Small molecule MarR modulators potentiate metronidazole antibiotic activity in aerobic E. coli by inducing activation by the nitroreductase NfsA.

Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.

Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae.

Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.

Regioselective and Stereoselective Synthesis of Parthenolide Analogs by Acyl Nitroso-Ene Reaction and Their Biological Evaluation against Mycobacterium tuberculosis.

Historically, natural products have played a major role in the development of antibiotics. Their complex chemical structures and high polarity give them advantages in the drug discovery process. In the broad range of natural products, sesquiterpene lactones are interesting compounds because of their diverse biological activities, their high-polarity, and sp3-carbon-rich chemical structures. Parthenolide (PTL) is a natural compound isolated from Tanacetum parthenium, of the family of germacranolide-type sesquiterpene lactones. In recent years, parthenolide has been studied for its anti-inflammatory, antimigraine, and anticancer properties. Recently, PTL has shown antibacterial activities, especially against Gram-positive bacteria. However, few studies are available on the potential antitubercular activities of parthenolide and its analogs. It has been demonstrated that parthenolide's biological effects are linked to the reactivity of α-exo-methylene-γ-butyrolactone, which reacts with cysteine in targeted proteins via a Michael addition. In this work, we describe the ene reaction of acylnitroso intermediates with parthenolide leading to the regioselective and stereoselective synthesis of new derivatives and their biological evaluation. The addition of hydroxycarbamates and hydroxyureas led to original analogs with higher polarity and solubility than parthenolide. Through this synthetic route, the Michael acceptor motif was preserved and is thus believed to be involved in the selective activity against Mycobacterium tuberculosis.

A Novel Natural Siderophore Antibiotic Conjugate Reveals a Chemical Approach to Macromolecule Coupling.

Inspired by natural sideromycins, the conjugation of antibiotics to siderophores is an attractive strategy to facilitate "Trojan horse" delivery of antibiotics into bacteria. Genome analysis of a soil bacterium, Dactylosporangium fulvum, found a "hybrid" biosynthetic gene cluster responsible for the production of both an antibiotic, pyridomycin, and a novel chlorocatechol-containing siderophore named chlorodactyloferrin. While both of these natural products were synthesized independently, analysis of the culture supernatant also identified a conjugate of both molecules. We then found that the addition of ferric iron to purified chlorodactyloferrin and pyridomycin instigated their conjugation, leading to the formation of a covalent bond between the siderophore-catechol and the pyridomycin-pyridine groups. Using model reactants, this iron-based reaction was found to proceed through a Michael-type addition reaction, where ferric iron oxidizes the siderophore-catechol group into its quinone form, which is then attacked by the antibiotic pyridyl-nitrogen to form the catechol-pyridinium linkage. These findings prompted us to explore if other "cargo" molecules could be attached to chlorodactyloferrin in a similar manner, and this was indeed confirmed with a pyridine-substituted TAMRA fluorophore as well as with pyridine-substituted penicillin, rifampicin, and norfloxacin antibiotic analogues. The resultant biomimetic conjugates were demonstrated to effectively enter a number of bacteria, with TAMRA-chlorodactyloferrin conjugates causing fluorescent labeling of the bacteria, and with penicillin and rifampicin conjugates eliciting antibiotic activity. These findings open up new opportunities for the design and facile synthesis of a novel class of biomimetic siderophore conjugates with antibiotic activity.

Characterization of pyridylpiperazine-based efflux pump inhibitors for Acinetobacter baumannii.

Objectives: In Acinetobacter baumannii, multidrug efflux pumps belonging to the resistance-nodulation-division (RND) superfamily result in decreased antibiotic susceptibility. Improving the activity of current antibiotics via efflux pump inhibitors (EPIs) represents an attractive alternative approach to control this bacterium. Pyridylpiperazines (PyrPips) are a new class of EPIs that can effectively inhibit the Escherichia coli RND efflux pump AcrAB-TolC and boost the activity of several antibiotics. Here we have evaluated and characterized whether the PyrPip chemical family is also able to boost antibiotic activity through inhibition of the RND efflux pumps in A. baumannii. Methods: Comparative structural modelling and docking, structure-activity relationship studies alongside molecular genetic approaches were deployed to improve, characterize and validate PyrPips' target. Results: We showed that two enhanced PyrPip EPIs are capable of rescuing the activity of different classes of antibiotics in A. baumannii. By expressing A. baumannii main efflux pumps (AdeB, AdeG and AdeJ) individually in E. coli recombinant strains, we could gain further insights about the EPIs' capacity to act upon each pump. Finally, we showed that PyrPip EPIs are mostly acting through AdeJ inhibition via interactions with two key charged residues, namely E959 and E963. Conclusions: Our work demonstrates that PyrPip EPIs are capable of inhibiting RND efflux pumps of A. baumannii, and thus may present a promising chemical scaffold for further development.

2,6-Disubstituted 7-(naphthalen-2-ylmethyl)-7H-purines as a new class of potent antitubercular agents inhibiting DprE1.

Phenotypic screening of an in-house library of small molecule purine derivatives against Mycobacterium tuberculosis (Mtb) led to the identification of 2-morpholino-7-(naphthalen-2-ylmethyl)-1,7-dihydro-6H-purin-6-one 10 as a potent antimycobacterial agent with MIC99 of 4 µM. Thorough structure-activity relationship studies revealed the importance of 7-(naphthalen-2-ylmethyl) substitution for antimycobacterial activity, yet opened the possibility of structural modifications at positions 2 and 6 of the purine core. As the result, optimized analogues with 6-amino or ethylamino substitution 56 and 64, respectively, were developed. These compounds showed strong in vitro antimycobacterial activity with MIC of 1 µM against Mtb H37Rv and against several clinically isolated drug-resistant strains, had limited toxicity to mammalian cell lines, medium clearance with respect to phase I metabolic deactivation (27 and 16.8 µL/min/mg), sufficient aqueous solubility (>90 µM) and high plasma stability. Interestingly, investigated purines, including compounds 56 and 64, lacked activity against a panel of Gram-negative and Gram-positive bacterial strains, indicating a specific mycobacterial molecular target. To investigate the mechanism of action, Mtb mutants resistant to hit compound 10 were isolated and their genomes were sequenced. Mutations were found in dprE1 (Rv3790), which encodes decaprenylphosphoryl-ß-d-ribose oxidase DprE1, enzyme essential for the biosynthesis of arabinose, a vital component of the mycobacterial cell wall. Inhibition of DprE1 by 2,6-disubstituted 7-(naphthalen-2-ylmethyl)-7H-purines was proved using radiolabelling experiments in Mtb H37Rv in vitro. Finally, structure-binding relationships between selected purines and DprE1 using molecular modeling studies in tandem with molecular dynamic simulations revealed the key structural features for effective drug-target interaction.

Optimization of pyridylpiperazine-based inhibitors of the Escherichia coli AcrAB-TolC efflux pump.

Multidrug-resistant Escherichia coli is a continuously growing worldwide public health problem, in which the well-known AcrAB-TolC tripartite RND efflux pump is a critical driver. We have previously described pyridylpiperazines as a novel class of allosteric inhibitors of E. coli AcrB which bind to a unique site in the protein transmembrane domain, allowing for the potentiation of antibiotic activity. Here, we show a rational optimization of pyridylpiperazines by modifying three specific derivatization points of the pyridine core to improve the potency and the pharmacokinetic properties of this chemical series. In particular, this work found that the introduction of a primary amine to the pyridine through ester (29, BDM91270) or oxadiazole (44, BDM91514) based linkers allowed for analogues with improved antibiotic boosting potency through AcrB inhibition. In vitro studies, using genetically engineered mutants, showed that this improvement in potency is mediated through novel interactions with distal acidic residues of the AcrB binding pocket. Of the two leads, compound 44 was found to have favorable physico-chemical properties and suitable plasma and microsomal stability. Together, this work expands the current structure-activity relationship data on pyridylpiperazine efflux pump inhibitors, and provides a promising step towards future in vivo proof of concept of pyridylpiperazines as antibiotic potentiators.

Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria.

Antimicrobial resistance (AMR) has become a major problem in public health leading to an estimated 4.95 million deaths in 2019. The selective pressure caused by the massive and repeated use of antibiotics has led to bacterial strains that are partially or even entirely resistant to known antibiotics. AMR is caused by several mechanisms, among which the (over)expression of multidrug efflux pumps plays a central role. Multidrug efflux pumps are transmembrane transporters, naturally expressed by Gram-negative bacteria, able to extrude and confer resistance to several classes of antibiotics. Targeting them would be an effective way to revive various options for treatment. Many efflux pump inhibitors (EPIs) have been described in the literature; however, none of them have entered clinical trials to date. This review presents eight families of EPIs active against Escherichia coli or Pseudomonas aeruginosa. Structure-activity relationships, chemical synthesis, in vitro and in vivo activities, and pharmacological properties are reported. Their binding sites and their mechanisms of action are also analyzed comparatively.

Tricyclic SpiroLactams Kill Mycobacteria In Vitro and In Vivo by Inhibiting Type II NADH Dehydrogenases.

It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.

Dactylosporolides: Glycosylated Macrolides from Dactylosporangium fulvum.

A series of novel macrolides were discovered from the culture supernatant of the rare soil actinobacteria Dactylosporangium fulvum and named dactylosporolides A-C. The structure and absolute configuration of these dactylosporolides were defined using a combination of NMR structural elucidation and analysis of the dactylosporolide biosynthetic gene cluster. Together these data revealed dactylosporolides to be composed of a central 22-membered macrolactone with an internal hemiketal ring and a protruding ketide tail that were (poly)glycosylated at two distal parts. While bearing no antibiotic activity, these dactylosporolides displayed activity against Plasmodium falciparum 3D7.

Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

Intragenic Distribution of IS6110 in Clinical Mycobacterium tuberculosis Strains: Bioinformatic Evidence for Gene Disruption Leading to Underdiagnosed Antibiotic Resistance.

Antibiotic resistance is a global challenge for tuberculosis control, and accelerating its diagnosis is critical for therapy decisions and controlling transmission. Genotype-based molecular diagnostics now play an increasing role in accelerating the detection of such antibiotic resistance, but their accuracy depends on the instructed detection of genetic variations. Genetic mobile elements such as IS6110 are established sources of genetic variation in Mycobacterium tuberculosis, but their implication in clinical antibiotic resistance has thus far been unclear. Here, we describe the discovery of an intragenic IS6110 insertion into Rv0678 that caused antibiotic resistance in an in vitro-selected M. tuberculosis isolate. The subsequent development of bioinformatics scripts allowed genome-wide analysis of intragenic IS6110 insertions causing gene disruptions in 6,426 clinical M. tuberculosis strains. This analysis identified 10,070 intragenic IS6110 insertions distributed among 333 different genes. Focusing on genes whose disruption leads to antibiotic resistance, 12 clinical isolates were identified with high confidence to be resistant to bedaquiline, clofazimine, pyrazinamide, ethionamide, and para-aminosalicylic acid because of an IS6110-mediated gene disruption event. A number of these IS6110-mediated resistant strains had identical genomic distributions of IS6110 elements and likely represent transmission events of a single resistant isolate. These data provide strong evidence that IS6110-mediated gene disruption is a clinically relevant mechanism of antibiotic resistance in M. tuberculosis that should be considered for molecular diagnostics. Concomitantly, this analysis provides a list of 333 IS6110-disrupted genes in clinical tuberculosis isolates that can be deemed nonessential for human infection. IMPORTANCE To help control the spread of drug-resistant tuberculosis and to guide treatment choices, it is important that rapid and accurate molecular diagnostic tools are used. Current molecular diagnostic tools detect the most common antibiotic-resistance-conferring mutations in the form of single nucleotide changes, small deletions, or insertions. Mobile genetic elements, named IS6110, are also known to move within the M. tuberculosis genome and cause significant genetic variations, although the role of this variation in clinical drug resistance remains unclear. In this work, we show that both in vitro and in data analyzed from 6,426 clinical M. tuberculosis strains, IS6110 elements are found that disrupt specific genes essential for the function of a number of pivotal antituberculosis drugs. By providing ample evidence of clinically relevant IS6110-mediated drug resistance, we believe that this shows that this form of genetic variation must not be overlooked in molecular diagnostics of drug resistance.

Rubrolone production by Dactylosporangium vinaceum: biosynthesis, modulation and possible biological function.

Rare actinomycetes are likely treasure troves for bioactive natural products, and it is therefore important that we enrich our understanding of biosynthetic potential of these relatively understudied bacteria. Dactylosporangium are a genus of such rare Actinobacteria that are known to produce a number of important antibacterial compounds, but for which there are still no fully assembled reference genomes, and where the extent of encoded biosynthetic capacity is not defined. Dactylosporangium vinaceum (NRRL B-16297) is known to readily produce a deep wine red-coloured diffusible pigment of unknown origin, and it was decided to define the chemical identity of this natural product pigment, and in parallel use whole genome sequencing and transcriptional analysis to lay a foundation for understanding the biosynthetic capacity of these bacteria. Results show that the produced pigment is made of various rubrolone conjugates, the spontaneous product of the reactive pre-rubrolone, produced by the bacterium. Genome and transcriptome analysis identified the highly expressed biosynthetic gene cluster (BGC) for pre-rubrolone. Further analysis of the fully assembled genome found it to carry 24 additional BGCs, of which the majority were poorly transcribed, confirming the encoded capacity of this bacterium to produce natural products but also illustrating the main bottleneck to exploiting this capacity. Finally, analysis of the potential environmental role of pre-rubrolone found it to react with a number of amine containing antibiotics, antimicrobial peptides and siderophores pointing to its potential role as a "minesweeper" of xenobiotic molecules in the bacterial environment. KEY POINTS: • D. vinaceum encodes many BGC, but the majority are transcriptionally silent. • Chemical screening identifies molecules that modulate rubrolone production. • Pre-rubrolone is efficient at binding and inactivating many natural antibiotics.

Synthesis and Structure-Activity Relationship Studies of C2-Modified Analogs of the Antimycobacterial Natural Product Pyridomycin.

A series of derivatives of the antimycobacterial natural product pyridomycin have been prepared with the C2 side chain attached to the macrocyclic core structure by a C-C single bond, in place of the synthetically more demanding enol ester double bond found in the natural product. Hydrophobic C2 substituents of sufficient size generally provide for potent anti-Mtb activity of these dihydropyridomycins (minimum inhibitory concentration (MIC) values around 2.5 µM), with several analogs thus approaching the activity of natural pyridomycin. Surprisingly, some of these compounds, in contrast to pyridomycin, are insensitive to overexpression of InhA in Mycobacterium tuberculosis (Mtb). This indicates that their anti-Mtb activity does not critically depend on the inhibition of InhA and that their overall mode of action may differ from that of the original natural product lead.

Pyrrolomycins Are Potent Natural Protonophores.

The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.

Characterization of new, efficient Mycobacterium tuberculosis topoisomerase-I inhibitors and their interaction with human ABC multidrug transporters.

Drug resistant tuberculosis (TB) is a major worldwide health problem. In addition to the bacterial mechanisms, human drug transporters limiting the cellular accumulation and the pharmacological disposition of drugs also influence the efficacy of treatment. Mycobacterium tuberculosis topoisomerase-I (MtTopo-I) is a promising target for antimicrobial treatment. In our previous work we have identified several hit compounds targeting the MtTopo-I by in silico docking. Here we expand the scope of the compounds around three scaffolds associated with potent MtTopo-I inhibition. In addition to measuring the effect of newly generated compounds on MtTopo-I activity, we characterized the compounds' antimicrobial activity, toxicity in human cells, and interactions with human multidrug transporters. Some of the newly developed MtTopo-I inhibitors have strong antimicrobial activity and do not harm mammalian cells. Moreover, our studies revealed significant human ABC drug transporter interactions for several MtTopo-I compounds that may modify their ADME-Tox parameters and cellular effects. Promising new drug candidates may be selected based on these studies for further anti-TB drug development.

Accelerated microfluidic native chemical ligation at difficult amino acids toward cyclic peptides.

Cyclic peptide-based therapeutics have a promising growth forecast that justifies the development of microfluidic systems dedicated to their production, in phase with the actual transitioning toward continuous flow and microfluidic technologies for pharmaceutical production. The application of the most popular method for peptide cyclization in water, i.e., native chemical ligation, under microfluidic conditions is still unexplored. Herein, we report a general strategy for fast and efficient peptide cyclization using native chemical ligation under homogeneous microfluidic conditions. The strategy relies on a multistep sequence that concatenates the formation of highly reactive S-(2-((2-sulfanylethyl)amino)ethyl) peptidyl thioesters from stable peptide amide precursors with an intramolecular ligation step. With very fast ligation rates (<5 min), even for the most difficult junctions (including threonine, valine, isoleucine, or proline), this technology opens the door toward the scale-independent, expedient preparation of bioactive macrocyclic peptides.

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 ß-chiral ß-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.

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.