The endosomal-lysosomal system is central for cell homeostasis and comprises the functions and dynamics of particular organelles including endosomes, lysosomes and autophagosomes. In previous studies, we found that the cysteinyl leukotriene receptor 1 (CysLTR1) regulates autophagy in the retinal pigment epithelial cell line ARPE-19 under basal cellular conditions. However, the underlying mechanism by which CysLTR1 regulates autophagy is unknown. Thus, in the present study, the effects of CysLTR1 inhibition on the endosomal-lysosomal system are analyzed in detail to identify the role of CysLTR1 in cell homeostasis and autophagy regulation. CysLTR1 inhibition in ARPE-19 cells by Zafirlukast, a CysLTR1 antagonist, depleted the lysosomal pool. Furthermore, CysLTR1 antagonization reduced endocytic capacity and internalization of epidermal growth factor and decreased levels of the transferrin receptor, CD71. Serum starvation abolished the effect of Zafirlukast on the autophagic flux, which identifies the endocytic regulation of serum components by CysLTR1 as an important autophagy-modulating mechanism. The role of CysLTR1 in inflammation and cell stress has been exceedingly studied, but its involvement in the endosomal-lysosomal pathway is largely unknown. This current study provides new insights into basal activity of CysLTR1 on cellular endocytosis and the subsequent impact on downstream processes like autophagy.

Chromosomal region maintenance protein 1 (CRM1) is a validated anticancer drug target, and its covalent inhibitor KPT-330 has been approved for marketing. However, the development of CRM1 inhibitors, especially the noncovalent ones, is still very limited. Drug repurposing is an effective strategy to develop drug leads for new targets. In this work, we virtually screened a library of marketed drugs and identified zafirlukast as a new CRM1 inhibitor. Biochemical and structural analysis revealed that zafirlukast was a noncovalent CRM1 inhibitor that bound to four subpockets in the nuclear-export-signal (NES) groove. Methylation of the sulfonamide group rendered zafirlukast completely inactive against CRM1. Zafirlukast inhibited the growth of a variety of cancer cells and worked synergistically with the drug doxorubicin. Taken together, these works laid a solid foundation for reshaping zafirlukast as a valuable lead compound for further design of noncovalent, specific, and potent CRM1 inhibitors toward the treatment of various cancers.

Montelukast and zafirlukast are cysteinyl leukotriene receptor antagonists indicated for the prevention and treatment of chronic asthma. This activity will highlight the mechanism of action, adverse event profile, and monitoring pertinent for members of the interprofessional team in the management of patients with asthma and related conditions with leukotriene receptor antagonists.

(1) Background: Cysteinyl leukotriene receptor antagonists (LTRAs), including montelukast and zafirlukast, are FDA-approved for treating pediatric asthma and allergic diseases. Tourette syndrome (TS), a common neuropsychiatric disorder in children, is associated with allergic diseases and asthma. In this study, we investigated the risk of TS following an LTRA prescription for pediatric allergic diseases. (2) Methods: Children younger than 18 years of age who were newly diagnosed with asthma, allergic rhinitis, or atopic dermatitis between 1 January 2005 and 31 December 2018 and who were registered in the Taiwan National Health Insurance Research Database, which comprises the medical records of nearly 23 million Taiwanese population, were enrolled. LTRA users were matched with randomly selected LTRA non-users by sex, age, asthma-diagnosis year, and urbanization level. In total, 26,984 participants with allergic disease and TS were enrolled and included in the Cox proportional hazards model analysis. (3) Results: Children with allergic disease and asthma treated with LTRAs had a higher risk for TS than LTRA non-users (adjusted hazard ratio 1.376 [95% CI: 1.232-1.536], p < 0.001). LTRA users had a significantly higher risk for TS than LTRA non-users with allergic disease. The cumulative incidence of TS was significantly higher in LTRA users than in non-users with allergic diseases and asthma (log-rank test, p < 0.0001). (4) Conclusion: A prescription of LTRAs, mainly montelukast, increased the risk of TS among children with asthma, allergic rhinitis, or atopic dermatitis. The mechanism underlying the neuropsychiatric effect of LTRAs needs further investigation.

The spike protein (SP) of SARS-CoV-2 (SC-2) is susceptible to high mutation and has contributed to the multiple waves of COVID-19 being experienced. Hence, targeting the SP remains a logical approach in the development of potent therapeutics against SARS-CoV-2. Here, a computational technique was adopted to identify broad-spectrum plant secondary metabolites with indigenous relevance in the management of respiratory infections against the SPs of the SC-2 wild- type (SC-2WT) and omicron variants. Following 100 ns molecular dynamic (MD) simulation and binding free energy calculation of the top five compounds identified through molecular docking, maysin (SC-2WT (-34.85 kcal/mol), omicron (-38.88 kcal/mol)) and geraniin (SC-2WT (-36.90 kcal/mol) omicron (-31.28 kcal/mol)) had better broad-spectrum activities for the investigated SPs than zafirlukast (SC-2WT (-33.73 kcal/mol) omicron (-22.38 kcal/mol)). Furthermore, 6-hydroxycyanidin-3-rutinoside (-42.97 kcal/mol) and kaempferol-7-glucoside (-37.11 kcal/mol) had the best affinity for the SPs of omicron and SC-2WT, respectively. Interestingly, except for Kaempferol-7-glucoside against omicron SP, all the top-ranked compounds were thermodynamically stable with the SP of both variants, and this observation was linked to the number, nature, and bond length in the resulting complexes in each case. Also, except for geraniin, all the top-ranked compounds had lower toxicity profiles compared to zafirlukast and this could be attributed to their phenolic moieties. Nevertheless, the in vitro and in vivo confirmation of the activities observed in this study is recommended, especially for maysin and geraniin with the best broad-spectrum activity, towards development of COVID-19 drug candidates.

Metastases are the main cause of death in cancer patients, and platelets are largely known for their contribution in cancer progression. However, targeting platelets is highly challenging given their paramount function in hemostasis. Using a high-throughput screening and platelet-induced breast tumor cell survival (PITCS) assay as endpoint, we identified the widely used anti-asthmatic drugs and cysteinyl leukotriene receptor 1 (CysLT1R) antagonists, zafirlukast and montelukast, as new specific blockers of platelet protumoral action. Here, we show that human MDA-B02 breast cancer cells produce CysLT through mechanisms involving microsomal glutathione-S-transferase 1/2/3 (MGST1/2/3) and that can modulate cancer cell-platelet interactions via platelet-CysLT1R. CysLT1R blockade with zafirlukast decreased platelet aggregation and adhesion on cancer cells and inhibited PITCS, migration, and invasion in vitro. Zafirlukast significantly reduced, by 90%, MDA-B02 cell dissemination to bone in nude mice and reduced by 88% 4T1 spontaneous lung metastasis formation without affecting primary tumor growth. Combined treatment of zafirlukast plus paclitaxel totally inhibited metastasis of 4T1 cells to the lungs. Altogether, our results reveal a novel pathway mediating the crosstalk between cancer cells and platelets and indicate that platelet CysLT1R represents a novel therapeutic target to prevent metastasis without affecting hemostasis.

Periodontal diseases are inflammatory diseases triggered by pathogenic oral bacterial species, such as Porphyromonas gingivalis. Zafirlukast (ZAF) has displayed antibacterial activity against P. gingivalis. Herein, we report the design, synthesis, and selective antibacterial activity of 14 novel third-generation ZAF derivatives. Two second-generation ZAF derivatives were tested as they were not previously tested against P. gingivalis ATCC 33277. Most third-generation derivatives displayed superior/selective antibacterial activity against P. gingivalis compared to ZAF and its second-generation derivatives. The compounds displayed bactericidal activity against P. gingivalis, inhibited biofilm growth, displayed no hemolytic activity, and displayed less cytotoxicity against mammalian cells than ZAF. The superior/selective antibacterial activity of ZAF derivatives against P. gingivalis, increased safety profile, and inhibition of biofilm growth compared to ZAF indicate that the compounds, especially 21a, 21b, and 24g, show promise as antibacterial agents for future studies to test their potential for preventing and treating P. gingivalis-induced periodontal diseases.

Torasemide is a loop diuretic with a molecule that is chemically similar to the sulphonamides described as eosinophilic granulomatosis with polyangiitis (EGPA) triggering drugs. The presented case is probably the first description of torasemide-induced vascular purpura in the course of EGPA. Any diagnosis of vasculitis should be followed by an identification of drugs that may aggravate the disease. A 74-year-old patient was admitted to the Department of Dermatology with purpura-like skin lesions on the upper, and lower extremities, including the buttocks. The lesions had appeared around the ankles 7 days before admission to the hospital and then started to progress upwards. The patient complained on lower limb paresthesia and pain. Other comorbidities included bronchial asthma, chronic sinusitis, ischemic heart disease, mild aortic stenosis, arterial hypertension, and degenerative thoracic spine disease. The woman had previously undergone nasal polypectomy twice. She was on a constant regimen of oral rosuvastatin 5 mg per day, spironolactone 50 mg per day, metoprolol 150 mg per day, inhaled formoterol 12 μg per day, and ipratropium bromide 20 μg per day. Ten days prior to admission, she was commenced on torasemide at a dose of 50 mg per day prescribed by a general practitioner due to high blood pressure. Doppler ultrasound upon admission to the hospital excluded deep venal thrombosis. The laboratory tests revealed leukocytosis (17.1 thousand per mm3) with eosinophilia (38.6%), elevated plasma level of C-reactive protein (119 mg per L) and D-dimers (2657 ng per mm3). Indirect immunofluorescent test identified a low titer (1:80) of antinuclear antibodies, but elevated (1:160) antineutrophil cytoplasmic antibodies (ANCA) in the patient's serum. Immunoblot found them to be aimed against myeloperoxidase (pANCA). A chest X-ray showed increased vascular lung markings, while high-resolution computed tomography revealed peribronchial glass-ground opacities. Microscopic evaluation of skin biopsy taken from the lower limbs showed perivascular infiltrates consisting of eosinophils and neutrophils, fragments of neutrophil nuclei, and fibrinous necrosis of small vessels. Electromyography performed in the lower limbs because of their weakness highlighted a loss of response from both sural nerves, as well as slowed conduction velocity of the right tibial nerve and in both common peroneal nerves. Both clinical characteristics of skin lesions and histopathology suggested a diagnosis of EGPA, which was later confirmed by a consultant in rheumatology. The patient was commenced on prednisone at a dose of 0.5 mg per kg of body weight daily and mycophenolate mofetil at a daily dose of 2 g. The antihypertensive therapy was modified, and torasemide was replaced by spironolactone 25 mg per day. The treatment resulted in a gradual regression of skin lesions within a few weeks. The first report of EGPA dates back to 1951. Its authors were Jacob Churg and Lotte Strauss. They described a case series of 13 patients who had severe asthma, fever, peripheral blood eosinophilia, and granulomatous vasculitis in microscopic evaluation of the skin. Three histopathological criteria were then proposed, and Churg-Strauss syndrome was recognized when eosinophilic infiltrates in the tissues, necrotizing inflammation of small and medium vessels, and the presence of extravascular granulomas were observed together in a patient (1). Only 17.4% of patients met all three histopathological criteria, and the diagnosis of the disease was frequently delayed despite of its overt clinical picture (2). In 1984, Lanham et al. proposed new diagnostic criteria which included the presence of bronchial asthma, eosinophilia in a peripheral blood smear >1.5 thousand per mm3, and signs of vasculitis involving at least two organs other than the lungs (3). Lanham's criteria could also delay the recognition of the syndrome before involvement of internal organs, and the American College of Rheumatology therefore established classification criteria in 1990. These included the presence of bronchial asthma, migratory infiltrates in the lungs as assessed by radiographs, the presence of abnormalities in the paranasal sinuses (polyps, allergic rhinitis, chronic inflammation), mono- or polyneuropathy, peripheral blood eosinophilia (>10% of leukocytes must be eosinophils), and extravascular eosinophilic infiltrates in a histopathological examination. Patients who met 4 out of 6 criteria were classified as having Churg-Strauss syndrome (4). The term EGPA was recommended to define patients with Churg-Strauss syndrome in 2012 (5). EGPA is a condition with low incidence (0.11-2.66 cases per million) and morbidity. It usually occurs in the fifth decade of life (6,7), although 65 cases reports of EGPA in people under 18 years of age could be found in the PubMed and Ovid Medline Database at the end of 2020 (8). The etiopathogenesis of the disease has not been fully explained so far. Approximately 40-60% of patients are positive to pANCA (9), but the role of these antibodies in the pathogenesis of EGPA remains unclear. They are suspected to mediate binding of the Fc receptor to MPO exposed on the surface of neutrophils. Subsequently, this may active neutrophils and contribute to a damage of the vascular endothelium (9,10). Glomerulonephritis, neuropathy, and vasculitis are more common in patients with EGPA who have detectable pANCA when compared with seronegative patients. There are at least several drugs which potentially may EGPA. The strongest association with the occurrence of EGPA was found with the use of leukotriene receptor antagonists (montelukast, zafirlukast, pranlukast), although they are commonly used in the treatment of asthma, which is paradoxically one of the complications of the syndrome (13). Although no relationship has been demonstrated so far between the occurrence of EGPA and the intake of drugs from the groups used by the presented patient, a clear time relationship can be observed between the commencement of torasemide and the onset of symptoms in our patient. To date, only three cases of leukocytoclastic vasculitis have been reported after the administration of torasemide. Both of them developed cutaneous symptoms of the disease within 24 hours of the administration of torasemide in patients with no previous history of drug hypersensitivity, but they disappeared quickly within 8-15 days after drug discontinuation (14,15). The chemical structure of torasemide is similar to the molecule of sulfonamides which were previously found to be a triggering factors for EGPA (12). This drug belongs to the group of loop diuretics classified as sulfonamide derivatives. A comparison of the chemical structure of torasemide and sulphanilamide molecules is presented in Figure 1. The clear time relationship between starting the administration of torasemide and the occurrence of purpura-like lesions suggests that it was an aggravating factor for EGPA in our patient. A coexistence of several disorders (asthma, nasal polyps, symptoms of peripheral neuropathy) in our patient suggest EGPA could have developed in her years before oral intake of torasemide. The sudden onset of skin symptoms shows torasemide to be possible inducing factor for the development of vascular purpura in patients suffering from EGPA but without previous cutaneous involvement.

To identify activation pathways and effector mechanisms of innate immunity in fish has become relevant for the sanitary management of intensive fish farming. However, little is known about the blocking of cysteinyl leukotrienes receptors (CysLTRs) and their effects in teleost fish. Our study evaluated the anti-inflammatory effect of 250 and 500 μg zafirlukast (antagonist of CysLTRs)/kg b.w., administered orally in the diet, during acute inflammatory reaction induced by Aeromonas hydrophila bacterins in Oreochromis niloticus. 80 tilapia were distributed in 10 aquariums (100L of water each, n = 8) to constitute three treatments: Control (inoculated with A. hydrophila bacterin and untreated); Treated with 250 μg or 500 μg of zafirlukast/kg b.w. and inoculated. To be evaluated in three periods: 6, 24 and 48 h post-inoculation (HPI), totaling nine aquariums. A tenth group was sampled without any stimulus to constitute reference values (Physiological standards). Tilapia treated with zafirlukast demonstrated dose-response effect in the decrease of accumulated inflammatory cells, strongly influenced by granulocytes and macrophages. Zafirlukast treated-tilapia showed decrease in blood leukocyte counts (mainly neutrophils, and monocytes) and reactive oxygen species production. Treatment with zafirlukast resulted in down-regulation of ceruloplasmin, complement 3, alpha2-macroglobulin, transferrin and apolipoprotein A1, as well as up-regulation of haptoglobin. Our study provided convincing results in the pathophysiology of tilapia inflammatory reaction, considering that treatment with zafirlukast, antagonist of cysteinyl leukotriene receptors, resulted in a dose-response effect by suppressing the dynamics between leukocytes in the bloodstream and cell accumulation in the inflamed focus, as well as modulated the leukocyte oxidative burst and the acute phase protein response.

Opposing findings have been published on the regulation of the sperm-specific Ca 2+ channel CatSper (cation channel of sperm) in human sperm cells by the plant triterpenoids lupeol and pristimerin. While the original study on this topic found these triterpenoids to act as potent inhibitors of human CatSper, subsequent studies have failed to replicate such an inhibitory effect. It has been suggested that these issues could in part be due to purity issues and/or batch variation between the plant-derived extracts of lupeol and pristimerin obtained for the studies. The aim of this study was to elucidate this controversy by investigating the batches of lupeol and pristimerin used in our previous study with state-of-the-art 1H-, 13C- and 2D-nuclear magnetic resonance (NMR) methods to reveal potential purity and/or batch variation issues. When comparing the NMR-spectra obtained from 1H-NMR and 13C-NMR with previously published NMR-spectra for lupeol and pristimerin, we could confirm that both the lupeol and pristimerin batch were ≥95 % pure. These results confirm the validity of the findings in our previous study for lupeol and pristimerin, showing that lupeol and pristimerin do not inhibit activation of CatSper in human sperm. In conclusion, using 1H-, 13C- and 2D-NMR methods, we confirm that the lupeol and pristimerin batches used in our previous study were ≥95 % pure and thereby fail to identify any purity issues and/or batch variation that could explain the observed inability of lupeol and pristimerin to inhibit activation of CatSper in human sperm.

Worldwide, obesity rates have doubled since the 1980s and in the USA alone, almost 40% of adults are obese, which is closely associated with a myriad of metabolic diseases such as type 2 diabetes and arteriosclerosis. Obesity is derived from an imbalance between energy intake and consumption, therefore balancing energy homeostasis is an attractive target for metabolic diseases. One therapeutic approach consists of increasing the number of brown-like adipocytes in the white adipose tissue (WAT). Whereas WAT stores excess energy, brown adipose tissue (BAT) can dissipate this energy overload in the form of heat, increasing energy expenditure and thus inhibiting metabolic diseases. To facilitate BAT production a high-throughput screening approach was developed on previously known drugs using human Simpson-Golabi-Behmel Syndrome (SGBS) preadipocytes. The screening allowed us to discover that zafirlukast, an FDA-approved small molecule drug commonly used to treat asthma, was able to differentiate adipocyte precursors and white-biased adipocytes into functional brown adipocytes. However, zafirlukast is toxic to human cells at higher dosages. Drug-Initiated Activity Metabolomics (DIAM) was used to investigate zafirlukast as a BAT inducer, and the endogenous metabolite myristoylglycine was then discovered to mimic the browning properties of zafirlukast without impacting cell viability. Myristoylglycine was found to be bio-synthesized upon zafirlukast treatment and was unique in inducing brown adipocyte differentiation, raising the possibility of using endogenous metabolites and bypassing the exogenous drugs to potentially alleviate disease, in this case, obesity and other related metabolic diseases.

Intestinal drug solubility is a key parameter controlling oral absorption but varies both intra and inter individuals and between the fasted and fed states, with food intake known to alter the bioavailability of many compounds. Intestinal solubility can be measured in vitro either using sampled fed human intestinal fluid (FeHIF) or simulated fed intestinal fluid (SIF) but neither approach is optimal. FeHIF is difficult to obtain and variable, whilst for fed SIF multiple recipes are available with no consensus on the ideal version. A recent study characterised FeHIF aspirates using a multidimensional approach and calculated nine simulated media recipes that covered over ninety percent of FeHIF compositional variability. In this study the equilibrium solubility of thirteen drugs have been measured using the nine simulated media recipes and compared to multiple previous design of experiment (DoE) studies, which have examined the impact of fed SIF media components on solubility. The measured nine media solubility data set is only statistically equivalent to the large scale 92 media DoE in 4 out of 13 drug comparisons, but has improved equivalence against small scale DoEs (9 or 10 media) with 6 out of 9 or 10 out of 12 (9 and 10 media respectively) equivalent. Selective removal of non-biorelevant compositions from the 92 media DoE improves statistical equivalence to 9 out of 13 comparisons. The results indicate that solubility equivalence is linked to media component concentrations and compositions, the nine media system is measuring a similar solubility space to previous systems, with a narrower solubility range than the 92 point DoE but equivalent to smaller DoE systems. Phenytoin and tadalafil display a narrow solubility range, a behaviour consistent with previous studies in fed and fasted states and only revealed through the multiple media approach. Custom DoE analysis of the nine media results to determine the most statistically significant component influencing solubility does not detect significant components. Indicating that the approach has a low statistical resolution and is not appropriate if determination of media component significance is required. This study demonstrates that it is possible to assess the fed intestinal equilibrium solubility envelope using the nine media recipes obtained from a multi-dimensional analysis of fed HIF. The derivation of the nine media compositions coupled with the results in this study indicate that the solubility results are more likely to reflect the fed intestinal solubility envelope than previous DoE studies and highlight that the system is worthy of further investigation.

Zafirlukast is a medication used in the management and treatment of chronic asthma. It is in the leukotriene receptor antagonist class. This activity reviews the indications, action, and contraindications for zafirlukast as an agent in the management and prophylaxis of chronic asthma. This activity will highlight the mechanism of action, adverse event profile, and other vital factors pertinent to the care of patients with chronic asthma.

In an earlier investigation, low-frequency Raman (LFR) spectroscopy was shown to detect the transition temperature of the β-relaxation (Tβ) in both amorphous celecoxib and various celecoxib amorphous solid dispersions [Be̅rziņš, K. Mol. Pharmaceutics 2021, 18(10), 3882-3893]. In this study, we further investigated the application of this technique to determine Tβ, an important parameter for estimating crystallization potency of amorphous drugs. Alongside commercially available amorphous drugs (zafirlukast and valsartan disodium salt), differently melt-quenched samples of cimetidine were also analyzed. Overall, the variable-temperature LFR measurements allowed for an easy access to the desired information, including the even lesser transition of the tertiary relaxation motions (Tγ). Thus, the obtained results not only highlighted the sensitivity, but also the practical usefulness of this technique to elucidate (subtle) changes in molecular dynamics within amorphous pharmaceutical systems.

Drug solubility in intestinal fluid is a key parameter controlling absorption after the administration of a solid oral dosage form. To measure solubility in vitro simulated intestinal fluids have been developed, but there are multiple recipes and the optimum is unknown. This situation creates difficulties during drug discovery and development research. A recent study characterised sampled fasted intestinal fluids using a multidimensional approach to derive nine bioequivalent fasted intestinal media that covered over 90% of the compositional variability. These media have been applied in this study to examine the equilibrium solubility of twenty one exemplar drugs (naproxen, indomethacin, phenytoin, zafirlukast, piroxicam, ibuprofen, mefenamic acid, furosemide, aprepitant, carvedilol, tadalafil, dipyridamole, posaconazole, atazanavir, fenofibrate, felodipine, griseofulvin, probucol, paracetamol, acyclovir and carbamazepine) to determine if consistent solubility behaviour was present. The bioequivalent media provide in the majority of cases structured solubility behaviour that is consistent with physicochemical properties and previous solubility studies. For the acidic drugs (pKa < 6.3) solubility is controlled by media pH, the profile is identical and consistent and the lowest and highest pH media identify the lowest and highest solubility in over 70% of cases. For weakly acidic (pKa > 8), basic and neutral drugs solubility is controlled by a combination of media pH and total amphiphile concentration (TAC), a consistent solubility behaviour is evident but with variation related to individual drug interactions within the media. The lowest and highest pH × TAC media identify the lowest and highest solubility in over 78% of cases. A subset of the latter category consisting of neutral and drugs non-ionised in the media pH range have been identified with a very narrow solubility range, indicating that the impact of the simulated intestinal media on their solubility is minimal. Two drugs probucol and atazanavir exhibit unusual behaviour. The study indicates that the use of two appropriate bioequivalent fasted intestinal media from the nine will identify in vitro the maximum and minimum solubility boundaries for drugs and due to the media derivation this is probably applicable in vivo. These media could be applied during discovery and development activities to provide a solubility range, which would assist placement of the drug within the BCS/DCS and rationalise drug and formulation decisions.

This study investigated the application of bio-ionic liquids (ILs) prepared from choline as cation and amino acid as anion for solubility enhancement of poorly water-soluble drug, Zafirlukast (ZFL). Herein, the solubility of ZFL in water and mixtures of water and ILs was assessed using UV spectroscopy at two temperature points 25°C and 37°C with increasing concentrations of IL. ZFL solubility was found to improve linearly with increasing concentration of [Ch][Pro] in water, representing 35- to 37-fold improvement in ZFL solubility at maximum concentration of [Ch][Pro] (1% w/v) compared to when only pure water was present. Also, the effect of IL on ZFL solubility was analyzed using NMR, DSC, and TGA. These results clearly suggest that ZFL solubility was increased by forming hydrogen bonds with selected [Ch][Pro] IL. Toxicity study of ILs was tested against gram-positive and gram-negative bacteria. Since IL solvent was found to increase the solubility of ZFL, this may serve as "functional excipient solvent" for solubility enhancement in its commercialized formulations.

Mutations in the Von Hippel-Lindau (VHL) gene are the driving force in many forms of clear cell renal cell carcinoma (ccRCC) and promote hypoxia-inducible factor (HIF)-dependent tumor proliferation, metastasis and angiogenesis. Despite the progress that has already been made, ccRCC generally remain resistant to conventional therapies and ccRCC patients suffer from metastasis and acquired resistance, highlighting the need for novel therapeutic options. Cysteinyl leukotriene receptor 1 (CysLTR1) antagonists, like zafirlukast, are administered in bronchial asthma to control eicosanoid signaling. Intriguingly, long-term use of zafirlukast decreases cancer risk and leukotriene receptor antagonists inhibit tumor growth, but the mechanisms still remain unexplored. Therefore, we aim to understand the mechanisms of zafirlukast-mediated cell death in ccRCC cells. We show that zafirlukast induces VHL-dependent and TNFα-independent non-apoptotic and non-necroptotic cell death in ccRCC cells. Cell death triggered by zafirlukast could be rescued with antioxidants and the PARP-1 inhibitor Olaparib, and additionally relies on HIF-2α. Finally, MG-132-mediated proteasome inhibition sensitized VHL wild-type cells to zafirlukast-induced cell death and inhibition of HIF-2α rescued zafirlukast- and MG-132-triggered cell death. Together, these results highlight the importance of VHL, HIF and proteasomal degradation in zafirlukast-induced oxidative cell death with potentially novel therapeutic implications for ccRCC.

Lung cancer has the highest mortality among cancers worldwide due to its high incidence and lack of the effective cures. We have previously demonstrated that the membrane ion channel TMEM16A is a potential drug target for the treatment of lung adenocarcinoma and have identified a pocket of inhibitor binding that provides the basis for screening promising new inhibitors. However, conventional drug discovery strategies are lengthy and costly, and the unpredictable side effects lead to a high failure rate in drug development. Therefore, finding new therapeutic directions for already marketed drugs may be a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library of over 1400 Food and Drug Administration-approved drugs through virtual screening and activity testing. We identified a drug candidate, Zafirlukast (ZAF), clinically approved for the treatment of asthma, that could inhibit the TMEM16A channel in a concentration-dependent manner. Molecular dynamics simulations and site-directed mutagenesis experiments showed that ZAF can bind to S387/N533/R535 in the nonselective inhibitor binding pocket, thereby blocking the channel pore. Furthermore, we demonstrate ZAF can target TMEM16A channel to inhibit the proliferation and migration of lung adenocarcinoma LA795 cells. In vivo experiments showed that ZAF can significantly inhibit lung adenocarcinoma tumor growth in mice. Taken together, we identified ZAF as a novel TMEM16A channel inhibitor with excellent anticancer activity, and as such, it represents a promising candidate for future preclinical and clinical studies.

For the last two years, the COVID-19 pandemic has continued to bring consternation on most of the world. According to recent WHO estimates, there have been more than 5.6 million deaths worldwide. The virus continues to evolve all over the world, thus requiring both vigilance and the necessity to find and develop a variety of therapeutic treatments, including the identification of specific antiviral drugs. Multiple studies have confirmed that SARS-CoV-2 utilizes its membrane-bound spike protein to recognize human angiotensin-converting enzyme 2 (ACE2). Thus, preventing spike-ACE2 interactions is a potentially viable strategy for COVID-19 treatment as it would block the virus from binding and entering into a host cell. This work aims to identify potential drugs using an in silico approach. Molecular docking was carried out on both approved drugs and substances previously tested in vivo. This step was followed by a more detailed analysis of selected ligands by molecular dynamics simulations to identify the best molecules that thwart the ability of the virus to interact with the ACE2 receptor. Because the SARS-CoV-2 virus evolves rapidly due to a plethora of immunocompromised hosts, the compounds were tested against five different known lineages. As a result, we could identify substances that work well on individual lineages and those showing broader efficacy. The most promising candidates among the currently used drugs were zafirlukast and simeprevir with an average binding affinity of -22 kcal/mol for spike proteins originating from various lineages. The first compound is a leukotriene receptor antagonist that is used to treat asthma, while the latter is a protease inhibitor used for hepatitis C treatment. From among the in vivo tested substances that concurrently exhibit promising free energy of binding and ADME parameters (indicating a possible oral administration) we selected the compound BDBM50136234. In conclusion, these molecules are worth exploring further by in vitro and in vivo studies against SARS-CoV-2.

Background: Despite the enormous efforts made towards combating tuberculosis (TB), the disease remains a major global threat. Hence, new drugs with novel mechanisms against TB are urgently needed. Fatty acid degradation protein D32 (FadD32) has been identified as a promising drug target against TB, the protein is required for the biosynthesis of mycolic acids, hence, essential for the growth and multiplication of the mycobacterium. However, the FadD32 mechanism upon the binding of FDA-approved drugs is not well established. Herein, we applied virtual screening (VS), molecular docking, and molecular dynamic (MD) simulation to identify potential FDA-approved drugs against FadD32. Methodology/Results: VS technique was found promising to identify four FDA-approved drugs (accolate, sorafenib, mefloquine, and loperamide) with higher molecular docking scores, ranging from -8.0 to -10.0 kcal/mol. Post-MD analysis showed that the accolate hit displayed the highest total binding energy of -45.13 kcal/mol. Results also showed that the accolate hit formed more interactions with FadD32 active site residues and all active site residues displayed an increase in total binding contribution. RMSD, RMSF, Rg, and DCCM analysis further supported that the presence of accolate exhibited more structural stability, lower bimolecular flexibility, and more compactness into the FadD32 protein. Conclusions: Our study revealed accolate as the best potential drug against FadD32, hence a prospective anti-TB drug in TB therapy. In addition, we believe that the approach presented in the current study will serve as a cornerstone to identifying new potential inhibitors against a wide range of biological targets.

Newly emerging roles of LRRC8 volume-regulated anion channels (VRAC) raise important questions about the therapeutic potential of VRAC in the treatment of epilepsy, type 2 diabetes, and other human diseases. A critical barrier to evaluating whether VRAC represents a viable drug target is the lack of potent and specific small-molecule inhibitors and activators of the channel. Here we review recent progress in developing the molecular pharmacology of VRAC made by screening a library of FDA-approved drugs for novel channel modulators. We discuss the discovery and characterization of cysteinyl leukotriene receptor antagonists Pranlukast and Zafirlukast as novel VRAC inhibitors, and zinc pyrithione (ZPT), which apparently activates VRAC through a reactive oxygen species (ROS)-dependent mechanism. These ongoing efforts set the stage for developing a pharmacological toolkit for probing the integrative physiology, molecular pharmacology, and therapeutic potential of VRAC.

Leukotriene antagonists constitute an important group of drugs in the therapeutic armamentarium of all dermatologists. It has been quite valuable in the management of various types of urticaria and atopic dermatitis. Recently, the role of zileuton in the management of acne has been elaborated, and in the near future it could be used as a first-line agent for the same, thereby preventing adverse effects and antibiotic resistance encountered following antibiotic use. This review will throw light on the dermatologic aspects of leukotriene antagonists.

Cancer remains a leading cause of death worldwide, despite many advances being made in recent decades. Changes in the tumor microenvironment, including dysregulated immunity, may contribute to carcinogenesis and cancer progression. The cysteinyl leukotriene (CysLT) pathway is involved in several signal pathways, having various functions in different tissues. We summarized major findings of studies about the roles of the CysLT pathway in cancer. Many in vitro studies suggested the roles of CysLTs in cell survival/proliferation via CysLT1 receptor (CysLT1R). CysLT1R antagonism decreased cell vitality and induced cell death in several types of cancer cells, such as colorectal, urological, breast, lung and neurological malignancies. CysLTs were also associated with multidrug resistance of cancer, and CysLT1R antagonism might reverse chemoresistance. Some animal studies demonstrated the beneficial effects of CysLT1R antagonist in inhibiting tumorigenesis and progression of some cancer types, particularly colorectal cancer and lung cancer. The expression of CysLT1R was shown in various cancer tissues, particularly colorectal cancer and urological malignancies, and higher expression was associated with a poorer prognosis. The chemo-preventive effects of CysLT1R antagonists were demonstrated in two large retrospective cohort studies. In summary, the roles of the CysLT pathway in cancer have been delineated, whereas further studies are still warranted.

Autophagy is an important cellular mechanism for maintaining cellular homeostasis, and its impairment correlates highly with age and age-related diseases. Retinal pigment epithelial (RPE) cells of the eye represent a crucial model for studying autophagy, as RPE functions and integrity are highly dependent on an efficient autophagic process. Cysteinyl leukotriene receptor 1 (CysLTR1) acts in immunoregulation and cellular stress responses and is a potential regulator of basal and adaptive autophagy. As basal autophagy is a dynamic process, the aim of this study was to define the role of CysLTR1 in autophagy regulation in a chronobiologic context using the ARPE-19 human RPE cell line. Effects of CysLTR1 inhibition on basal autophagic activity were analyzed at inactive/low and high lysosomal degradation activity with the antagonists zafirlukast (ZTK) and montelukast (MTK) at a dosage of 100 nM for 3 hours. Abundances of the autophagy markers LC3-II and SQSTM1 and LC3B particles were analyzed in the absence and presence of lysosomal inhibitors using western blot analysis and immunofluorescence microscopy. CysLTR1 antagonization revealed a biphasic effect of CysLTR1 on autophagosome formation and lysosomal degradation that depended on the autophagic activity of cells at treatment initiation. ZTK and MTK affected lysosomal degradation, but only ZTK regulated autophagosome formation. In addition, dexamethasone treatment and serum shock induced autophagy, which was repressed by CysLTR1 antagonization. As a newly identified autophagy modulator, CysLTR1 appears to be a key player in the chronobiological regulation of basal autophagy and adaptive autophagy in RPE cells.

Recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen is the hallmark of an acute immunopathologic inflammatory response during vulvovaginal candidiasis (VVC) caused by Candida albicans. Recurrent VVC (RVVC) remains a chronic health burden in affected women worldwide despite the use of antifungal therapy. Based on the role leukotrienes (LTs) play in promoting inflammation, leukotriene receptor antagonists (LTRAs) targeted for LTB4 (etalocib) or LTC4, LTD4, and LTE4 (zafirlukast or montelukast) have been shown to reduce inflammation of epithelial tissues. An open-label pilot study using long-term regimens of zafirlukast in women with RVVC indicated the potential for some relief from recurrent episodes. To investigate this clinical observation further, we evaluated the effects of LT antagonistic agents and LT deficiency on the immunopathogenic response in a mouse model of VVC. Results showed that mice given daily intraperitoneal injections of individual LTRAs, starting 2days prior to vaginal inoculation with C. albicans and continuing through 14days post-inoculation, had no measurable reduction in PMN migration. The LTRAs were also ineffective in reducing levels of the hallmark vaginal inflammatory markers (S100A8, IL-1β) and tissue damage (LDH) associated with the immunopathogenic response. Finally, LT-deficient 5-lipoxygenase knockout mice showed comparable levels of vaginal fungal burden and PMN infiltration to wild-type mice following inoculation with a vaginal (ATCC 96113) or laboratory (SC5314) C. albicans isolate. These results indicate that despite some clinical evidence suggestive of off-target efficacy of LTRAs in RVVC, LTs and associated signaling pathways appear to be dispensable in the immunopathogenesis of VVC.

This study investigated the effect of solid dispersions (SD) on solubility and release of Zafirlukast (ZA) by physical mixture (PM), solvent evaporation (SE) and kneading method (KM) with Eudragit EPO (EPO) as binary component and Poloxamer 188 (P188) and Poloxamer 407 (P407) as ternary components. The binary and ternary systems caused an increase of 322 folds and 356 folds in aqueous solubility of ZA, respectively. Formulations were characterized for solubility, FTIR, PXRD, DSC, SEM and dissolution studies. P407 was found to be an excellent solubility booster in combination with EPO. It was concluded that solubility and dissolution rate of ZA increased significantly when SD of the ZA was prepared by solvent evaporation method (1:7 ratio) using 15% P407 as ternary component.