Development of Pathoblockers by Targeting Secreted Bacterial Proteases

The alarming rise of antibiotic resistant pathogens poses a serious threat to public health and urgently necessitates the development of new antibiotics. In order to reduce the selection pressure on bacteria, this study aims at the development of ‘pathoblockers’ which target bacterial virulence instead of killing the pathogens. In this context, secreted bacterial collagenases represent highly attractive targets because of their pivotal roles in the infection process and their advantageous extracellular localization. In this thesis, the development of inhibitors of ColH from Clostridium histolyticum and LasB from Pseudomonas aeruginosa is described. A class of N-aryl mercaptoacetamides displays promising activity on both targets, with distinct differences in structure-activity relationships and the binding mode to the respective protease. Synthetic efforts further led to the development of a hydroxamate derivative of the lead LasB inhibitor. The inhibitors described herein share a high selectivity toward mammalian matrix metalloproteases, which was rationalized by combining the information obtained from the co-crystal structures with computational approaches. Further biological assays shed light on the impact of LasB inhibition on key resistance mechanisms of P. aeruginosa and demonstrate in vivo efficacy in a Galleria mellonella infection model.Der alarmierende Anstieg von Antibiotika-resistenten Bakterien stellt eine ernstzunehmende Bedrohung für die menschliche Gesundheit dar. Aus diesem Grund ist Entwicklung neuer Antibiotika dringend notwendig. Um den Selektionsdruck auf Bakterien zu reduzieren, hat diese Arbeit zum Ziel, „Pathoblocker“ zu entwickeln, welche die bakterielle Virulenz hemmen anstatt die Pathogene zu töten. In diesem Kontext stellen sezernierte bakterielle Kollagenasen sehr attraktive Targets dar, da sie eine zentrale Rolle im Infektionsprozess spielen und den Vorteil mit sich bringen, dass die bakterielle Zellwand nicht überwunden werden muss. In dieser Dissertation ist die Entwicklung von Inhibitoren der Enzyme ColH aus Clostridium histolyticum und LasB aus Pseudomonas aeruginosa beschrieben. Eine Klasse von N-Arylmercaptoacetamiden zeigt vielversprechende Aktivität an beiden Targets, allerdings mit klaren Unterschieden in den Struktur-Wirkungs-Beziehungen und im Bindungsmodus. Über chemische Synthese wurde weiterhin ein Hydroxamsäure-Derivat des besten LasB-Inhibitors entwickelt. Die hier beschriebenen Inhibitoren haben eine hohe Selektivität gegenüber humanen Matrix-Metalloproteasen gemein, die über die Kristallstrukturen und Computer-basierte Methoden rational erklärt werden konnte. Weitere biologische Assays beleuchten die Auswirkung der LasB-Hemmung auf zentrale Resistenzmechanismen von P. aeruginosa und zeigen in vivo-Effekte in einem Galleria mellonella-Infektionsmodell

Disrupting Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Latent Replication with a Small Molecule Inhibitor

Kaposi's sarcoma-associated herpesvirus (KSHV) can establish latent lifelong infections in infected individuals. During viral latency, the latency-associated nuclear antigen (LANA) mediates the replication of the latent viral genome in dividing cells and tethers them to mitotic chromosomes, thus ensuring their partitioning into daughter cells during mitosis. This study aims to inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) latent replication by targeting the LANA-DNA interaction using small molecular entities. Drawing from first-generation inhibitors and using growth vectors identified through STD-NMR, we expanded these compounds using Suzuki-Miyaura cross-coupling. This led to a deeper understanding of SAR achieved by microscale thermophoresis (MST) measurements and cell-free tests via electrophoretic mobility shift assays (EMSA). Our most potent compounds successfully inhibit LANA-mediated replication in cell-based assays and demonstrate favorable in vitro ADMET-profiles, including suitable metabolic stability, Caco-2 permeability, and cytotoxicity. These compounds could serve as qualified leads for the future refinement of small molecule inhibitors of KSHV latent replication

Tackling pseudomonas aeruginosa virulence by a hydroxamic acid-absed LasB inhibitor

In search of novel antibiotics to combat the challenging spread of resistant pathogens, bacterial proteases represent promising targets for pathoblocker development. A common motif for protease inhibitors is the hydroxamic acid function, yet this group has often been related to unspecific inhibition of various metalloproteases. In this work, the inhibition of LasB, a harmful zinc metalloprotease secreted by Pseudomonas aeruginosa, through a hydroxamate derivative is described. The present inhibitor was developed based on a recently reported, highly selective thiol scaffold. Using X-ray crystallography, the lack of inhibition of a range of human matrix metalloproteases could be attributed to a distinct binding mode sparing the S1′ pocket. The inhibitor was shown to restore the effect of the antimicrobial peptide LL-37, decrease the formation of P. aeruginosa biofilm and, for the first time for a LasB inhibitor, reduce the release of extracellular DNA. Hence, it is capable of disrupting several important bacterial resistance mechanisms. These results highlight the potential of protease inhibitors to fight bacterial infections and point out the possibility to achieve selective inhibition even with a strong zinc anchor

Micro-rheological properties of lung homogenates correlate with infection severity in a mouse model of Pseudomonas aeruginosa lung infection

Lung infections caused by Pseudomonas aeruginosa pose a serious threat to patients suffering from, among others, cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis, often leading to life-threatening complications. The establishment of a chronic infection is substantially related to communication between bacteria via quorum-sensing networks. In this study, we aimed to assess the role of quorum-sensing signaling molecules of the Pseudomonas quinolone signal (PQS) and to investigate the viscoelastic properties of lung tissue homogenates of PA-infected mice in a prolonged acute murine infection model. Therefore, a murine infection model was successfully established via intra-tracheal infection with alginate-supplemented Pseudomonas aeruginosa NH57388A. Rheological properties of lung homogenates were analyzed with multiple particle tracking (MPT) and quorum-sensing molecules were quantified with LC–MS/MS. Statistical analysis of bacterial load and quorum-sensing molecules showed a strong correlation between these biomarkers in infected lungs. This was accompanied by noticeable changes in the consistency of lung homogenates with increasing infection severity. Furthermore, viscoelastic properties of the lung homogenates strongly correlated with bacterial load and quorum sensing molecules. Considering the strong correlation between the viscoelasticity of lung homogenates and the aforementioned biomarkers, the viscoelastic properties of infected lungs might serve as reliable new biomarker for the evaluation of the severity of P. aeruginosa infections in murine models

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

Flexible Fragment Growing Boosts Potency of Quorum Sensing Inhibitors against Pseudomonas aeruginosa Virulence.

Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-amino pyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa . We pursue an innovative treatment strategy by interfering with the Pseudomonas Quinolone Signal (PQS) Quorum Sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency

Unlocking the Antiviral Arsenal: Multiparametric Optimization of Small-Molecule Inhibitors against RSV and hCoV-229E

Acute respiratory diseases in humans can be caused by various viral pathogens such as respiratory syncytial virus (RSV), human coronavirus 229E (hCoV-229E), and severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2). To prevent severe cases by an early treatment, one effective strategy is to inhibit viral infection at the entry stage of the replication cycle. However, there is a lack of efficient, FDA-approved small molecule drugs targeting these pathogens. Previously, we identified two dual RSV/hCoV-229E small molecule inhibitors with activity in the single-digit micromolar range. In this study, we focused on optimizing the more promising starting point using a multiparametric hit optimization approach. Here, we present the results, including valuable insights into the structure activity relationship (SAR), and report the discovery of a submicromolar RSV entry inhibitor and a highly potent compound against hCoV-229E

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.Helmholtz Association HVF0054German Centre for Infection Research FlexFundTTU09.908, FlexFundTTU09.916, TTU09.719Federal Ministry of Education and Research (BMBF) 16GW0304

Micro-rheological properties of lung homogenates correlate with infection severity in a mouse model of Pseudomonas aeruginosa lung infection.

Lung infections caused by Pseudomonas aeruginosa pose a serious threat to patients suffering from, among others, cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis, often leading to life-threatening complications. The establishment of a chronic infection is substantially related to communication between bacteria via quorum-sensing networks. In this study, we aimed to assess the role of quorum-sensing signaling molecules of the Pseudomonas quinolone signal (PQS) and to investigate the viscoelastic properties of lung tissue homogenates of PA-infected mice in a prolonged acute murine infection model. Therefore, a murine infection model was successfully established via intra-tracheal infection with alginate-supplemented Pseudomonas aeruginosa NH57388A. Rheological properties of lung homogenates were analyzed with multiple particle tracking (MPT) and quorum-sensing molecules were quantified with LC-MS/MS. Statistical analysis of bacterial load and quorum-sensing molecules showed a strong correlation between these biomarkers in infected lungs. This was accompanied by noticeable changes in the consistency of lung homogenates with increasing infection severity. Furthermore, viscoelastic properties of the lung homogenates strongly correlated with bacterial load and quorum sensing molecules. Considering the strong correlation between the viscoelasticity of lung homogenates and the aforementioned biomarkers, the viscoelastic properties of infected lungs might serve as reliable new biomarker for the evaluation of the severity of P. aeruginosa infections in murine models