A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development

Natural compounds as pathoblockers of streptococcal infections

Worldwide millions of people are affected by streptococcal diseases ranging from mild to severe. Lethality rates up to 30 % for invasive diseases and recurrent infections often leading to post-infection sequelae like rheumatic heart disease show that current antibiotic treatment is not sufficient. Therefore, a new concept has to be applied: It is assumed that resistance will not arise if conserved virulence mechanisms are targeted. To influence them small molecules are needed. Myxobacteria are proven producers of small molecules offering new chemical scaffolds and unknown modes-of-action. Consequently, by combining microbiological, cell-biological and analytical methods myxobacterial extracts and compounds were screened for activity with regard to two virulence mechanisms of S.pyogenes: invasion into epithelial cells and exploitation of the host’s plasmin(ogen) system. Active compounds were identified by linking a bioactivity-guided isolation strategy with analytical methods. This work demonstrates that myxobacterial compounds interfere successfully with two virulence mechanisms of S.pyogenes: (1) Two compounds inhibit streptococcal invasion by targeting a protein not known to be involved in this process so far. (2) Several compounds can serve as new inhibitors of plasmin(ogen) showing better inhibitory activity than known antifibrinolytics. Moreover, two of these compounds significantly prolonged survival in an in vivo skin infection model usually leading to systemic severity. There is rising evidence that the new class of anti-infectives targeting virulence - the ‘pathoblockers’ -will be enriched by myxobacterial compounds and improve the therapy of diseases caused by S.pyogenes.Millionen Menschen weltweit sind von Krankheiten betroffen, die durch Streptokokken verursacht werden und bisweilen schwere Verlaufsformen aufweisen. Letalitätsraten von bis zu 30 % für schwere Verläufe und wiederkehrende Infektionen, die oft zu Spätkomplikationen wie der rheumatischen Herzkrankheit führen, zeigen, dass die derzeitigen antibiotischen Möglichkeiten nicht ausreichen. Daher wird nun ein neuer Ansatz verfolgt: Diesem liegt die These zugrunde, dass das Angreifen an konservierten Virulenzmechanismen das Auftreten von Resistenzen vermeidet. Für diesen Ansatz werden so genannte „kleine Moleküle“ benötigt. Myxobakterien haben sich als verlässliche Produzenten solcher „kleinen Moleküle“ erwiesen, wobei sie zudem oft neu chemische Grundstrukturen und neuartige Wirkmechanismen bieten. Daher wurden myxobakterielle Extrakte und Substanzen auf ihre Aktivität in Bezug auf zwei Virulenzmechanismen von S.pyogenes untersucht: die Invasion in Epithelzellen und die Ausnutzung des menschlichen Plasmin(ogen)-Systems. Dabei wurden mikro-, zellbiologische und analytische Methoden kombiniert. Aktive Substanzen wurden mittels einer bioaktivitätsgeleiteten Isolierungsstrategie in Verbindung mit analytischen Methoden identifiziert. Diese Arbeit zeigt, dass myxobakterielle Substanzen erfolgreich mit zwei Virulenzmechanismen von S.pyogenes interferieren können: (1) Zwei Substanzen hemmen die Invasion, indem sie an einem Protein angreifen, dessen Rolle in diesem Prozess bisher unbekannt war. (2) Mehrere Substanzen können als neue Hemmstoffe des Plasmin(ogen)s dienen und zeigen zudem eine bessere Aktivität als bisher bekannte Antifibrinolytika. Des Weiteren verlängerten zwei dieser Substanzen signifikant das Überleben in einem Maus-Hautinfektionsmodell, welches (sonst) zu einer Sepsis führt. Diese Ergebnisse zeigen, dass diese neue Klasse von Anti-Infektiva, die Virulenzmechanismen angreift - die „Pathoblocker“ - durch die myxobakteriellen Substanzen bereichert und die Therapiemöglichkeiten von durch Streptokokken hervorgerufenen Krankheiten verbessern wird

All Roads Lead to Rome: Enhancing the Probability of Target Attainment with Different Pharmacokinetic/Pharmacodynamic Modelling Approaches

In light of rising antimicrobial resistance and a decreasing number of antibiotics with novel modes of action, it is of utmost importance to accelerate development of novel treatment options. One aspect of acceleration is to understand pharmacokinetics (PK) and pharmacodynamics (PD) of drugs and to assess the probability of target attainment (PTA). Several in vitro and in vivo methods are deployed to determine these parameters, such as time-kill-curves, hollow-fiber infection models or animal models. However, to date the use of in silico methods to predict PK/PD and PTA is increasing. Since there is not just one way to perform the in silico analysis, we embarked on reviewing for which indications and how PK and PK/PD models as well as PTA analysis has been used to contribute to the understanding of the PK and PD of a drug. Therefore, we examined four recent examples in more detail, namely ceftazidime-avibactam, omadacycline, gepotidacin and zoliflodacin as well as cefiderocol. Whereas the first two compound classes mainly relied on the ‘classical’ development path and PK/PD was only deployed after approval, cefiderocol highly profited from in silico techniques that led to its approval. Finally, this review shall highlight current developments and possibilities to accelerate drug development, especially for anti-infectives

Lectin-Targeted Prodrugs Activated by Pseudomonas aeruginosa for Self-Destructive Antibiotic Release

Chronic Pseudomonas aeruginosa infections are characterized by biofilm formation, a major virulence factor of P. aeruginosa and cause of extensive drug resistance. Fluoroquinolones are effective antibiotics but are linked to severe side effects. The two extracellular P. aeruginosa-specific lectins LecA and LecB are key structural biofilm components and can be exploited for targeted drug delivery. In this work, several fluoroquinolones were conjugated to lectin probes by cleavable peptide linkers to yield lectin-targeted prodrugs. Mechanistically, these conjugates therefore remain non-toxic in the systemic distribution and will be activated to kill only once they have accumulated at the infection site. The synthesized prodrugs proved stable in the presence of host blood plasma and liver metabolism but rapidly released the antibiotic cargo in the presence of P. aeruginosa in a self-destructive manner in vitro. Furthermore, the prodrugs showed good absorption, distribution, metabolism, and elimination (ADME) properties and reduced toxicity in vitro, thus establishing the first lectin-targeted antibiotic prodrugs against P. aeruginosa

Lectin-Targeted Prodrugs Activated by Pseudomonas aeruginosa for Self-Destructive Antibiotic Release

Chronic Pseudomonas aeruginosa infections are characterized by biofilm formation, a major virulence factor of P. aeruginosa and cause of extensive drug resistance. Fluoroquinolones are effective antibiotics but are linked to severe side effects. The two extracellular P. aeruginosa-specific lectins LecA and LecB are key structural biofilm components and can be exploited for targeted drug delivery. In this work, several fluoroquinolones were conjugated to lectin probes by cleavable peptide linkers to yield lectin-targeted prodrugs. Mechanistically, these conjugates therefore remain non-toxic in the systemic distribution and will be activated to kill only once they have accumulated at the infection site. The synthesized prodrugs proved stable in the presence of host blood plasma and liver metabolism but rapidly released the antibiotic cargo in the presence of P. aeruginosa in a self-destructive manner in vitro. Furthermore, the prodrugs showed good absorption, distribution, metabolism, and elimination (ADME) properties and reduced toxicity in vitro, thus establishing the first lectin-targeted antibiotic prodrugs against P. aeruginosa

Zirconyl Clindamycinphosphate Antibiotic Nanocarriers for Targeting Intracellular Persisting Staphylococcus aureus

[ZrO]²⁺[CLP]^2– (CLP: clindamycinphosphate) inorganic–organic hybrid nanoparticles (IOH-NPs) represent a novel strategy to treat persisting, recurrent infections with multiresistant Staphylococcus aureus. [ZrO]²⁺[CLP]^2– is prepared in water and contains the approved antibiotic with unprecedented high load (82 wt % CLP per nanoparticle). The IOH-NPs result in 70–150-times higher antibiotic concentrations at difficult-to-reach infection sites, offering new options for improved drug delivery for chronic and difficult-to-treat infections

A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors

Trimethylamine-N-oxide (TMAO) has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases (CVDs). Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs, TMAO determinations are not clinical routine yet. The current methodology relies on isotope-labeled internal standards, which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine, betaine or choline. Here, we report a liquid chromatography-tandem mass spectrometry based method that is fast (throughput up to 240 samples/day), consumes low sample volumes (e.g., from a finger prick), and does not require isotope-labeled standards. We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for calibration. We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO, carnitine, betaine and choline were accurately quantified in ‘real-life’ human plasma samples from healthy volunteers, obtained either from a finger prick or from venous puncture. Additionally, we assessed the stability of samples stored at −20 °C and room temperature. Whereas all metabolites were stable at −20 °C, increasing concentrations of choline were determined when stored at room temperature. Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development, or to monitor disease progression and intervention effects.German Centre for Infection Researc

Synthesis and Characterization of DOTAM-Based Sideromycins for Bacterial Imaging and Antimicrobial Therapy

The rise of antimicrobial resistance, especially in Gram-negative bacteria, calls for novel diagnostics and antibiotics. To efficiently penetrate their double-layered cell membrane, we conjugated the potent antibiotics daptomycin, vancomycin, and sorangicin A to catechol siderophores, which are actively internalized by the bacterial iron uptake machinery. LC–MS/MS uptake measurements of sorangicin derivatives verified that the conjugation led to a 100- to 525-fold enhanced uptake into bacteria compared to the free drug. However, the transfer to the cytosol was insufficient, which explains their lack of antibiotic efficacy. Potent antimicrobial effects were observed for the daptomycin conjugate 7 (∼1 μM) against multidrug-resistant Acinetobacter baumannii. A cyanin-7 label aside the daptomycin warhead furnished the theranostic 13 that retained its antibiotic activity and was also able to label ESKAPE bacteria, as demonstrated by microscopy and fluorescence assays. 13 and the cyanin-7 imaging conjugate 14 were stable in human plasma and had low plasma protein binding and cytotoxicity