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Aktivitätsbasierte Sonden sind wertvolle Werkzeuge in der chemischen Biologie. Nach wie vor ist es jedoch eine Herausforderung, molekulare Sonden zu entwickeln, die spezifisch an das aktive Zentrum eines bestimmten Enzyms binden. Wir stellen hier eine Strategie zur Ligandenselektion vor, die es ermöglicht, rasch elektrophile Sonden auf ausgewählte Enzyme zuzuschneiden, und zeigen in einer Machbarkeitsstudie ihre Anwendung für die beiden Cysteinproteasen von SARS‐CoV‐2. Die resultierenden Sonden markieren spezifisch die aktiven Zentren von 3CLpro und PLpro mit hinreichender Selektivität sowohl in einem lebenden Zellmodell als auch vor dem Hintergrund eines nativen menschlichen Proteoms. Durch die Nutzung der Sonden als Werkzeuge für das kompetitive Screening einer Bibliothek von Naturstoffen wurden Salvianolsäurederivate als vielversprechende 3CLpro‐Inhibitoren identifiziert. Unsere Strategie zur Ligandenselektion wird für die schnelle Entwicklung von maßgeschneiderten Sonden von großem Nutzen sein und die Entdeckung von Inhibitoren für eine Vielzahl von Zielproteinen ermöglichen, die auch über Coronavirus‐Proteasen hinausgehen. published

The mechanisms underlying interactions between diatoms and bacteria are crucial to understand diatom behaviour and proliferation, and can result in far‐reaching ecological consequences. Recently, 2‐alkyl‐4‐quinolones have been isolated from marine bacteria, both of which (the bacterium and isolated chemical) inhibited growth of microalgae, suggesting these compounds could mediate diatom–bacteria interactions. The effects of several quinolones on three diatom species have been investigated. The growth of all three was inhibited, with half‐maximal inhibitory concentrations reaching the sub‐micromolar range. By using multiple techniques, dual inhibition mechanisms were uncovered for 2‐heptyl‐4‐quinolone (HHQ) in Phaeodactylum tricornutum. Firstly, photosynthetic electron transport was obstructed, primarily through inhibition of the cytochrome b 6 f complex. Secondly, respiration was inhibited, leading to repression of ATP supply to plastids from mitochondria through organelle energy coupling. These data clearly show how HHQ could modulate diatom proliferation in marine environments., Double trouble: In the light, 2‐heptyl‐4‐quinolone (HHQ) primarily blocks photosynthetic electron flow at the cytochrome b 6 f complex in the diatom P. tricornutum. In darkness, HHQ inhibits mitochondrial respiration, resulting in a decreased ATP supply, which ultimately leads to a decreased electric field strength in the thylakoid membranes inside the chloroplast of diatoms.

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete

Activity‐based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS‐CoV‐2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases., Tuning the ligand space allows to rapidly customize chemical probes for efficacy and active site specificity for a protease of interest. The application of this ligand selection approach is exemplified by generating probes for the two non‐homologous yet essential proteases of the new corona virus SARS‐CoV‐2 providing complementary chemical tools for proteomic labeling and inhibitor discovery.

The compound class of 2-alkyl-4(1H)-quinolones represents a unique group of bacterial secondary metabolites that have been the subject of extensive research since their first discovery over 70 years ago. New insights into their structural diversity and their role in the complex interactions in bacterial ecology and human pathogenicity are still being discovered. In parallel with the ongoing discovery of new 2-alkyl-4(1H)-quinolones and derivatives produced by microbes, synthetic methods were developed to facilitate access to these structurally diverse bioactive compounds. Here we present a detailed overview of the historical development and recent advances in their chemical synthesis. published

We synthesized all major saturated and unsaturated 2-alkyl-4(1H)-quinolone N-oxides of Pseudomonas and Burkholderia, quantified their native production levels and characterized their antibiotic activities against competing Staphylococcus aureus. We demonstrate that quinolone core methylation and position of unsaturation in the alkyl-chain dictate antibiotic potency which supports the proposed mechanism of action.

Various bacterial species are known to produce metabolites of the 4-quinolone N-oxide class. These compounds have antibiotic ­activities, and are believed to target the respiration of competing ­microbes by interfering with menaquinone-binding sites in enzymes of the electron-transport chain. Interestingly, even minor structural changes in related N-oxides produced by the same organism can produce a dramatic difference in antibiotic activity, suggesting possible functional specialization.

Bacteria engage in numerous collaborative and competitive interactions, which are often mediated by small molecule metabolites. Bacterial competition involves for example the production of compounds that effectively kill or inhibit growth of their neighbours but also the secretion of siderophores that allow securing the essential and fiercely embattled resource of ferric iron. Yet, the enormous diversity of metabolites produced has remained puzzling in many cases. We here present examples of both types of competition from our recent work. These include the human pathogen Pseudomonas aeruginosa producing HQNO derived 4-quinolone N-oxides varying in chain length and saturation as antibiotics against Staphylococcus aureus and two marine bacteria, Shewanella algae and Vibrio alginolyticus competing for iron acquisition via homodimeric and heterodimeric cyclic hydroxamate siderophores. In each case, bacteria not only produce one but a whole set of closely related metabolites encoded by a single biosynthetic gene cluster. Our recent work has demonstrated that individual metabolites can have significantly different biological activities and we speculate on the reasons for maintaining this metabolite diversity from the perspective of interspecies competition.

Das Humanpathogen Pseudomonas aeruginosa produziert uber 50 verschiedene Chinolone, von denen 16 zur Klasse der 2-Alkyl-4-chinolon-N-oxide (AQNOs) gehoren, die in Lange und Sattigung der Seitenkette variieren. Wir berichten hier uber die erste Synthese einer zuvor vermuteten ungesattigten AQNO-Variante, die wir in Kulturextrakten von P. aeruginosa bestatigen und deren Struktur als trans-Δ1-2-(Non-1-enyl)-4-chinolon-N-oxid aufklaren konnten. Wahrend das cis-Isomer inaktiv ist, zeigt die trans-Verbindung Aktivitat gegen S. aureus, einschlieslich MRSA, die um mehr als eine Grosenordnung hoher ist als die der ubrigen AQNOs. Bei niedrigen Konzentrationen induziert die Verbindung das Wachstum kleiner Kolonievarianten von S. aureus und inhibiert dessen Virulenz durch Hemmung der Hamolyse sowie unter anaeroben Bedingungen die Aktivitat der Nitratreduktase. Unsere Untersuchungen lassen vermuten, dass das ungesattigte AQNO einer der Hauptwirkstoffe von P. aeruginosa ist, durch den es konkurrierende Bakterienspezies moduliert.

The Pseudomonas quinolone signal (PQS) is an important quorum sensing signal of the pathogen Pseudomonas aeruginosa. We discovered an additional activity of PQS as a narrow spectrum antibiotic. Exploiting the privileged structure of PQS by the synthesis of heteroatom-substituted analogues led to a class of 2-alkyl-3-hydroxythiochromen-4-ones with highly potent antibiotic activity against the nasopharyngeal pathogen Moraxella catarrhalis. Synthetic optimization resulted in minimum inhibitory concentrations in the nanomolar range even for clinical isolates of M. catarrhalis. Surprisingly, the growth of other human pathogens and commensals, including closely related Moraxella species, was not inhibited, indicating exceptional species selectivity. Mechanistic studies revealed that the antibiotic was bactericidal and likely inhibits a target in the primary energy metabolism causing rapid depletion of the cellular ATP pool. published

Expanding the chemical space of quinolones led to a tandem quinolone-alkyne-cyclisation reaction allowing chemoselective control of the synthesis of tricyclic pyrrolo[1,2-a]quinolin-5-ones. Importantly, we discovered anti-protozoal activity against Plasmodium and Toxoplasma with specific potency of one of the compounds against the liver stage of the malaria parasite in the nanomolar range.

Scaffold engineering of the Pseudomonas quinolone signal results in a highly potent antibiotic with unprecedented species selectivity., The Pseudomonas quinolone signal (PQS) is an important quorum sensing signal of the pathogen Pseudomonas aeruginosa. We discovered an additional activity of PQS as a narrow spectrum antibiotic. Exploiting the privileged structure of PQS by the synthesis of heteroatom-substituted analogues led to a class of 2-alkyl-3-hydroxythiochromen-4-ones with highly potent antibiotic activity against the nasopharyngeal pathogen Moraxella catarrhalis. Synthetic optimization resulted in minimum inhibitory concentrations in the nanomolar range even for clinical isolates of M. catarrhalis. Surprisingly, the growth of other human pathogens and commensals, including closely related Moraxella species, was not inhibited, indicating exceptional species selectivity. Mechanistic studies revealed that the antibiotic was bactericidal and likely inhibits a target in the primary energy metabolism causing rapid depletion of the cellular ATP pool.

We explore the chemical space of Pseudomonas quinolone signal analogs as privileged structures and report the discovery of a thioquinolone as a potent inhibitor of the important virulence factor elastase of the human pathogen Pseudomonas aeruginosa. We provide evidence that the derivative binds to the active site zinc of elastase and additionally acts as a fluorescent zinc sensor. published

Entscheidend dafur, ob Bakterien Menschen krank machen, ist ihre Virulenz, also ihre Infektionskraft. Dabei spielen nicht nur Enzyme eine Rolle, sondern auch kleine organische Molekule: darunter Siderophore, die Eisen komplexieren, und Signalstoffe, die den Angriff auf den Wirt steuern.

Only a small fraction of bacteria can autonomously initiate growth on agar plates. Nongrowing bacteria typically enter a metabolically inactive dormant state and require specific chemical trigger factors or signals to exit this state and to resume growth. Micrococcus luteus has become a model organism for this important yet poorly understood phenomenon. Only a few resuscitation signals have been described to date, and all of them are produced endogenously by bacterial species. We report the discovery of a novel type of resuscitation signal that allows M. luteus to grow on agar but not agarose plates. Fractionation of the agar polysaccharide complex and sulfation of agarose allowed us to identify the signal as highly sulfated saccharides found in agar or carrageenans. Purification of hydrolyzed κ-carrageenan ultimately led to the identification of the signal as a small fragment of a large linear polysaccharide, i.e., an oligosaccharide of five or more sugars with a repeating disaccharide motif containing d -galactose-4-sulfate (G4S) 1,4-linked to 3,6-anhydro-α- d -galactose (DA), G4S-(DA-G4S) n ≥2 . IMPORTANCE Most environmental bacteria cannot initiate growth on agar plates, but they can flourish on the same plates once growth is initiated. While there are a number of names for and manifestations of this phenomenon, the underlying cause appears to be the requirement for a molecular signal indicating safe growing conditions. Micrococcus luteus has become a model organism for studying this growth initiation process, often called resuscitation, because of its apparent connection with the persistent or dormant form of Mycobacterium tuberculosis , an important human pathogen. In this report, we identify a highly sulfated saccharide from agar or carrageenans that robustly resuscitates dormant M. luteus on agarose plates. We identified and characterized the signal as a small repeating disaccharide motif. Our results indicate that signals inherent in or absent from the polysaccharide composition of solid growth media can have major effects on bacterial growth.

Triazoles are interesting templates for novel chemotherapeutic drugs. We synthesized here 17 1,3,4-substituted-1,2,3-triazoles that differed in their 1'-substituent (variable alkyl chain lengths C3-C12), the 3'-substituent (no substituent, -methyl or -propyl) or the salt form obtained. Several of the compounds were cytotoxic (μM range) for tumor cells (HL-60, Jurkat, MCF-7, HCT-116), and when the effect was compared to non-transformed cells (Vero), selectivity ratios of up to 23-fold were obtained. To estimate the liability of these potential drug candidates for triggering neurotoxicity, we used the LUHMES cell-based NeuriTox assay. This test quantifies damage to the neurites of human neurons. The four most potent tumoricidal compounds were found to be neurotoxic in a concentration range similar to the one showing tumor cell toxicity. As the neurites of the LUHMES neurons were affected at >4-fold lower concentrations than the overall cell viability, the novel triazoles were classified as specific neurotoxicants. The structure-activity relationship (SAR) for neurotoxicity was sharply defined and correlated with the one for anti-neoplastic activity. Based on this SAR, two non-neurotoxic compounds were predicted, and testing in the NeuriTox assay confirmed this prediction. In summary, the panel of novel triazoles generated and characterized here, allowed to define structural features associated with cytotoxicity and neurotoxicity. Moreover, the study shows that potential neurotoxic side effects may be predicted early in drug development if highly sensitive test methods for neurite integrity are applied.

The pathogen Pseudomonas aeruginosa produces over 50 different quinolones, 16 of which belong to the class of 2-alkyl-4-quinolone N-oxides (AQNOs) with various chain lengths and degrees of saturation. We present the first synthesis of a previously proposed unsaturated compound that is confirmed to be present in culture extracts of P. aeruginosa, and its structure is shown to be trans-Δ1-2-(non-1-enyl)-4-quinolone N-oxide. This compound is the most active agent against S. aureus, including MRSA strains, by more than one order of magnitude whereas its cis isomer is inactive. At lower concentrations, the compound induces small-colony variants of S. aureus, reduces the virulence by inhibiting hemolysis, and inhibits nitrate reductase activity under anaerobic conditions. These studies suggest that this unsaturated AQNO is one of the major agents that are used by P. aeruginosa to modulate competing bacterial species.

The human pathogen Pseudomonas aeruginosa uses the pqs quorum sensing system to coordinate the production of its broad spectrum of virulence factors to facilitate colonization and infection of its host. Hereby, the enzyme PqsD is a virulence related quorum sensing signal synthase that catalyzes the central step in the biosynthesis of the Pseudomonas quinolone signals HHQ and PQS. We developed a library of cysteine reactive chemical probes with an alkyne handle for fluorescence tagging and report the selective and highly sensitive in vitro labelling of the active site cysteine of this important enzyme. Interestingly, only one type of probe, with a reactive α-chloroacetamide was capable of covalently reacting with the active site. We demonstrated the potential of our probes in a competitive labelling platform where we screened a library of synthetic HHQ and PQS analogues with heteroatom replacements and found several inhibitors of probe binding that may represent promising scaffolds for the development of customized PqsD inhibitors as well as a chemical toolbox to investigate the activity and active site specificity of the enzyme.