicating that RNAP β' subunit residue 622 and the RNAP ω subunit are required for the antibacterial activity of MRL-436. Similarity between the resistance determinant for MRL-436 and the resistance determinant for the cellular alarmone ppGpp suggests a possible similarity in binding site and/or induced conformational state for MRL-436 and ppGpp.

Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening

Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (βS531L) containing a mutation (β′V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifRE. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.

Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening

Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (βS531L) containing a mutation (β'V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifR E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function.