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.

Endothelial dysfunction plays a critical role in the initiation and progression of vascular remodeling and pulmonary arterial hypertension (PAH). Sine oculis homeobox 1 (SIX1) is a developmentally restricted transcription factor, and its expression ceases upon the completion of embryonic development. Deletion of Six1 impairs the differentiation of pulmonary vascular endothelial cells, resulting in pulmonary vascular defects and postnatal death. In this study, we found that SIX1 was increased in the endothelia of pulmonary arteries from patients with PAH and from patients with idiopathic pulmonary fibrosis-associated pulmonary hypertension (PH). Silencing SIX1 using siRNA inhibited hypoxia-induced endothelial dysfunction in vitro, including proliferation, endothelial-to-mesenchymal transition, and endothelin-1 release. Endothelial-specific Six1 knockout improved pulmonary hemodynamics, endothelial dysfunction, pulmonary artery remodeling, and right ventricular function in SU5416/hypoxia (SuHx)-induced PH mice. Moreover, endothelial-specific Six1 overexpression mediated by adeno-associated virus induced a spontaneous PH in wild-type C57BL/6 mice. Structure-based virtual screening and surface plasmon resonance analysis demonstrated that zafirlukast was an inhibitor of SIX1 transcriptional activity. Zafirlukast suppressed hypoxia-induced endothelial dysfunction in vitro and reversed SuHx-induced PH in Sprague-Dawley rats. Mechanistically, multiomics profiling of hypoxia-induced endothelial dysfunction in vitro revealed that microtubule-associated serine/threonine kinase family member 4 (MAST4) was a target of SIX1. MAST4 phosphorylated the Thr[185/202] sites of mitogen-activated protein kinases 1 and 3 (MAPK1/3), resulting in endothelial dysfunction. Overall, we found that SIX1 was a driver for endothelial dysfunction and PH through regulating MAST4 transcription and subsequently MAPK1/3 activation. Targeting SIX1 may be a promising strategy for PAH treatment and drug development.

Cancer-associated thrombosis (CAT), encompassing both venous thromboembolism and arterial thrombosis, contributes to up to 14% of cancer-related mortality and remains difficult to treat due to the bleeding risks of conventional anticoagulants. Protein disulfide isomerase (PDI) and its family member ERp57 (PDIA3) are thiol isomerases that regulate both arterial and venous thrombosis and are also upregulated in tumors, where they promote growth, metastasis, and immune evasion. Here, we evaluated the therapeutic potential of two thiol isomerase inhibitors-isoquercetin (ISOQ), a selective PDI inhibitor, and zafirlukast (ZAF), a broad-spectrum inhibitor of thiol isomerases such as PDI and ERp57-individually and in combination, in a xenograft model of ovarian cancer. ISOQ inhibited both platelet aggregation and Factor Xa generation induced by tumor cells and significantly suppressed tumor growth, thromboinflammatory markers, and expression of tissue factor, VEGF, TMEM176B, and PD-L1. ISOQ also potentiated standard cisplatin/gemcitabine chemotherapy. Notably, the combination of low-dose ISOQ plus ZAF achieved ≥ 80% inhibition of key tumor-associated markers at one-third the monotherapy dose and outperformed either agent alone. These findings support ISOQ and ZAF as promising agents for the treatment of cancer and CAT and establish thiol isomerase inhibition as a strategy to simultaneously target thrombosis, tumor progression, and immune escape.

The main goal of this study is the identification of existing drugs that could be repurposed as antagonists of the V2R, a GPCR controlling renal water balance and involved in abnormal cell proliferation, cancer, and renal cyst enlargement. Given its clinical importance, we carried out the reverse screening of a collection of 1882 existing drugs to repurpose them towards V2R by employing PLATO, a home-built target fishing AI-based platform. Five drugs were shortlisted as promising candidates for V2R: cabergoline, clopidogrel, cloxacillin, perphenazine, and zafirlukast. Renal collecting duct MCD4 cells, stably expressing human V2R and AQP2, were used for experimentally testing the effects of the prioritized drugs on V2R responses. FRET studies were conducted to assess whether these drugs affect the DDAVP-induced cAMP responses. Interestingly, zafirlukast, at single-digit nanomolar concentration significantly reduced the DDAVP-dependent cAMP production and water reabsorption, with effects comparable to tolvaptan, a well-known selective V2R antagonist. The molecular rationale behind the observed binding was explained by mapping on the V2R a molecular cleft superimposable to CysLTR1 binding site of zafirlukast. Induced-fit docking simulations demonstrated that zafirlukast engages V2R by adopting a binding conformation closely resembling that of X-ray solved vasopressin. Taken together, our results support the repurposing of zafirlukast as a promising V2R antagonist candidate.

Prostate cancer (PCa) is a major malignancy affecting men and is a significant contributor to global male mortality. Over the past decade, several new treatments for advanced PCa have been approved; however, opportunities remain for the development of novel therapeutic strategies. Therefore, in this study, we developed an integrated bioinformatics pipeline to identify potential therapeutic targets and repurposed drugs using RNA-seq datasets, aiming to advance treatment options for PCa. Using the LIMMA approach, 458 common differentially expressed genes (cDEGs) were analyzed from three publicly available microarray datasets, leading to the identification of 15 hub genes (HubGs) through a protein-protein interaction (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed their critical roles in PCa, and lower expressions of five HubGs (BIRC5, CDCA5, CENPF, NUSAP1, and TK1) correlated with better survival. All of these genes could potentially serve as biomarkers for the detection and therapy of PCa. Following that, we considered these possible genes as targets for drugs, performed docking analysis with 255 meta-drug agents, and identified the top 10 candidate drugs (adapalene, ergotamine, imatinib, dutasteride, vistusertib, risperidone, zafirlukast, irinotecan hydrochloride, drospirenone, and telmisartan). Finally, we evaluated the binding stability of the top-ranked three complexes-BIRC5-adapalene, BIRC5-imatinib, and TK1-ergotamine-through a 100 nanoseconds (ns) molecular dynamics (MD) simulation conducted using NAMD. The analysis revealed consistent stability across all complexes. This study uniquely combines multidataset transcriptomic integration, HubG prioritization, and MD validation to propose novel biomarker-drug pairings for PCa. The findings offer promising leads for future experimental and clinical validation.

The inhibition of a specific protein-protein interaction is often difficult to achieve in targeted drug design. We report the development and optimization of a general-purpose, readily implemented enzyme-linked immunosorbent assay (ELISA) for high-throughput screening to identify small-molecule inhibitors of protein interactions. This ELISA does not involve the use of any capture antibodies, probes, or compounds coated on the plate and represents a general strategy to identify inhibitors of a given protein-protein interaction. We demonstrate its utility in blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook by targeting the native form of the FlgE protein, which differs from the strategies used in previous assays. The flagellar hook protein FlgE self-catalyzes the formation of a lysinoalanine (Lal) inter-subunit crosslink that is essential for the motility, and thus, infectivity of spirochetes. Prevention of Lal crosslinking through inhibition with small molecules thus represents an avenue for therapeutic development against spirochete-related diseases, such as Lyme and syphilis. Screening a library of ∼700 compounds with the ELISA confirmed that hexachlorophene, currently the only known inhibitor of Lal crosslinking in FlgE, effectively inhibits the crosslinking reaction. In addition, the assay identified two new potential inhibitors, honokiol and zafirlukast, and several activators which belong to well-known classes of antibiotics.

The inhibition of a specific protein-protein interaction is often difficult to achieve in targeted drug design. We report the development and optimization of a general-purpose, readily implemented enzyme-linked immunosorbent assay (ELISA) for high-throughput screening to identify small- molecule inhibitors of protein interactions. This ELISA does not involve the use of any capture antibodies, probes, or compounds coated on the plate and represents a general strategy to identify inhibitors of a given protein-protein interaction. We demonstrate its utility in blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook by targeting the native form of the FlgE protein, which differs from the strategies used in previous assays. The flagellar hook protein FlgE self-catalyzes the formation of a lysinoalanine (Lal) inter-subunit crosslink that is essential for the motility, and thus, infectivity of spirochetes. Prevention of Lal crosslinking through inhibition with small molecules thus represents an avenue for therapeutic development against spirochete-related diseases, such as Lyme and syphilis. Screening a library of ∼700 compounds with the ELISA confirmed that hexachlorophene, currently the only known inhibitor of Lal crosslinking in FlgE, effectively inhibits the crosslinking reaction. In addition, the assay identified two new potential inhibitors, honokiol and zafirlukast, and several activators which belong to well-known classes of antibiotics.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide. Current treatments are often limited by multidrug resistance and significant side effects, underscoring the urgent need for novel therapeutic strategies. Leukotriene receptor antagonists (LTRAs), including montelukast, zafirlukast, and pranlukast, have shown promising anticancer potential; however, their underlying molecular mechanisms remain insufficiently understood. In this study, the antiproliferative effects of montelukast, zafirlukast, and pranlukast on CRC cell lines (HCT-116 and Caco-2) were evaluated using cell viability and proliferation assays. Montelukast and zafirlukast were further examined using a liquid chromatography-mass spectrometry (LC-MS)-based metabolomics to assess any possible metabolic alterations in HCT-116 cells, along with flow cytometry to determine their effects on cell death. Montelukast and zafirlukast were found to significantly inhibit the cell proliferation of HCT-116 in a dose-dependent manner, with IC50 values of 57.4 μM and 73.28 μM, respectively. Flow cytometry demonstrated that apoptosis was the predominant mode of cell death with both drugs. Neither montelukast nor zafirlukast showed any significant in vitro antioxidant effects. LC-MS Metabolomics revealed that montelukast and zafirlukast induced distinct metabolic changes with 47 and 34 significantly altered metabolites, respectively. Montelukast notably affected mannosamine, 5-oxo-L-proline, acetyl-phenylalanine, acetoin, and taurine, implicating pathways related to amino acid metabolism, redox homeostasis, and mitochondrial function. In contrast, zafirlukast impacted nucleotide-related metabolites, including inosine, adenine, cytidine, deoxyguanosine, and itaconate, highlighting nucleotide biosynthesis and immunometabolism disruptions. These findings demonstrate the antiproliferative potential of montelukast and zafirlukast in CRC and provide new insights into their distinct molecular mechanisms of action. This supports their potential repurposing as adjunctive therapies in CRC treatment.