Background: Antimicrobial resistance is at alarming levels. Methicillin-resistant Staphylococcus aureus is a clinical concern and has been classified as one of the so-called ESKAPE pathogens. The increase in resistance coupled with the lack of new agents in development requires innovative ways of developing new antimicrobials. Methods: Here, we investigate an approved drug (Zafirlukast), which has been previously reported to have activity against Mycobacterium, for activities against other bacterial strains. Antibacterial activity was assessed using standard MIC testing along with checkerboard analysis to investigate potential synergies/antagonisms. In order to define the mechanism of action (MOA), a number of Zafirlukast-resistant mutant strains of MRSA (generated via serial passage) were sequenced. Biochemical analysis was performed on the target pathway to confirm the putative target of Zafirlukast in Staphylococcus aureus. Results: Zafirlukast showed modest direct-acting antimicrobial activity (0.5-32 μg/mL) against a panel of Gram-positive bacteria, including MRSA. It was also found that Zafirlukast synergised with a number of existing antibiotics. Remarkably, mutants forced to be resistant to Zafirlukast became resensitised to the β-lactams that they were originally resistant to. This is potentially a very useful property of a potentiator drug. Mutational analysis suggested the target of Zafirlukast in Staphylococci is LtaS (Lipoteichoic acid synthase), of which there are no currently approved inhibitors. Teichoic acid pathway involvement was further confirmed via biochemical analysis. Conclusions: Here, we establish that Zafirlukast has activities beyond Mycobacterium and Gingivalis, encompassing many Gram-positive bacteria including MRSA, and, for the first time, we report its putative target as LtaS. The repurposing of safe-to-use drugs is an attractive strategy to derisk drug discovery programmes. Zafirlukast has many attractive antimicrobial activities, which may encourage further investigations in this field. However, it should be noted that it may represent a safe starting scaffold, and further chemistry is likely to be needed for it to be used in the antimicrobial setting.

This hypothesis-generating in silico screening study aimed to prioritize candidate ST2-binding bioactive compounds for severe Type 2-high asthma using an integrated workflow comprising molecular docking, protein-ligand interaction analysis, predicted ADMET profiling, molecular dynamics simulations, MM-GBSA binding-energy estimation, and density functional theory calculations. Sixteen compounds, including fifteen phytochemicals and one reference drug Zafirlukast, were screened against the ST2 receptor. Docking results indicated that several flavonoids, particularly Fisetin, Myricetin, Hesperetin, and Luteolin, showed more favorable predicted docking scores than the reference compound. Interaction analysis identified hydrogen-bonding and hydrophobic contacts with residues located within the selected ST2 binding pocket. Predicted ADMET profiling identified generally acceptable physicochemical and oral drug-likeness characteristics among the top-ranked compounds, although these predictions did not evaluate pulmonary delivery or inhalation pharmacokinetics. Among all candidates, Hesperetin was prioritized as a putative ST2-binding candidate based on its favorable predicted docking score, generally acceptable SwissADME profile, and recurring docking-derived interaction pattern within the selected ST2 pocket. Across three independently initialized molecular dynamics trajectories, the Hesperetin-ST2 complex showed dynamic persistence after an initial adjustment phase, whereas the Zafirlukast complex generally equilibrated earlier and exhibited lower RMSD values. Density functional theory analysis indicated a moderate HOMO-LUMO energy gap, providing supportive information on molecular reactivity and electronic stability. Hence, the findings prioritize Hesperetin for further experimental investigation as a candidate ST2-binding compound; however, receptor binding, functional IL-33/ST2 pathway inhibition, efficacy, and safety require validation through in vitro and in vivo studies.

Acute orchitis and other inflammatory testicular diseases lack delivery platforms that enable label-free fluorescence tracking of drug distribution in inflamed testes. Here, we show that three clinically approved leukotriene receptor antagonists, namely Pranlukast, Zafirlukast, and Montelukast can be transformed into self-reporting, self-delivering, and self-therapeutic nanotheranostics. These drugs intrinsically possess aggregation-induced emission (AIE): extensive intermolecular halogen and hydrogen bonds simultaneously restrict intramolecular motion to generate bright solid-state fluorescence and drive spontaneous self-assembly into uniform, PEG-stabilized nanocrystals. Unlike conventional dye-labeled nanocarriers, in which dye leakage causes false signals, AIE fluorescence faithfully reports the aggregated nanocrystal state, enabling monitoring of drug integrity. Beyond their canonical anti-leukotriene activity, these drugs directly suppress NF-κB signaling and scavenge reactive oxygen species-mechanisms highly relevant to acute orchitis pathogenesis, which involves TLR4/MyD88/NF-κB activation and oxidative stress. In LPS-stimulated cells, the nanocrystals suppress NF-κB activation and scavenge reactive oxygen species. In a rat model of acute orchitis, AIE imaging reveals efficient testicular accumulation, which correlates with attenuated histopathological damage, restored oxidative stress balance, and downregulation of the TLR4/MyD88/NF-κB and PK2/PKR1 pathways, with no obvious adverse effects on the measured short-term reproductive indices (sperm morphology and testosterone levels) observed within the experimental timeframe. This strategy converts old drugs into minimal-excipient, self-monitoring AIE nanotheranostics for imaging-guided orchitis therapy.

Folic acid and antifolates rely on SLC19A1 and SLC46A1 for membrane transport, which is critical for their biological functions and therapeutic efficacy. Leukotriene receptor antagonists (LTRAs) are widely used for long-term management of asthma and allergic rhinitis. This study aimed to explore the effects of four LTRAs (MK571, montelukast, zafirlukast, pranlukast) on the activities of SLC19A1 and SLC46A1. In vitro studies showed that MK571, montelukast, zafirlukast, and pranlukast concentration-dependently inhibited SLC19A1 and SLC46A1-mediated uptake of folic acid, methotrexate, and pemetrexed, with IC50 values ranging from 2.34 to 31.8 μM for SLC19A1 and 2.79 to 45.4 μM for SLC46A1. Kinetic analysis revealed distinct, transporter-specific inhibition mechanisms: LTRAs acted as noncompetitive inhibitors of SLC19A1 (Kᵢ = 5.09-6.71 μM) and as uncompetitive inhibitors of SLC46A1 (Kᵢ = 4.12-95.1 μM). In vivo pharmacokinetic studies in mice showed that co-administration of montelukast or zafirlukast significantly increased the systemic exposure (AUC and Cmax) and prolonged the half-life of folic acid, while reducing its hepatic accumulation. Taken together, this study identified LTRAs as novel and potent inhibitors of the key folate transporters SLC19A1 and SLC46A1, and uncovered previously unrecognized transporter-mediated drug-drug interactions between LTRAs and folate-related agents. These findings highlight the necessity of careful clinical monitoring and potential dose adjustments during concurrent administration to ensure therapeutic safety and efficacy.

It is crucial to determine equilibrium and kinetics of drug-receptor interactions for understanding thoroughly drug mechanism and drug efficacy. In this work, three antagonist drugs toward cysteinyl leukotriene receptor 1 (CysLTR1) were exemplified to measure the binding parameters by multiple affinity chromatographic approaches like frontal chromatography, injection amount-dependent method and nonlinear chromatography. The association equilibrium constants were measured as montelukast < zafirlukast < pranlukast and the dissociation rate constants were in the rank of montelukast > zafirlukast > pranlukast, which were correlated to the inhibition potencies of montelukast < zafirlukast < pranlukast to CysLTR1. Additionally, thermodynamic analysis in combination with molecular docking indicated that the three antagonists were spontaneously bound to CysLTR1 and their bindings were mainly driven by hydrogen bonds and van der Waals forces. Our work demonstrated that affinity chromatography can be utilized to evaluate the binding characteristics of ligand-receptor based on their retention behaviors, aiding the selection of the best ligands for further studies to become a drug molecule.

Tumor necrosis factor receptor 1 (TNFR1) signaling regulates cell fate in inflammation, immune responses, and tumorigenesis. While TNF-α-mediated TNFR1 pathways are well known, the role of receptor clustering remains unclear. Utilizing homo-FRET using fluorescence anisotropy, we show that intra-cluster receptor density (IRD) governs TNFR1 signaling outcomes. Soluble TNF-α (sTNF-α) increases IRD at cluster cores but decreases it at rims via receptor reorganization. Reducing IRD through membrane tension, zafirlukast, actin depolymerization, or cholesterol depletion suppresses sTNF-α signaling, whereas increasing IRD by lowering membrane tension or exposing cells in a 3D gel-like microenvironment triggers ligand-independent activation. These findings reveal IRD as a key regulator of receptor signaling, with potential relevance across related receptor families and innovative strategies in modulating TNFR1 signaling.

Epilepsy is one of the most prevalent neurological disorders worldwide, and approximately 25% of patients remain refractory to pharmacological treatment. Blood-brain barrier (BBB) disruption and reactive gliosis are key mechanisms implicated in early epileptogenesis. This study investigated the effects of zafirlukast, a leukotriene receptor antagonist, on BBB permeability, reactive gliosis, and behavioral seizure activity in a pentylenetetrazol (PTZ)-induced early epileptogenesis model in C57BL/6 mice. Zafirlukast was administered twice daily at a dose of 10 mg/kg. Seizure activity was evaluated by behavioral observation in terms of seizure severity, latency, duration, and frequency. BBB permeability was assessed using the Evans Blue assay, and brain tissues were analyzed by biochemical and immunohistochemical methods. The PTZ + ZAFIR group exhibited more severe seizures, characterized by increased seizure frequency and duration, shorter latency, and a higher kindling rate (80% vs. 27%). BBB permeability was also increased, whereas MMP-9 levels remained, suggesting disruption may be linked to direct mechanical effects of recurrent seizures rather than inflammation. Clues suggest that zafirlukast may exert paradoxical effects on two prominent cell types involved in reactive gliosis. While increased GFAP and TGF-β1 expression may reflect enhanced astrocyte activation, changes in IL-1β and Iba1 expression suggest suppression of microglial activation. Notably, pro-inflammatory and oxidative stress markers remained unchanged despite the increase in seizure severity. The observed reduction in neurodegeneration may be attributable to the suppressive effects of zafirlukast on microglial activation and the subsequent reduction in pro-inflammatory cytokine release. These findings indicate a complex role for leukotriene signaling during early epileptogenesis. Further studies using different doses, vehicles, and experimental models are warranted to clarify the effects of zafirlukast on the mechanisms underlying early epileptogenesis.

WD40 repeat-containing protein 5 (WDR5) plays a critical role in chromatin-related processes, and its overexpression is associated with poor prognosis in multiple cancers, making it an attractive therapeutic target. Screening of an approved drug library identified zafirlukast (ZAF), an anti-asthmatic agent, as a binder to the WIN site of WDR5, inhibiting the histone methyltransferase activity of the MLL1 complex with an IC50 of 22.96 μM. Structure-based optimization yielded the candidate HLC40, which not only abolished its original CysLT1 receptor antagonism (IC50 > 5 μM) but also exhibited a 30-fold enhancement in MLL1 histone methyltransferase (HMT) inhibitory activity (IC50 = 0.82 μM). Consistent with these findings, HLC40 exhibited potent antiproliferation efficacy (IC50 = 7-8 μM) in AML cells and demonstrated significant efficacy in the MV4-11 xenograft model (TGI = 49.1% @ 30 mg/kg). Collectively, these results highlight that HLC40 is a promising WDR5-targeting candidate with high therapeutic potential for hematologic malignancies.

In this study, a drug repurposing strategy was implemented with the aim of identifying new trypanocidal agents against Trypanosoma cruzi (T. cruzi). A total of 924 Food and Drug Administration (FDA)-approved drugs were screened by molecular docking on three sites of trypanothione synthetase (TS), including the catalytic site, a blind docking site, and a potential allosteric site. Selected compounds were further evaluated through in vitro and in vivo assays. Tadalafil, Zafirlukast, Raltegravir, and Olmesartan had better trypanocidal activity than the reference drugs Benznidazole and Nifurtimox in the in vitro evaluation against the trypomastigote form. Additionally, these drugs were able to decrease parasitemia by 20-50% in mice in an acute treatment. Molecular dynamics simulations (MDS) at 120 ns helped link findings from in vitro/in vivo experiments to a potential mechanism of action targeting T. cruzi trypanothione synthetase (TcTS). Therefore, the results encourage the use of these drugs to develop new anti-T. cruzi agents.

Blast disease is a severe fungal infection that primarily impacts cereal crops caused by pathogenic fungus. e.g., Magnaporthe oryzae (M. oryzae) that uses autophagy for its pathogenecity. Experimental studies show the importance of autophagy for programmed cell death of filamentous fungi and the inhibition process of Atg4-mediated Atg8 cleaving by obtaining potential drug molecules to inhibit autophagy process in blast causing fungus. However, the molecular level details of the drug-Atg4 complex remains elusive. In this study, we use virtual screening and Molecular Dynamics (MD) simulations to repurpose drugs which are able to kill the fungus by targeting the Atg4-mediated cleaving of Atg8 in M. oryzae. Of the 3800 FDA-approved drugs, we identify six promising candidates- 31 h-Phthalocyanine, rebastinib, flumatinib, zafirlukast, beigene-283, and radotinib. A total of 18 µs all-atom MD simulations is conducted to further identify the stability of these molecules and their complexes with Atg4 with the help of RMSD, RMSF, Rg, and binding energy. The compound stability is further validated with the number of hydrogen bonds formed between Atg4 and drug molecules during the complete trajectory. Together with binding energy, the stability results indicate distinct ligand binding to Atg4. Our work provides pharmacokinetic properties of the screened compounds, the molecular level details of the key residues involved in Atg4-drug interactions, and dynamic stability of Atg4::drug complexes, establishing a basis for drug repurposing for the inhibition of the Atg4 mediated Atg8 cleavage in M. oryzae.

TMEM175 is an AKT-activated lysosomal potassium- and proton-permeable channel that functions to dissipate voltage and pH gradients generated by the V-type H+-ATPase. Loss-of-function variants in TMEM175 have been identified as genetic risk factors for Parkinson's disease, highlighting the potential of small-molecule activators as a novel therapeutic strategy for this disease. We developed a high-throughput screening (HTS) assay using HEK-293 cells stably overexpressing TMEM175 at the cell surface and screened 960 Food and Drug Administration (FDA)-approved drugs for TMEM175 potentiators. The screen identified 71 activators, including the cysteinyl leukotriene 1 receptor (CysLT1R) antagonists, pranlukast and montelukast. Because HEK-293 cells lack CysLT1R expression, we suspected these drugs may be direct channel activators. Fluorescence and automated patch-clamp assays were used to evaluate the dose dependency of pranlukast, montelukast, zafirlukast, and the known TMEM175 activator, (2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB). These experiments revealed rank-order potencies and efficacies of DCPIB ∼ zafirlukast > montelukast ≫ pranlukast. DCPIB, zafirlukast, and pranlukast activated TMEM175 independently of AKT activation, whereas the AKT inhibitor MK2206 partially inhibited montelukast-dependent TMEM175 activation. Computer modeling revealed a conformation-dependent solvent-accessible cavity near T119 and H449 that could participate in drug-induced activation, prompting us to examine these sites with mutagenesis. Not only did T119A and H449A mutations decrease apparent potencies of DCPIB, zafirlukast, and montelukast, but the T119A mutation produced a constitutively open channel phenotype. This study adds zafirlukast to the short list of moderately potent TMEM175 activators and identifies a region of the channel that contributes to activation gating.NEW & NOTEWORTHY TMEM175 regulates lysosomal voltage and pH and is genetically linked to Parkinson's disease. Our study identifies zafirlukast and related drugs as activators, defines their AKT dependency, and maps structural determinants of gating. These results open new avenues for targeting TMEM175 in neurodegenerative diseases.

Severe type-2-high asthma is driven by upstream epithelial alarmins, notably interleukin-33 signaling through the ST2 receptor, and many patients remain inadequately controlled despite optimized inhaled therapy. This study employed a comprehensive in silico discovery workflow to explore small-molecule binding at the IL-33 interaction surface of human ST2. The experimentally resolved ST2-IL-33 complex structure (PDB ID: 4KC3) was curated, and IL-33 was removed to define the receptor interaction interface, followed by binding-site identification and network-level functional analysis. A focused set of clinically relevant small molecules, together with a reference ST2-directed comparator, was retrieved from PubChem and screened using molecular docking, followed by drug-likeness, ADMET, and toxicity profiling. Zafirlukast emerged as the top-ranked scaffold with a docking score of - 7.3 kcal/mol. A structurally optimized Zafirlukast analogue exhibited a comparable docking score of approximately - 7.0 kcal/mol and was further examined using molecular dynamics simulations to characterize dynamic interaction behavior at the ST2 interface. MM-GBSA analysis provided qualitative insight into interaction energetics across representative simulation frames. Collectively, these results prioritize a non-biologic, ST2-directed small-molecule scaffold for further investigation and provide a computational framework to support subsequent experimental validation, including biochemical and cellular studies, in the context of severe type-2-high asthma. As this is a purely in silico study, the findings are hypothesis-generating and do not demonstrate functional inhibition of the IL-33/ST2 interaction; experimental validation is required.