Functionalization of Chlorotonils: Dehalogenil as Promising Lead Compound for In Vivo Application.

The natural product chlorotonil displays high potency against multidrug-resistant Gram-positive bacteria and Plasmodium falciparum. Yet, its scaffold is characterized by low solubility and oral bioavailability, but progress was recently made to enhance these properties. Applying late-stage functionalization, we aimed to further optimize the molecule. Previously unknown reactions including a sulfur-mediated dehalogenation were revealed. Dehalogenil, the product of this reaction, was identified as the most promising compound so far, as this new derivative displayed improved solubility and in vivo efficacy while retaining excellent antimicrobial activity. We confirmed superb activity against multidrug-resistant clinical isolates of Staphylococcus aureus and Enterococcus spp. and mature transmission stages of Plasmodium falciparum. We also demonstrated favorable in vivo toxicity, pharmacokinetics and efficacy in infection models with S. aureus. Taken together, these results identify dehalogenil as an advanced lead molecule.

Regio- and Stereoselective Epoxidation and Acidic Epoxide Opening of Antibacterial and Antiplasmodial Chlorotonils Yield Highly Potent Derivatives.

The rise of antimicrobial resistance poses a severe threat to public health. The natural product chlorotonil was identified as a new antibiotic targeting multidrug resistant Gram-positive pathogens and Plasmodium falciparum. Although chlorotonil shows promising activities, the scaffold is highly lipophilic and displays potential biological instabilities. Therefore, we strived towards improving its pharmaceutical properties by semisynthesis. We demonstrated stereoselective epoxidation of chlorotonils and epoxide ring opening in moderate to good yields providing derivatives with significantly enhanced solubility. Furthermore, in vivo stability of the derivatives was improved while retaining their nanomolar activity against critical human pathogens (e.g. methicillin-resistant Staphylococcus aureus and P. falciparum). Intriguingly, we showed further superb activity for the frontrunner molecule in a mouse model of S. aureus infection.

New terpenoids from the fermentation broth of the edible mushroom Cyclocybe aegerita.

The strophariaceous basidiomycete Cyclocybe aegerita (synonyms Agrocybe aegerita and A. cylindracea) is one of the most praised cultivated edible mushrooms and is being cultivated at large scale for food production. Furthermore, the fungus serves as a model organism to study fruiting body formation and the production of secondary metabolites during the life cycle of Basidiomycota. By studying the secondary metabolite profiles of C. aegerita, we found several terpenoids in submerged cultures. Aside from the main metabolite, bovistol (1), two new bovistol derivatives B and C (2, 3) and pasteurestin C as a new protoilludane (4) were isolated by preparative HPLC. Their structures were elucidated by mass spectrometry and NMR spectroscopy. The relative configurations of 2-4 were assigned by ROESY correlations, and 3 J H,H coupling constants in the case of 4. Applying quantitative PCR for gene expression validation, we linked the production of bovistol and its derivatives to the respective biosynthesis gene clusters.

Nematicidal Cyclic Lipodepsipeptides and a Xanthocillin Derivative from a Phaeosphariaceous Fungus Parasitizing Eggs of the Plant Parasitic Nematode Heterodera filipjevi.

The new cyclic lipodepsipeptide ophiotine (1), two new arthrichitin derivatives named arthrichitins B (4) and C (5), a new xanthocillin-like alkaloid, xanthomide Z (2), and the previously described arthrichitin (3) were isolated from the liquid culture broth of a nematode-associated fungus with affinities to the genus Ophiosphaerella. The structural elucidation and determination of the absolute configuration of the new molecules were accomplished using a combination of spectroscopic and chemical techniques, including 1D and 2D NMR, HRMS, and Marfey's analysis. Opiotine (1) displayed moderate nematicidal activity against the host nematode ( Heterodera filipjevi), while xanthomide Z (2) exhibited very weak activity. Arthrichitin C (5) showed very weak cytotoxic effects on several cancer cell lines, with IC50 values in the range of 24-33 µM. Xanthomide Z is among few xanthocillin derivatives that comprise formamide functions instead of the cyano functions that are usually observed in this class of fungal alkaloids.

Pinensins: the first antifungal lantibiotics.

Lantibiotics (lanthionine-containing antibiotics) from Gram-positive bacteria typically exhibit activity against Gram-positive bacteria. The activity and structure of pinensin A (1) and B (2), lantibiotics isolated from a native Gram-negative producer Chitinophaga pinensis are described. Surprisingly, the pinensins were found to be highly active against many filamentous fungi and yeasts but show only weak antibacterial activity. To the best of our knowledge, lantibiotic fungicides have not been described before. An in-depth bioinformatic analysis of the biosynthetic gene cluster established the ribosomal origin of these compounds and identified candidate genes encoding all of the enzymes required for post-translational modification. Additional encoded functions enabled us to build up a hypothesis for the biosynthesis, export, sensing, and import of this intriguing lantibiotic.

Indothiazinone, an indolyl thiazolyl ketone from a novel myxobacterium belonging to the Sorangiineae.

Indothiazinone (1), an indolyl thiazolyl ketone, was discovered in cultures of novel myxobacterial strain 706, recently isolated from compost in Germany. Molecular phylogenetic studies based on 16S rRNA gene sequences revealed strain 706 to be a representative of a new family of the Sorangiineae. A screening of the culture broth for antimicrobial metabolites followed by isolation and characterization of these compounds revealed six indole derivatives and a 1,4-naphthoquinone derivative. The structures were determined to be indothiazinone (1; 1H-indol-3-yl(1,3-thiazol-2-yl)methanone) and three 3-methylbuta-1,3-dien-1-yl-substituted indoles, indolyl ethanol 2 and the E- and Z-isomers of indolyl ethylidenehydroxylamine 4 and 5 by MS and NMR spectroscopic analyses. In the indolyl ethanol derivative 3 the unsaturated methylene group of the butadienyl residue was replaced by an oxygen atom to give the keto group of the butanone side chain. Further 1H-indol-3-ylacetonitrile (6) was identified, which was already known as a myxobacterial metabolite. 2-Hydroxyethyl-3-methyl-1,4-naphthoquinone (7) was recognized during dereplication as an antibiotic previously isolated from Actinoplanes capillaceus. Whereas 1, 4, 5, and 7 showed weak activity against yeasts and filamentous fungi, isomers 4 and 5 were weakly active against Gram-positive bacteria and mouse fibroblasts. Compound 6 is volatile, and 2 and 3 showed no activity in a number of assays.

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.

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.

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.

Large Scale Production and Downstream Processing of Labyrinthopeptins from the Actinobacterium Actinomadura namibiensis.

A method was established for the production of 1.2-fold and 4.2-fold increased amounts of the antiviral and central nervous system-active lantipeptides, labyrinthopeptins A1 and A2, respectively, isolated from the actinobacterium Actinomadura namibiensis, to enable production in gram scale. We then performed in vivo characterization of this promising compound class. The labyrinthopeptins A1 and A2 have similar chemical structures and physical properties but differ drastically in their bioactivities. Therefore, large quantities of highly pure material are required for pharmacological studies. An effective methodology was established for the first time for their production in bioreactors, their separation involving gel permeation chromatography on LH20 material, followed by reversed phase-high performance liquid chromatography. With an optimized methodology, 580 mg of labyrinthopeptin A1 and 510 mg of labyrinthopeptin A2 were quantitatively isolated with recovery rates of 72.5% and 42.3% from 7.5 L of culture broth, respectively. However, the fermentation that had already resulted in maximum yields of over 100 mg/L of both target molecules after 300 h in a 10-L scale bioreactor, still requires further optimisation.

Linoleic and palmitoleic acid block streptokinase-mediated plasminogen activation and reduce severity of invasive group A streptococcal infection.

In contrast to mild infections of Group A Streptococcus (GAS) invasive infections of GAS still pose a serious health hazard: GAS disseminates from sterile sites into the blood stream or deep tissues and causes sepsis or necrotizing fasciitis. In this case antibiotics do not provide an effective cure as the bacteria are capable to hide from them very quickly. Therefore, new remedies are urgently needed. Starting from a myxobacterial natural products screening campaign, we identified two fatty acids isolated from myxobacteria, linoleic and palmitoleic acid, specifically blocking streptokinase-mediated activation of plasminogen and thereby preventing streptococci from hijacking the host's plasminogen/plasmin system. This activity is not inherited by other fatty acids such as oleic acid and is not attributable to the killing of streptococci. Moreover, both fatty acids are superior in their inhibitory properties compared to two clinically used drugs (tranexamic or ε-amino caproic acid) as they show 500-1000 fold lower IC50 values. Using a humanized plasminogen mouse model mimicking the clinical situation of a local GAS infection that becomes systemic, we demonstrate that these fatty acids ameliorate invasive GAS infection significantly. Consequently, linoleic and palmitoleic acid are possible new options to combat GAS invasive diseases.

Botryane, noreudesmane and abietane terpenoids from the ascomycete Hypoxylon rickii.

In the course of our screening for new bioactive natural products, a culture of Hypoxylon rickii, a xylariaceous ascomycete collected from the Caribbean island Martinique, was identified as extraordinary prolific producer of secondary metabolites. Ten metabolites of terpenoid origin were isolated from submerged cultures of this species by preparative HPLC. Their structures were elucidated using spectral techniques including 2D NMR and HRESIMS. Three of the compounds were elucidated as new botryanes (1-3) along with three known ones, i.e. (3aS)-3a,5,5,8-tetramethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[de]isochromen-1-one (4), (3aS,8R)-3a,5,5,8-tetramethyl-3,3a,4,5,7,8-hexahydro-1H-cyclopenta[de]isochromen-1-one (5) and botryenanol (6). Further three new sesquiterpenoids featured a 14-noreudesmane-type skeleton and were named hypoxylan A-C (7-9); the diterpenoid rickitin A (10) contains an abietane-type backbone. Compounds 1, 2, 3, 7, and 10 showed cytotoxic effects against murine cells.