Tuft cells (TCs) act as crucial airway sentinels that detect bacterial metabolites and initiate immune responses, yet the underlying mechanisms remain poorly understood. Here, we identify tracheal TCs as the initial source of leukotrienes (LTs), released during bacterial infection. Tracheal TCs discriminate pathogenic from commensal bacteria by sensing extracellular ATP (eATP) released by pathogens, including Pseudomonas aeruginosa and Rodentibacter pneumotropicus, within 4 h of infection, through the transient receptor potential cation channel subfamily M member 5 (Trpm5). This induces the LT release, including LTB4, and promotes rapid recruitment of neutrophils and macrophages to the trachea and alveolar spaces. Trpm5-/- mice failed to detect bacterial eATP, exhibited neutrophil sequestration in the spleen, and became colonized following R. pneumotropicus infection, while Trpm5+/+ mice efficiently cleared the pathogen. These findings uncover a critical TC-dependent sensing mechanism in pneumonia, establishing TCs as both ATP sensors and triggers of acute innate immune responses.

Nα-Aroyl-N-aryl-phenylalanine amides (AAPs) are a class of antimycobacterial substances that inhibit the RNA polymerase and are effective against various pathogenic and opportunistic mycobacteria, including Mycobacterium tuberculosis, Mycobacterium abscessus, and Mycobacterium avium. Further development of these promising compounds, however, has been hindered by their low microsomal stability, leading to insufficient bioavailability. The present study investigates the mechanism by which microsomal enzymes metabolically degrade AAPs and identifies the resulting metabolites using LC-MS/MS. Rapid oxidation of the ortho-phenylenediamine structure, present in various substances in this class, plays a key role in this process. Additionally, we demonstrated in vitro and in vivo that cytochrome P450 enzyme inhibitors significantly slow the degradation of AAPs. Identification of metabolites will inform further chemical modification of AAPs to achieve metabolic stability.

Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2/IMP2) plays a crucial role in the posttranscriptional regulation of gene expression and influences various cellular processes including cell growth, differentiation, and metabolism. Dysregulation of IMP2 has been associated with several diseases, including cancer and metabolic disorders. Targeting IMP2 with small molecules is a promising therapeutic strategy. However, the structural diversity of IMP2-targeting compounds remains limited. In this study, we present a comprehensive screening approach with the aim of identifying new structural classes of compounds that can inhibit IMP2 activity, particularly its binding to the KH34 domain. Screening of a chemically diverse library comprising 10,240 compounds using fluorescence polarization-based assays led to the identification of ten primary actives belonging to five distinct structural classes. After rigorous resynthesis and hit validation, only one compound comprising a sulfonamide scaffold reproducibly inhibited the KH34-RNA interaction in vitro. This hit was further characterized by STD-NMR and in vitro ADME profiling, including solubility, lipophilicity, metabolic stability, plasma protein binding, and cellular permeability. While this sulfonamide-based inhibitor exhibits clear biochemical activity and a defined binding mode at the KH34 RNA-binding interface, its limited cellular permeability and high plasma protein binding currently preclude cellular efficacy. This work identifies a new structural class of IMP2 KH34 inhibitors serving as a starting point for an ongoing hit-to-lead optimization campaign towards next-generation anti-cancer drugs.

Chronic infection with Pseudomonas aeruginosa is a major driver of airway inflammation, which plays a central role in the progression of cystic fibrosis (CF) lung disease. During long-term colonization, P. aeruginosa adapts to the CF lung by downregulating virulence factors and adopting a biofilm-associated, mucoid lifestyle. Despite the expected reduction in immune activation due to these adaptations, excessive inflammation persists, a paradox that remains poorly understood. Our objective was to identify novel bacterial mediators sustaining persistent inflammation by P. aeruginosa in the CF lung. To this end, we analyzed clinical P. aeruginosa CF isolates, cultured them in synthetic CF sputum medium, and exposed 3D lung epithelial cell cultures to the resulting cell-free supernatants. There was considerable variability in pro-inflammatory activity among the isolates, with a subset of the isolates inducing strong IL-8 secretion by the 3D cells despite low production of known virulence factors. Comparative proteomics analysis of the cell-free supernatants of pro-inflammatory and immunosuppressive isolates revealed several mediators not previously linked to inflammation. Thirteen of these candidate pro-inflammatory mediators were selected for further analysis. Using P. aeruginosa transposon mutants lacking the respective mediators, DksA (a transcription factor) was confirmed as an immunomodulatory mediator in the 3D lung model. Finally, analysis of existing transcriptomes of P. aeruginosa in CF sputum revealed that dksA was found to be one of the most strongly expressed genes in this patient population, highlighting the relevance of our findings. In conclusion, we identified a novel P. aeruginosa mediator that may contribute to CF airway inflammation.

To tackle the emerging resistance against existing antibiotics, we screened natural-product (NP) libraries against two underexploited target enzymes from the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, namely, Mycobacterium tuberculosis DXPS and Escherichia coli IspD. We have chosen these two enzymes due to the availability of the crystal structures that helped to elucidate the putative binding modes of the NPs identified. The screening of a NP collection led to the discovery of myxobacteria-derived maracen A and Streptomyces-derived polyketomycin as the first NPs targeting these enzymes.

Making use of the first published X-ray structure of the Parathyroid Hormone 1 Receptor, we employed small-molecule docking calculations in order to identify novel ligands. Besides directly using the experimental structure, we also generated several models through homology modeling or by reverting stabilizing mutations present in the experimental structure to wild-type. Our calculations yielded three ligands, two of which we further developed into two series of compounds through similarity searches and docking calculations. The set of ligands presented here enriches the landscape of Parathyroid Hormone 1 Receptor ligands, and introduces novel starting points for further development of compounds targeting disorders of calcium homeostasis and bone metabolism.

Catechol-modified polymers, such as DOPA-functionalized systems, have recently gained significant interest for a variety of biomedical applications, particularly in their role as antibacterial adjuvants due to their oxidative activity and ability to generate reactive oxygen species (ROS). Current catechol-functionalized polymers, however, often suffer from a restricted number of catechol groups, limited biocompatibility and solubility, and low stability due to the rapid oxidation under physiological conditions. In this study, we developed a water-soluble, biocompatible DOPA-modified biodynamer (DOPA-BD), leveraging the principles of constitutional dynamic chemistry (CDC). DOPA-BD was synthesized via polycondensation of DOPA-hydrazide and the hexaethylene glycol-conjugated carbazole dialdehyde (CA-HG), forming dynamic imine and acylhydrazone linkages between the monomers. As a result of its dynamic covalent backbone, DOPA-BD exhibits biodegradability and undergoes pH-responsive degradation under mildly acidic conditions typically found at infection sites, leading to a more than 3-fold increase in DOPA-hydrazide release compared to physiological pH. Interestingly, driven by CDC, DOPA-BD folds into a nanorod structure with a hydrodynamic diameter of ∼7.8 nm, surrounded by HG chains that offer water solubility and biocompatibility. Moreover, the incorporation of the DOPA-derivative in each repeating unit yields a polymer with exceptionally high catechol content, which remains stable and resistant to oxidation for 72 h in physiological buffer conditions. Regarding its antibacterial applicability, DOPA-BD demonstrated synergistic antibacterial activity with Azithromycin (AZM) against AZM-resistant E. coli, enhancing the antibiotic’s efficacy by 4-fold. Our study indicates that DOPA-BD induces ROS production in the respective bacterial strain, suggesting ROS generation as one of the possible mechanisms contributing to the observed synergy. Overall, DOPA-BD represents a promising alternative strategy to potentiate antibacterial activity against resistant strains, holding strong potential for future antibacterial applications.

The increasing prevalence of drug-resistant Staphylococcus aureus infections highlights the urgent need for innovative treatments. In this study, we present MCZ-038, a bactericidal thiourea derivative from a targeted chemical optimization campaign. MCZ-038 showed potent antimicrobial activity against multidrug-resistant clinical strains of S. aureus and Enterococcus faecium, including methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant Enterococcus faecium (VRE) with a minimal inhibitory concentration (MIC) of 4-8 μg/mL. MCZ-038 exhibited concentration-dependent bactericidal kill kinetics against S. aureus and synergized with gentamicin, enhancing its potential for combination therapies. Importantly, MCZ-038 demonstrated superior efficacy against biofilms and intracellular bacteria residing in macrophages as compared to FDA approved antibiotics. Furthermore, prolonged exposure of S. aureus to MCZ-038 did not lead to detectable resistance, underscoring its potential as a durable therapeutic option. MCZ-038 targeted membrane fluidity, an unconventional target, which explains lack of generated resistance. In a murine thigh and skin infection model with MDR MRSA NRS119, MCZ-038 effectively reduced bacterial load both alone and in combination with gentamicin. These findings position MCZ-038 as a promising candidate for further optimization of the current liabilities such as metabolic stability and in vitro cytotoxicity and further development against challenging, drug-resistant S. aureus infections.

Pseudomonas aeruginosa, a major cause of pulmonary infections, poses significant clinical challenges due to its virulence and rising antimicrobial resistance. We investigated the role of LasB, a key virulence factor and elastase of P. aeruginosa, in disrupting the lung epithelial barrier. LasB cleaves the junctional protein E-cadherin, alters Claudin-4 localization, and reduces levels of immunomodulatory cytokines including GM-CSF and G-CSF. Using bronchial and alveolar cell models, we demonstrate that LasB induces dose-dependent barrier damage in both systems. Transcriptomic analysis reveals widespread gene expression changes, including the upregulation of DUSP2 and FGFBP1 associated with stress signaling and immune modulation. LasB inhibitors mitigate barrier disruption and partially restore cytokine levels. In a live bacterial infection model, LasB inhibition supports antibiotic treatment, enhancing bacterial clearance and preserving epithelial integrity. These findings establish LasB as a pivotal factor in P. aeruginosa pathogenesis and highlight the therapeutic potential of antivirulence strategies targeting LasB as promising adjuncts to conventional antibiotics.

Virtual screening remains a critical step in structure-based drug design, yet variability in docking algorithms and scoring functions often limits its reliability. To address this challenge, we introduce DockM8, an open-source platform for consensus virtual screening that integrates pocket detection, ligand preparation, docking, rescoring, and consensus ranking within a single modular workflow. DockM8 supports 5 docking engines, 17 pose-selection methods, 17 scoring functions, and 5 consensus methods, all accessible through both a graphical user interface (GUI) and a Python application programming interface (API). Systematic evaluation on the DEKOIS 2.0, DUD-E, and Lit-PCBA datasets yielded median relative enrichments of 100%, 86.05%, and 8.675%, respectively, frequently outperforming state-of-the-art approaches and highlighting the robustness and adaptability of DockM8 across diverse targets. Our findings further reveal that no pose-selection or consensus strategy universally excels, emphasizing the need for tailored, target-specific workflows. DockM8 is freely available under the GNU General Public License at https://github.com/DrugBud-Suite/DockM8 . Scientific Contribution DockM8 is, to our knowledge, the first open-source platform to expose the complete consensus structure-based virtual screening (SBVS) pipeline within a single tool, yielding over 55 million configurable protocols, a breadth unmatched by previously published consensus docking software. Through systematic multi-benchmark evaluation, we demonstrate that no single pose-selection or consensus strategy generalizes across targets, motivating a target-specific workflow-selection paradigm in place of prevailing one-size-fits-all heuristics. By delivering this tunability through a graphical interface rather than scripts, DockM8 brings rigorous, reproducible consensus virtual screening within reach of non-specialist medicinal chemists.

A targeted sequencing and genome mining approach for delftibactin-like biosynthetic pathways revealed three distinct biosynthetic gene cluster architectures (BGC del, dlc and dlp) encoded in genomes of members of the genus Delftia. Comparative metabolomic analysis guided the isolation and characterization of a yet unreported metallophore, delftichelin A from Delftia deserti DSM1621 (previously named Delftia acidovorans DSM1621). Prediction of BGC architecture and A domain specificity was in line with the structure analysis uncovering previously unreported differences in amino acid composition and modifications. Analysis of bioactivity and metal-binding characteristics demonstrated that delftichelin A shows a preferential affinity for ferric iron, while also exhibiting heavy metal detoxification mechanisms via oxidative degradation, analogous to those reported for the delftibactin family of compounds.

Antimicrobial resistance is a global crisis driven by a scarce pipeline of new antibiotics. A major contributor is the intrinsic resistance conferred by the bacterial envelope, highlighting the need for innovative molecules for improved therapies. In this study, TAT-ArgBD, a conjugate of the cell-penetrating TAT peptide and arginine biodynamer (ArgBD), serves in vitro as a multivalent macromolecular antibiotic and synergist. TAT-ArgBD rapidly kills 99.9% of Pseudomonas aeruginosa at 32 µg/mL within 1 h, outperforming colistin, and shows minimum inhibitory concentrations (MICs) of 2-8 µg/mL against Acinetobacter baumannii and Staphylococcus aureus. Notably, it potentiates antibiotics such as novobiocin, chloramphenicol, and imipenem, leading to lowered MICs up to 256-fold. Notably, novobiocin, typically active only against Gram-positive bacteria, showed activity against Gram-negative bacteria when combined with TAT-ArgBD. Mechanistic studies suggest TAT-ArgBD antimicrobial and synergistic actions result from preferential binding to POPG and cardiolipin. This interaction induces bacterial membrane pore formation by adopting an α-helical conformation in the presence of bacterial lipids. With a favorable in vitro safety profile, a membranolytic index > 64 and low mammalian cell toxicity at effective bactericidal concentrations, TAT-ArgBD's potential to enhance antibiotic efficacy, as well as function as a stand-alone treatment, supports further preclinical evaluation as an antimicrobial adjuvant.

Structure-based virtual screening (SBVS) is a cornerstone of computer-aided drug design, yet its success depends on selecting a combination of docking tools, scoring function (SF), and ranking strategies. MolDockLab addresses this challenge with an automated, data-driven framework that optimizes SBVS workflows for a protein target, balancing predictive performance and computational efficiency. It systematically explores combinations of five docking engines, 15 SF, and three consensus ranking strategies using a calibration set of ≈ 200 compounds with known bioactivity, and applies the best-correlating workflow to the larger screening library. Final hit selection from the top 1% integrates protein-ligand interaction profiler (PLIP)-derived interaction fingerprints, structural-diversity assessment, and expert visual inspection. In a retrospective evaluation on the epidermal growth factor receptor (EGFR), the chosen pipeline achieved a Spearman correlation of 0.36 and an enrichment factor (EF) at 10% of 1.57, consistent with calibration. Prospectively, for the energy coupling factor transporters (ECF-T)-a challenging transmembrane target with a cryptic binding site and no co-crystallized ligand-the pipeline reached a correlation of 0.45 and enrichment of 3.13. Post-processing enabled in vitro confirmation of two chemically novel inhibitors rivaling the most potent ECF-T inhibitors reported to date.

Targeting the extracellular protease elastase (LasB) of the high-priority pathogen Pseudomonas aeruginosa is a promising strategy to develop second-generation, narrow-spectrum antibiotics with a novel mode of action. P. aeruginosa is responsible for a variety of infections, particularly of the lung. Herein, we report the structure-based optimization of a previously reported potent and selective phosphonate-based LasB inhibitor scaffold. Having improved the activity while maintaining high selectivity and favorable ADMET properties, we also demonstrate, for the first time within this scaffold, that intravenous administration leads to favorable lung retention. We could rationally align this with in vitro plasma protein binding. We further observed a link between physicochemical properties like logD7.4 and protein binding, including surfactant proteins that can impair compound activity in the lung. This multiparameter optimization paves the way for the exploration of additional indications requiring systemic treatment, such as hospital-acquired or ventilator-associated pneumonia.

Multiparameter optimization of a previously identified class of inhibitors of the energy-coupling factor (ECF) transporters enabled the confirmation of in vivo efficacy. ECFs are a class of transmembrane proteins that play a vital role in the active translocation of essential nutrients across cell membranes and are therefore important in the fight against antimicrobial resistance. Aiming to improve the drug-like properties of our inhibitory class, we performed a focused structure-activity relationship study around the Eastern part of our starting molecule 3 by exploiting click chemistry. Our multiparameter optimization resulted in compounds with enhanced metabolic stability and solubility, potent activity against both a panel of Gram-positive bacteria, and against the ECF transporters. We further demonstrate rapid bacterial killing using Enterococcus faecium as a model organism and confirmed in vivo efficacy of the best compounds in Galleria mellonella larvae and Danio rerio (zebrafish) infection models, highlighting the therapeutic potential of our approach.

Energy-Coupling Factor (ECF) transporters are a hitherto underexplored target involved in the uptake of several micronutrients in bacteria and are absent in humans. Here, in Streptococcus pneumoniae , we demonstrate that the genes encoding ECF transporters are highly conserved and their expression is crucial for causing bacterial infection in both murine (in vivo) and human (ex vivo) infection models. Next, we demonstrated that the antimicrobial activity of our inhibitors against S. pneumoniae is related to the level of the ECF transporter expressed by the bacterium, confirming the target engagement of this chemical class in S. pneumoniae . The pharmacokinetic studies conducted revealed high peroral bioavailability for 4, which was also assessed in a murine neutropenic lung-infection model with S. pneumoniae , and observed a one log10 reduction in bacterial load compared to vehicle. This work sets the stage for an innovative approach to combat S. pneumonia -derived infections by targeting the ECF transporters with the novel chemotype reported.

The methylerythritol phosphate (MEP) pathway is essential for isoprenoid biosynthesis in many pathogenic bacteria but is absent in humans, making its enzymes attractive antibacterial targets. IspE catalyzes the ATP-dependent phosphorylation of 4-diphosphocytidyl-2-C-methylerythritol, a key step in this pathway. Using a previously identified optimized hit as a starting point, we designed and synthesized a focused library of twelve simplified analogues that retained essential pharmacophoric features while improving synthetic accessibility. Docking studies with Escherichia coli IspE guided the design and predicted binding orientations consistent with known ligand interactions. Biochemical evaluation of the library against E. coli and Klebsiella pneumoniae IspE revealed several low-micromolar inhibitors, confirming the predicted binding interactions. Structure-activity relationships indicated that the hydrophobic pocket adjacent to the cytidine-binding region is a key determinant of potency. Although the compounds showed limited whole-cell activity, these results demonstrate that simplified amide analogues can effectively engage the IspE active site and highlight the importance of the hydrophobic pocket in ligand binding. Overall, this work combines structure-based design, synthesis, and biochemical validation to provide a foundation for further optimization of simplified IspE inhibitors as potential antibacterial leads.

Abstract Large language models and generative protein design promise to accelerate biotechnology, but it remains unclear whether they can engineer dynamic megasynth(et)ases whose activity depends on transient, context-specific domain interfaces. Non-ribosomal peptide synthetases (NRPSs) are an especially demanding target, yet a high-value one because they produce many clinically important natural products and offer a route to analogs that are often difficult or impractical to access by chemical synthesis. Here we integrate pretrained generative models (ESM3, ProteinMPNN and EvoDiff) with design–build–test–learn cycles and data-guided prioritization to generate 76 de novo thiolation (T) domains. We built and tested 578 recombinant NRPS variants in vivo spanning minimal, full-length and hybrid assembly lines. AI-designed T-domains supported product formation across architectures, enabled catalytically active hybrids at recombined junctions and increased yields by up to ∼3-fold relative to NRPSs carrying the native T-domain. A representative design showed improved soluble expression, refolding, and a 12 °C higher melting temperature, while molecular dynamics simulations indicated preserved global stability but reshaped, state-dependent interdomain contact networks. Together, these results establish generative design as an effective route to context-conditioned optimization and reprogramming of biosynthetic assembly lines.

Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positive thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) among the reported aromatic rings, which is the best? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can improve selectivity.

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 (SARS-CoV-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 a structure-guided optimization approach of the more promising prototype addressing activity, cell viability, selectivity, solubility and metabolic stability. We present valuable insights into the structure-activity relationship (SAR), and report the discovery of a sub-micromolar RSV entry inhibitor, a dual RSV/CoV-229E inhibitor and a highly potent compound against hCoV-229E.