Interaction of the Atypical Tetracyclines Chelocardin and Amidochelocardin with Renal Drug Transporters.

Antimicrobial resistance is expected to increase mortality rates by up to several million deaths per year by 2050 without new treatment options at hand. Recently, we characterized the pharmacokinetic (PK) and pharmacodynamic properties of two atypical tetracyclines, chelocardin (CHD) and amidochelocardin (CDCHD) that exhibit no cross-resistance with clinically used antibacterials. Both compounds were preferentially renally cleared and demonstrated pronounced effects in an ascending urinary tract infection model against E. coli. Renal drug transporters are known to influence clearance into the urine. In particular, inhibition of apical transporters in renal tubular epithelial cells can lead to intracellular accumulation and potential cell toxicity, whereas inhibition of basolateral transporters can cause a higher systemic exposure. Here, selected murine and human organic cation (Oct), organic anion (Oat), and efflux transporters were studied to elucidate interactions with CHD and CDCHD underlying their PK behavior. CHD exhibited stronger inhibitory effects on mOat1 and mOat3 and their human homologues hOAT1 and hOAT3 compared to CDCHD. While CHD was a substrate of mOat3 and mOct1, CDCHD was not. By contrast, no inhibitory effect was observed on Octs. CDCHD rather appeared to foster enhanced substrate transport on mOct1. CHD and CDCHD inhibited the efflux transporter hMRP2 on the apical side. In summary, the substrate nature of CHD in conjunction with its autoinhibition toward mOat3 rationalizes the distinct urine concentration profile compared to CDCHD that was previously observed in vivo. Further studies are needed to investigate the accumulation in renal tubular cells and the nephrotoxicity risk.

Pharmacokinetic and pharmacodynamic evaluation of the atypical tetracyclines chelocardin and amidochelocardin in murine infection models.

IMPORTANCE: There is a strong need to find novel treatment options against urinary tract infections associated with antimicrobial resistance. This study evaluates two atypical tetracyclines, namely chelocardin (CHD) and amidochelocardin (CDCHD), with respect to their pharmacokinetics and pharmacodynamics. We show CHD and CDCHD are cleared at high concentrations in mouse urine. Especially, CDCHD is highly effective in an ascending urinary tract infection model, suggesting further preclinical evaluation.

Tartrolon sensing and detoxification by the Listeria monocytogenes timABR resistance operon.

Listeria monocytogenes is a foodborne bacterium that naturally occurs in the soil. Originating from there, it contaminates crops and infects farm animals and their consumption by humans may lead to listeriosis, a systemic life-threatening infectious disease. The adaptation of L. monocytogenes to such contrastive habitats is reflected by the presence of virulence genes for host infection and other genes for survival under environmental conditions. Among the latter are ABC transporters for excretion of antibiotics produced by environmental competitors; however, most of these transporters have not been characterized. Here, we generated a collection of promoter-lacZ fusions for genes encoding ABC-type drug transporters of L. monocytogenes and screened this reporter strain collection for induction using a library of natural compounds produced by various environmental microorganisms. We found that the timABR locus (lmo1964-lmo1962) was induced by the macrodiolide antibiotic tartrolon B, which is synthesized by the soil myxobacterium Sorangium cellulosum. Tartrolon B resistance of L. monocytogenes was dependent on timAB, encoding the ATPase and the permease component of a novel ABC transporter. Moreover, transplantation of timAB was sufficient to confer tartrolon B resistance to Bacillus subtilis. Expression of the timABR locus was found to be auto-repressed by the TimR repressor, whose repressing activity was lost in the presence of tartrolon B. We also demonstrate that tartrolon sensitivity was suppressed by high external potassium concentrations, suggesting that tartrolon acts as potassium ionophore. Our results help to map the ecological interactions of an important human pathogen with its co-residing species within their joint natural reservoir.

Revision of the Absolute Configurations of Chelocardin and Amidochelocardin.

Even with the aid of the available methods, the configurational assignment of natural products can be a challenging task that is prone to errors, and it sometimes needs to be corrected after total synthesis or single-crystal X-ray diffraction (XRD) analysis. Herein, the absolute configuration of amidochelocardin is revised using a combination of XRD, NMR spectroscopy, experimental ECD spectra, and time-dependent density-functional theory (TDDFT)-ECD calculations. As amidochelocardin was obtained via biosynthetic engineering of chelocardin, we propose the same absolute configuration for chelocardin based on the similar biosynthetic origins of the two compounds and result of TDDFT-ECD calculations. The evaluation of spectral data of two closely related analogues, 6-desmethyl-chelocardin and its semisynthetic derivative 1, also supports this conclusion.

The Disorazole Z Family of Highly Potent Anticancer Natural Products from Sorangium cellulosum: Structure, Bioactivity, Biosynthesis, and Heterologous Expression.

Myxobacteria serve as a treasure trove of secondary metabolites. During our ongoing search for bioactive natural products, a novel subclass of disorazoles termed disorazole Z was discovered. Ten disorazole Z family members were purified from a large-scale fermentation of the myxobacterium Sorangium cellulosum So ce1875 and characterized by electrospray ionization-high-resolution mass spectrometry (ESI-HRMS), X-ray, nuclear magnetic resonance (NMR), and Mosher ester analysis. Disorazole Z compounds are characterized by the lack of one polyketide extension cycle, resulting in a shortened monomer in comparison to disorazole A, which finally forms a dimer in the bis-lactone core structure. In addition, an unprecedented modification of a geminal dimethyl group takes place to form a carboxylic acid methyl ester. The main component disorazole Z1 shows comparable activity in effectively killing cancer cells to disorazole A1 via binding to tubulin, which we show induces microtubule depolymerization, endoplasmic reticulum delocalization, and eventually apoptosis. The disorazole Z biosynthetic gene cluster (BGC) was identified and characterized from the alternative producer S. cellulosum So ce427 and compared to the known disorazole A BGC, followed by heterologous expression in the host Myxococcus xanthus DK1622. Pathway engineering by promoter substitution and gene deletion paves the way for detailed biosynthesis studies and efficient heterologous production of disorazole Z congeners. IMPORTANCE Microbial secondary metabolites are a prolific reservoir for the discovery of bioactive compounds, which prove to be privileged scaffolds for the development of new drugs such as antibacterial and small-molecule anticancer drugs. Consequently, the continuous discovery of novel bioactive natural products is of great importance for pharmaceutical research. Myxobacteria, especially Sorangium spp., which are known for their large genomes with yet-underexploited biosynthetic potential, are proficient producers of such secondary metabolites. From the fermentation broth of Sorangium cellulosum strain So ce1875, we isolated and characterized a family of natural products named disorazole Z, which showed potent anticancer activity. Further, we report on the biosynthesis and heterologous production of disorazole Z. These results can be stepping stones toward pharmaceutical development of the disorazole family of anticancer natural products for (pre)clinical studies.

The natural product chlorotonil A preserves colonization resistance and prevents relapsing Clostridioides difficile infection.

Clostridioides difficile infections (CDIs) remain a healthcare problem due to high rates of relapsing/recurrent CDIs (rCDIs). Breakdown of colonization resistance promoted by broad-spectrum antibiotics and the persistence of spores contribute to rCDI. Here, we demonstrate antimicrobial activity of the natural product class of chlorotonils against C. difficile. In contrast to vancomycin, chlorotonil A (ChA) efficiently inhibits disease and prevents rCDI in mice. Notably, ChA affects the murine and porcine microbiota to a lesser extent than vancomycin, largely preserving microbiota composition and minimally impacting the intestinal metabolome. Correspondingly, ChA treatment does not break colonization resistance against C. difficile and is linked to faster recovery of the microbiota after CDI. Additionally, ChA accumulates in the spore and inhibits outgrowth of C. difficile spores, thus potentially contributing to lower rates of rCDI. We conclude that chlorotonils have unique antimicrobial properties targeting critical steps in the infection cycle of C. difficile.

In Vitro-In Vivo Relationship in Mini-Scale-Enabling Formulations of Corallopyronin A.

In vivo studies in mice provide a valuable model to test novel active pharmaceutical ingredients due to their low material need and the fact that mice are frequently used as a species for early efficacy models. However, preclinical in vitro evaluations of formulation principles in mice are still lacking. The development of novel in vitro and in silico models supported the preclinical formulation evaluation for the anti-infective corallopyronin A (CorA). To this end, CorA and solubility-enhanced amorphous solid dispersion formulations, comprising povidone or copovidone, were evaluated regarding biorelevant solubilities and dissolution in mouse-specific media. As an acidic compound, CorA and CorA-ASD formulations showed decreased solubilities in mice when compared with human-specific media. In biorelevant biphasic dissolution experiments CorA-povidone showed a three-fold higher fraction partitioned into the organic phase of the biphasic dissolution, when compared with CorA-copovidone. Bioavailabilities determined by pharmacokinetic studies in BALB/c mice correlated with the biphasic dissolution prediction and resulted in a Level C in vitro-in vivo correlation. In vitro cell experiments excluded intestinal efflux by P-glycoprotein or breast cancer resistance protein. By incorporating in vitro results into a physiologically based pharmacokinetic model, the plasma concentrations of CorA-ASD formulations were predicted and identified dissolution as the limiting factor for bioavailability.

The RNA Polymerase Inhibitor Corallopyronin A Has a Lower Frequency of Resistance Than Rifampicin in Staphylococcus aureus.

Corallopyronin A (CorA) is active against Gram-positive bacteria and targets the switch region of RNA polymerase. Because of the high frequency of mutation (FoM) leading to rifampicin resistance, we determined the CorA FoM in S. aureus using fluctuation analysis at 4 × minimum inhibitory concentration (MIC). Resistant mutants were characterized. S. aureus strains HG001, Mu50, N315, and USA300 had an MIC of 0.25 mg/L. The median FoM for CorA resistance was 1.5 × 10−8, 4.5-fold lower than the median FoM of 6.7 × 10−8 for rifampicin, and was reflected in a 4-fold lower mutation rate for CorA than rifampicin (6 × 10−9 for CorA vs. 2.5 × 10−8 for rifampicin). In CorA-resistant/rifampicin-sensitive strains, the majority of amino acid exchanges were S1127L in RpoB or K334N in RpoC. S. aureus Mu50, a rifampicin-resistant clinical isolate, yielded two further exchanges targeting amino acids L1131 and E1048 of the RpoB subunit. The plating of >1011 cells on agar containing a combination of 4 × MIC of rifampicin and 4 × MIC of CorA did not yield any growth. In conclusion, with proper usage, e.g., in combination therapy and good antibiotic stewardship, CorA is a potential antibiotic for treating S. aureus infections.

Corallopyronin A: antimicrobial discovery to preclinical development.

Covering: August 1984 up to January 2022Worldwide, increasing morbidity and mortality due to antibiotic-resistant microbial infections has been observed. Therefore, better prevention and control of infectious diseases, as well as appropriate use of approved antibacterial drugs are crucial. There is also an urgent need for the continuous development and supply of novel antibiotics. Thus, identifying new antibiotics and their further development is once again a priority of natural product research. The antibiotic corallopyronin A was discovered in the 1980s in the culture broth of the Myxobacterium Corallococcus coralloides and serves, in the context of this review, as a show case for the development of a naturally occurring antibiotic compound. The review demonstrates how a hard to obtain, barely water soluble and unstable compound such as corallopyronin A can be developed making use of sophisticated production and formulation approaches. Corallopyronin A is a bacterial DNA-dependent RNA polymerase inhibitor with a new target site and one of the few representatives of this class currently in preclinical development. Efficacy against Gram-positive and Gram-negative pathogens, e.g., Chlamydia trachomatis, Orientia tsutsugamushi, Staphylococcus aureus, and Wolbachia has been demonstrated. Due to its highly effective in vivo depletion of Wolbachia, which are essential endobacteria of most filarial nematode species, and its robust macrofilaricidal efficacy, corallopyronin A was selected as a preclinical candidate for the treatment of human filarial infections. This review highlights the discovery and production optimization approaches for corallopyronin A, as well as, recent preclinical efficacy results demonstrating a robust macrofilaricidal effect of the anti-Wolbachia candidate, and the solid formulation strategy which enhances the stability as well as the bioavailability of corallopyronin A.

Pharmacokinetics and Pharmacodynamics (PK/PD) of Corallopyronin A against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a World Health Organization's high priority pathogen organism, with an estimated > 100,000 deaths worldwide in 2019. Thus, there is an unmet medical need for novel and resistance-breaking anti-infectives. The natural product Co-rallopyronin A (CorA), currently in preclinical development for filariasis, is efficacious against MRSA in vitro. In this study, we evaluated the pharmacokinetics of CorA after dosing in mice. Furthermore, we determined compound concentrations in target compartments, such as lung, kidney and thigh tissue, using LC-MS/MS. Based on the pharmacokinetic results, we evaluated the pharmacodynamic profile of CorA using the standard neutropenic thigh and lung infection models. We demonstrate that CorA is effective in both standard pharmacodynamic models. In addition to reaching effective levels in the lung and muscle, CorA was detected at high levels in the thigh bone. The data presented herein encourage the further exploration of the additional CorA indications treatment of MRSA- and methicillin-sensitive S. aureus- (MSSA) related infections.

Total Synthesis of Thuggacin cmc-A and Its Structure Determination.

The first total synthesis of thuggacin cmc-A and the determination of the absolute structure are described. The thuggacin family of antibiotics is of great interest due to the antibiotic activity against Mycobacterium tuberculosis. Based on the assumption that seven stereogenic centers in thuggacin cmc-A would share the same stereochemistry as thuggacin-A, all stereogenic centers of thuggacin cmc-A were strictly constructed in a stereocontrolled manner. The total synthesis allowed its stereostructure to be fully confirmed.

Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.

Solubility and Stability Enhanced Oral Formulations for the Anti-Infective Corallopyronin A.

Novel-antibiotics are urgently needed to combat an increase in morbidity and mortality due to resistant bacteria. The preclinical candidate corallopyronin A (CorA) is a potent antibiotic against Gram-positive and some Gram-negative pathogens for which a solid oral formulation was needed for further preclinical testing of the active pharmaceutical ingredient (API). The neat API CorA is poorly water-soluble and instable at room temperature, both crucial characteristics to be addressed and overcome for use as an oral antibiotic. Therefore, amorphous solid dispersion (ASD) was chosen as formulation principle. The formulations were prepared by spray-drying, comprising the water-soluble polymers povidone and copovidone. Stability (high-performance liquid chromatography, Fourier-transform-infrared spectroscopy, differential scanning calorimetry), dissolution (biphasic dissolution), and solubility (biphasic dissolution, Pion's T3 apparatus) properties were analyzed. Pharmacokinetic evaluations after intravenous and oral administration were conducted in BALB/c mice. The results demonstrated that the ASD formulation principle is a suitable stability- and solubility-enhancing oral formulation strategy for the API CorA to be used in preclinical and clinical trials and as a potential market product.

Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.

Production optimization and biosynthesis revision of corallopyronin A, a potent anti-filarial antibiotic.

Corallopyronins (COR) are α-pyrone antibiotics from myxobacteria representing highly promising lead structures for the development of antibacterial therapeutic agents. Their ability to inhibit RNA polymerase through interaction with the "switch region", a novel target, distant from binding sites of previously characterized RNA polymerase inhibitors (e.g. rifampicin), makes them particularly promising as antibiotic candidates. Corallopyronin A is currently also investigated as a lead compound for the treatment of lymphatic filariasis because of its superb activity against the nematode symbiont Wolbachia. As total synthesis is not a valid production option biotechnological optimization of compound supply is of utmost importance to further develop this highly potent compound class. Here we describe decisive improvements of the previously reported heterologous COR production and engineering platform yielding production of ~100 mg/L COR A. Furthermore, we provide a revised model of COR biosynthesis shedding light on the function of several biosynthetic proteins, including an unusual ECH-like enzyme providing dehydration functionality in trans and an uncharacterized protein conferring COR self-resistance in the myxobacterial heterologous host Myxococcus xanthus DK1622. We also report two new COR derivatives, COR D and oxyCOR A discovered in genetically engineered strains.

Kenalactams A-E, Polyene Macrolactams Isolated from Nocardiopsis CG3.

In our screening program for new biologically active secondary metabolites, a new strain, Nocardiopsis CG3 (DSM 106572), isolated from the saltpan of Kenadsa, was found to produce five new polyene macrolactams, the kenalactams A-E (1-5). Their structures were elucidated by spectral methods (NMR and HRESIMS), and the absolute configuration was derived by chemical derivatization (Mosher's method). Through a feeding experiment, alanine was proven to be the nitrogen-bearing starter unit involved in biosynthesis of the polyketide kenalactam A (1). Kenalactam E (5) was cytotoxic against human prostate cancer PC-3 cells with an IC50 value of 2.1 µM.

Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding.

A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex1-3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed4-6. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.

Lanyamycin, a macrolide antibiotic from Sorangium cellulosum, strain Soce 481 (Myxobacteria).

Lanyamycin (1/2), a secondary metabolite occurring as two epimers, was isolated from the myxobacterium Sorangium cellulosum, strain Soce 481. The structures of both epimers were elucidated from HRESIMS and 1D and 2D NMR data and the relative configuration of their macrolactone ring was assigned based on NOE and vicinal 1H NMR coupling constants and by calculation of a 3D model. Lanyamycin inhibited HCV infection into mammalian liver cells with an IC50 value of 11.8 µM, and exhibited a moderate cytotoxic activity against the mouse fibroblast cell line L929 and the human nasopharyngeal cell line KB3 with IC50 values of 3.1 and 1.5 µM, respectively, and also suppressed the growth of the Gram-positive bacterium Micrococcus luteus.

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on ß-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Six Heterocyclic Metabolites from the Myxobacterium Labilithrix luteola.

Two new secondary metabolites, labindole A [2-methyl-3-(2-nitroethyl)-3H-indole] (1) and labindole B [2-methyl-3-(2-nitrovinyl)-3H-indole] (2), were isolated from the myxobacterium Labilithrixluteola (DSM 27648T). Additionally, four metabolites 3, 4, 5 and 6 already known from other sources were obtained. Their structures were elucidated from high resolution electrospray ionisation mass spectrometry (HRESIMS) and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy data and their relative configuration was assigned based on nuclear Overhauser effect (NOE) and vicinal ¹H-NMR coupling data. The compounds where tested for biological activities; labindoles A (1) and B (2) exhibited significant activity against Hepatitis C Virus, 9H-carbazole (3), 3-chloro-9H-carbazole (4) and 4-hydroxymethyl-quinoline (5) showed antifungal activities. Moreover, compound 3 had weak to moderate antibacterial activities, while labindoles A (1) and B (2) were devoid of significant antifungal and antibacterial effects.