Studies on two compound classes from actinobacteria exhibiting new antibacterial mechanisms of action : chelocardins and telomycins

Due to the increasing emergence of multidrug- resistant bacterial species and a concurrent decline in the development of new potent antibacterial drugs, there is an urgent need for new potent compounds with novel mechanisms of action and even new target molecules. Within this thesis, two known but rather underexploited microbial natural product classes were characterized in terms of their mechanism of action and resistance mechanisms: chelocardins and telomycins. Furthermore, derivatives of both natural products were available through semi-synthesis or genetic engineering of the producer strains, which were used to establish structure-activity-relationships. Chelocardin and its amidated analogue amidochelocardin were shown to act bactericidal on a broad-spectrum of bacterial species and the compounds possess resistance-breaking properties. Both molecules were shown to exert a cell membrane-based mechanism, even though the target molecule was not identified. Moreover, the resistance mechanism of CHD was identified and characterized to rely on efflux mediated by mutations of the repressor protein RamR. Telomycin was characterised to act strongly bactericidal on Gram-positive bacteria in a calcium-dependent manner. Two acylated derivatives (TM-Dodec, TM-N-Oct) were shown to exhibit an improved and calcium-independent activity pattern compared to the parent molecule. Studies as part of this thesis led to the assumption that the compounds interfere mainly with the cellular membrane and cardiolipin was characterized as main interaction partner, particularly for telomycin. However, the antibacterial activity of both acylated derivatives does not exclusively rely on a binding to this phospholipid. This hints towards interaction with additional target molecules, which might also hold true for telomycin itself and hints towards a mechanism of action beyond membrane activity. Potential protein targets were studied but the mechanism of action of telomycins could not be elucidated in detail.Aufgrund des vermehrten Aufkommens von multiresistenten Bakterienspezies und der gleichzeitigen Abnahme in der Verfügbarkeit von wirksamen antibakteriellen Arzneimittel, ist es von größter Wichtigkeit neue potente Verbindungen zu entwickeln, die idealerweise neue Wirkmechanismen und Zielstrukturen aufweisen. Im Rahmen dieser Arbeit wurden zwei bekannte, aber noch wenig untersuchte mikrobielle Naturstoffklassen hinsichtlich ihres Wirk- und Resistenzmechanismus charakterisiert: Chelocardine und Telomycine. Von beiden Naturstoffen lagen zudem Derivate vor, die über Semisynthese oder gentechnische Veränderung der Produzentenstämme gewonnen wurden. Dies ermöglichte die Charakterisierung von Struktur-Aktivitäts-Beziehungen beider Antibiotikaklassen. Chelocardin und sein amidiertes Derivat Amidochelocardin wurden als bakterizide Substanzen charakterisiert, die sich gegen ein weites Spektrum von Bakterienspezies wirksam zeigten und zudem Resistenz-brechende („resistance-breaking“) Eigenschaften aufweisen. Es konnte gezeigt werden, dass Chelocardine einen Einfluss auf bakterielle Membranen ausüben, ein spezifisches Targetmolekül konnte jedoch nicht identifiziert werden. Des Weiteren wurde gezeigt, dass der Resistenzmechanismus gegenüber Chelocardin auf Efflux-Mechanismen beruht, die durch Mutationen im Repressorprotein RamR vermittelt werden. Telomycin wurde als stark bakterizides, Calcium-abhängiges Antibiotikum mit Wirksamkeit gegen Gram-positive Bakterien charakterisiert. Zwei semisynthetische acylierte Telomycin-Derivate (TM-Dodec, TM-N-Oct) zeigten eine verbesserte und Calcium-unabhängige antibakterielle Aktivität. Die durchgeführten Studien führten zu der Schlussfolgerung, dass die Verbindungen in erster Linie mit der Zellmembran interagieren, wobei das Phospholipid Cardiolipin insbesondere für Telomycin einen wichtigen Bindepartner darstellt. Im Gegensatz dazu wurde für beide acylieten Derivate gezeigt, dass ihre antibakterielle Wirkung nicht ausschließlich auf einer Bindung an Cardiolipin beruht, was auf eine Interaktion mit weiteren Targetmolekülen hindeutet. Dies ist auch für Telomycin möglich, was einen Wirkmechanismus jenseits der Membranaktivität denkbar macht. Potentielle Proteintargets wurden im Rahmen dieser Arbeit untersucht, jedoch konnte keine eindeutige Schlussfolgerung auf den Wirkmechanismus der Telomycine gezogen werden

Semisynthesis and biological evaluation of amidochelocardin derivatives as broad-spectrum antibiotics.

To address the global challenge of emerging antimicrobial resistance, the hitherto most successful strategy to new antibiotics has been the optimization of validated natural products; most of these efforts rely on semisynthesis. Herein, we report the semisynthetic modification of amidochelocardin, an atypical tetracycline obtained via genetic engineering of the chelocardin producer strain. We report modifications at C4, C7, C10 and C11 by the application of methylation, acylation, electrophilic substitution, and oxidative C-C coupling reactions. The antibacterial activity of the reaction products was tested against a panel of Gram-positive and Gram-negative pathogens. The emerging structure-activity relationships (SARs) revealed that positions C7 and C10 are favorable anchor points for the semisynthesis of optimized derivatives. The observed SAR was different from that known for tetracyclines, which underlines the pronounced differences between the two compound classes

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

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