Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is ...responsible for the biosynthesis of the oximidine anticancer agents, oxime‐substituted benzolactone enamides that inhibit vacuolar H+‐ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O‐methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans‐AT PKSs and identify potential strategies for the production of novel oximidine analogues.
The anticancer agent oximidine I and three novel variants were discovered as products of a cryptic trans‐AT PKS/NRPS in Pseudomonas baetica. By manipulating the biosynthetic pathway, a key intermediate was identified that retains potent anticancer properties. A combination of bioinformatics analysis and genetic and biochemical experiments illuminated the oximidine biosynthetic pathway, including a novel mechanism for O‐methyloxime formation.
Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal ...product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.
Tricyclic scaffolds structurally related to the well‐known benzodiazepine class of drugs show diverse biological activities strikingly different from those of their benzodiazepine counterparts. ...Interested by this scaffold‐hopping perspective, we previously developed a continuous‐flow method for the conversion of benzodiazepinediones into oxazoloquinolinones. Attempted extension of this synthetic route to the corresponding oxazolonaphthyridinone scaffolds met with limited success, however. This encouraged us to develop a different approach to pyridine‐based tricyclic motifs. In line with our interest in scaffold hopping, in this paper we describe a general, convergent 3+3 cyclocondensation approach to 1,3oxazolo4,5‐c‐1‐naphthyridin‐4(5H)‐ones. The key synthetic steps in this approach are: (1) the construction of an amide linkage connecting two peripheral heterocycles; and (2) a palladium‐catalysed intramolecular C–H arylation to complete the tricyclic scaffold.
A general, convergent 3+3 cyclocondensation approach giving access to four isomeric 1,3oxazolo4,5‐c‐1‐naphthyridin‐4(5H)‐ones was established. The key synthetic steps in this approach are: (1) the construction of a challenging amide linker connecting the two peripheral heterocycles; and (2) a palladium‐catalysed intramolecular C–H arylation to form the key heterocycle.
Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is ...responsible for the biosynthesis of the oximidine anticancer agents, oxime‐substituted benzolactone enamides that inhibit vacuolar H+‐ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O‐methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans‐AT PKSs and identify potential strategies for the production of novel oximidine analogues.
The anticancer agent oximidine I and three novel variants were discovered as products of a cryptic trans‐AT PKS/NRPS in Pseudomonas baetica. By manipulating the biosynthetic pathway, a key intermediate was identified that retains potent anticancer properties. A combination of bioinformatics analysis and genetic and biochemical experiments illuminated the oximidine biosynthetic pathway, including a novel mechanism for O‐methyloxime formation.
Leucyl-tRNA synthetase (LeuRS) is a clinically validated target for the development of antimicrobials. This enzyme catalyzes the formation of charged tRNALeu molecules, an essential substrate for ...protein translation. In the first step of catalysis LeuRS activates leucine using ATP, forming a leucyl-adenylate intermediate. Bi-substrate inhibitors that mimic this chemically labile phosphoanhydride-linked nucleoside have proven to be potent inhibitors of different members of the aminoacyl-tRNA synthetase family but, to date, they have demonstrated poor antibacterial activity. We synthesized a small series of 1,5-anhydrohexitol-based analogues coupled to a variety of triazoles and performed detailed structure-activity relationship studies with bacterial LeuRS. In an in vitro assay, Kiapp values in the nanomolar range were demonstrated. Inhibitory activity differences between the compounds revealed that the polarity and size of the triazole substituents affect binding. X-ray crystallographic studies of N. gonorrhoeae LeuRS in complex with all the inhibitors highlighted the crucial interactions defining their relative enzyme inhibitory activities. We further examined their in vitro antimicrobial properties by screening against several bacterial and yeast strains. While only weak antibacterial activity against M. tuberculosis was detected, the extensive structural data which were obtained could make these LeuRS inhibitors a suitable starting point towards further antibiotic development.
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•Eleven anhydrohexitol analogues targeting leucyl-tRNA synthetase (LeuRS).•Several inhibitors showed enzymatic inhibitory activity in low nanomolar range.•Crystal structures of all compounds in complex with LeuRS determined.•SAR study revealed crucial interactions for obtaining enzymatic inhibitory activity.
Abstract
S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PM
Background
The current armamentarium of antifungal drugs and the restricted variety in antifungal drug classes combined ...with the ever-rising threat of resistant fungal pathogens highlighted the urgent need for novel antifungal compounds. Natural antifungal secondary metabolites have always been the prevalent source for drug development, exemplified by the echinocandin and polyene drug classes. Yet, the golden age discovery platforms were abandoned due to compound rediscovery and its paired economic cost.
Study
In an effort to revive the original success stories, we combined the traditional approach of sampling and screening for active secondary metabolites with present-day advances in sequencing, genome mining, impedance spectroscopy, HPLC, LCMS, and NMR.
Soil bacteria and fungi were isolated through in situ cultivation via the iChip method. After application of the OSMAC approach, 389 broths were identified with activity against Candida albicans. To prioritize active extracts, several criteria were set up; low to absent mammalian host cell toxicity, activity against a broad spectrum of fungal pathogens including wild-type reference strains, and established antifungal drug-resistant variants and species identification of the producing strain. Continuing, Lead hits were purified utilizing bioactivity-based semi-preparative HPLC. The resulting pure fractions were analyzed by tandem LCMS-MS, and proposed structures were later confirmed with NMR. In vitro and in vivo validation of the purified compounds will be performed.
Additionally, aside from discovering a novel antifungal compound, another project goal is to gauge if impedance spectroscopy can provide an early suggestion regarding the mode of action of the present antifungal agent. For this, a POC study was performed which showed that different antifungal drug classes provide distinct signature response profiles by which they can be classified. As such, when active secretion broths show unique impedance profiles, in comparison with the signature profiles of established antifungal drugs, it suggests that they work through a different mode of action.
Results
Several species were identified as producing antifungal secondary metabolites that are currently absent in the literature. Either the compound itself was unknown or literature never described the species as a producer of a known, or variant of a known antimycotic compound. Moreover, several species are novel based on Illumina sequencing. Genera producing our current lead hits include bacteria: Pseudomonas, Tsukamurella, Paraburkholderia, and fungi: Athelia, Penicillium. Within the collection, the Pseudomonas species appear to produce variants of the antimycotic iron-chelating pyoverdine class.
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•Eight new CB 168 analogues targeting isoleucyl- or tyrosyl-tRNA synthetase.•New IleRS-targeting compounds with 3-carbon linker showed good inhibitory activity.•Docking approach for ...11 revealed a different binding mode compared to CB 168.•Activity decrease was due to loss of crucial pyran hydroxyl group interactions.•Enhanced lipophilic/hydrophilic balance suggested an improved bacterial uptake.
Antimicrobial resistance is considered as one of the major threats for the near future as the lack of effective treatments for various infections would cause more deaths than cancer by 2050. The development of new antibacterial drugs is considered as one of the cornerstones to tackle this problem. Aminoacyl-tRNA synthetases (aaRSs) are regarded as good targets to establish new therapies. Apart from being essential for cell viability, they are clinically validated. Indeed, mupirocin, an isoleucyl-tRNA synthetase (IleRS) inhibitor, is already commercially available as a topical treatment for MRSA infections. Unfortunately, resistance developed soon after its introduction on the market, hampering its clinical use. Therefore, there is an urgent need for new cellular targets or improved therapies. Follow-up research by Cubist Pharmaceuticals led to a series of selective and in vivo active aminoacyl-sulfamoyl aryltetrazole inhibitors targeting IleRS (e.g. CB 168).
Here, we describe the synthesis of new IleRS and TyrRS inhibitors based on the Cubist Pharmaceuticals compounds, whereby the central ribose was substituted for a tetrahydropyran ring. Various linkers were evaluated connecting the six-membered ring with the base-mimicking part of the synthesized analogues. Out of eight novel molecules, a three-atom spacer to the phenyltriazole moiety, which was established using azide-alkyne click chemistry, appeared to be the optimized linker to inhibit IleRS. However, 11 (Ki,app = 88 ± 5.3 nM) and 36a (Ki,app = 114 ± 13.5 nM) did not reach the same level of inhibitory activity as for the known high-affinity natural adenylate-intermediate analogue isoleucyl-sulfamoyl adenosine (IleSA, CB 138; Ki,app = 1.9 ± 4.0 nM) and CB 168, which exhibit a comparable inhibitory activity as the native ligand. Therefore, 11 was docked into the active site of IleRS using a known crystal structure of T. thermophilus in complex with mupirocin. Here, we observed the loss of the crucial 3′- and 4′- hydroxyl group interactions with the target enzyme compared to CB 168 and mupirocin, which we suggest to be the reason for the limited decrease in enzyme affinity. Despite the lack of antibacterial activity, we believe that structurally optimizing these novel analogues via a structure-based approach could ultimately result in aaRS inhibitors which would help to tackle the antibiotic resistance problem.
The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed ...phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Å crystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.
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•Improved synthesis strategy of N-acylated sulfonamide adenosines developed.•Two aminoacylated compounds evaluated as aminoacyl-tRNA synthetase (aaRS) inhibitors.•Inhibitory activity depends on the aaRS structural class.•Structural studies suggest an unfavorable pose of the compound in class II aaRS.•Modelling suggests a general targeting potential towards adenylate-forming enzymes.
To compare dose to organs at risk (OARs) and dose-escalation possibility for 24 stage I non-small cell lung cancer (NSCLC) patients in a ROCOCO (Radiation Oncology Collaborative Comparison) trial.
...For each patient, 3 photon plans Intensity-modulated radiotherapy (IMRT), volumetric modulated arc therapy (VMAT) and CyberKnife, a double scattered proton (DSP) and an intensity-modulated carbon-ion (IMIT) therapy plan were created. Dose prescription was 60 Gy (equivalent) in 8 fractions.
The mean dose and dose to 2% of the clinical target volume (CTV) were lower for protons and ions compared with IMRT (p < 0.01). Doses to the lungs, heart, and mediastinal structures were lowest with IMIT (p < 0.01), doses to the spinal cord were lowest with DSP (p < 0.01). VMAT and CyberKnife allowed for reduced doses to most OARs compared with IMRT. Dose escalation was possible for 8 patients. Generally, the mediastinum was the primary dose-limiting organ.
On average, the doses to the OARs were lowest using particles, with more homogenous CTV doses. Given the ability of VMAT and CyberKnife to limit doses to OARs compared with IMRT, the additional benefit of particles may only be clinically relevant in selected patients and thus should be carefully weighed for every individual patient.
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