Single and multiple inhibitors of the biosynthesis of 5-, 12-, 15-lipoxygenase products derived from cinnamyl-3,4-dihydroxy-α-cyanocinnamate: Synthesis and structure-activity relationship.

The involvement of lipoxygenases in various pathologies, combined with the unavailability of safe and effective inhibitors of the biosynthesis of their products, is a source of inspiration for the development of new inhibitors. Based on a structural analysis of known inhibitors of lipoxygenase products biosynthesis, a comprehensive structure-activity study was carried out, which led to the discovery of several novel compounds (16a-c, 17a) demonstrating promising potency to inhibit the biosynthesis of products of 5-, 12- and 15-LO. Compounds 16b and 16c outperformed zileuton (1), the only FDA-approved 5-LO inhibitor, as well as known inhibitors such as caffeic acid phenethyl ester (CAPE (2)) and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC (4)). However, the introduction of a cyano group at the α-position of the carbonyl abolished the activity. Compounds 16a and 17a also inhibited the biosynthesis of 12- and 15-LO products. Compounds 16a, 17a far surpassed baicalein, a known 12-LO inhibitor, as inhibitors of 12-LO products biosynthesis. Compound 17a and CDC (4) showed equivalent inhibition of LO products, proposing that the double bond in the ester moiety is not necessary for the inhibitory activity. The introduction of the cyano group, as in compound 17a, at the α-position of the carbonyl in compound 16a significantly reduced the inhibitory activity against the biosynthesis of 15-LO products. In addition to the interactions with residues His372 and Phe421 also found with zileuton and CAPE, compounds 16a and 16c each interact with residue His367 as shown by molecular docking. This new interaction may explain their high affinity with the 5-LO active site.

Evaluation of mucosal immune profile associated with Zileuton nanocrystal-formulated BCS-II drug upon oral administration in Sprague Dawley rats.

Nanocrystal drug formulation involves several critical manufacturing procedures that result in complex structures to improve drug solubility, dissolution, bioavailability, and consequently the efficacy of poorly soluble Biopharmaceutics Classification System (BCS) II and IV drugs. Nanocrystal formulation of an already approved oral drug may need additional immunotoxic assessment due to changes in the physical properties of the active pharmaceutical ingredient (API). In this study, we selected Zileuton, an FDA-approved drug that belongs to BCS-II for nanocrystal formulation. To evaluate the efficacy and mucosal immune profile of the nanocrystal drug, 10-week-old rats were dosed using capsules containing either API alone or nanocrystal formulated Zileuton (NDZ), or with a physical mixture (PM) using flexible oral gavage syringes. Control groups consisted of untreated, or placebo treated animals. Test formulations were administrated to rats at a dose of 30 mg/kg body weight (bw) once a day for 15 days. The rats treated with NDZ or PM had approximately 4.0 times lower (7.5 mg/kg bw) API when compared to the micron sized API treated rats. At the end of treatment, mucosal (intestinal tissue) and circulating cytokines were measured. The immunological response revealed that NDZ decreased several proinflammatory cytokines in the ileal mucosa (Interleukin-18, Tumor necrosis Factor-α and RANTES [regulated upon activation, normal T cell expressed and secreted]). A similar pattern in the cytokine profile was also observed for the micron sized API and PM treated rats. The cytokine production revealed that there was a significant increase in the production of IL-1ß and IL-10 in the females in all experimental groups. Additionally, NDZ showed an immunosuppressive effect on proinflammatory cytokines both locally and systemically, which was similar to the response in micron sized API treated rats. These findings indicate that NDZ significantly decreased several proinflammatory cytokines and it displays less immunotoxicity, probably due to the nanocrystal formulation. Thus, the nanocrystal formulation is more suitable for oral drug delivery, as it exhibited better efficacy, safety, and reduced toxicity.

Eeyarestatin I, an inhibitor of the valosin-containing protein, exhibits potent virucidal activity against the flaviviruses.

Cellular responses to stress generally lead to the activation of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Several lines of study support that ERAD may be playing a proviral role during flaviviral infection. A key host factor in ERAD is the valosin-containing protein (VCP), an ATPase which ushers ubiquitin-tagged proteins to degradation by the proteasome. VCP exhibits different proviral activities, such as engaging in the biogenesis of viral replication organelles and facilitating flavivirus genome uncoating after the viral particle entry. To investigate the possible antiviral value of drugs targeting VCP, we tested two inhibitors: eeyarestatin I (EEY) and xanthohumol (XAN). Both compounds were highly effective in suppressing Zika virus (ZIKV) and Usutu virus (USUV) replication during infection in cell culture. Further analysis revealed an unexpected virucidal activity for EEY, but not for XAN. Preincubation of ZIKV or USUV with EEY before inoculation to cells resulted in significant decreases in infectivity in a dose- and time-dependent manner. Viral genomes in samples previously treated with EEY were more sensitive to propidium monoazide, an intercalating agent, with 10- to 100-fold decreases observed in viral RNA levels, supporting that EEY affects viral particle integrity. Altogether, these results support that EEY is a strong virucide against two unrelated flaviviruses, encouraging further studies to investigate its potential use as a broad-acting drug or the development of improved derivatives in the treatment of flaviviral infection.

Zileuton Alleviates Radiation-Induced Cutaneous Ulcers via Inhibition of Senescence-Associated Secretory Phenotype in Rodents.

Radiation-induced cutaneous ulcers are a challenging medical problem for patients receiving radiation therapy. The inhibition of cell senescence has been suggested as a prospective strategy to prevent radiation ulcers. However, there is no effective treatment for senescent cells in radiation ulcers. In this study, we investigated whether zileuton alleviated radiation-induced cutaneous ulcer by focusing on cell senescence. We demonstrate increased cell senescence and senescence-associated secretory phenotype (SASP) in irradiated dermal fibroblasts and skin tissue. The SASP secreted from senescent cells induces senescence in adjacent cells. In addition, 5-lipoxygenase (5-LO) expression increased in irradiated dermal fibroblasts and skin tissue, and SASP and cell senescence were regulated by 5-LO through p38 phosphorylation. Finally, the inhibition of 5-LO following treatment with zileuton inhibited SASP and mitigated radiation ulcers in animal models. Our results demonstrate that inhibition of SASP from senescent cells by zileuton can effectively mitigate radiation-induced cutaneous ulcers, indicating that inhibition of 5-LO might be a viable strategy for patients with this condition.

Zileuton, a 5-Lipoxygenase Inhibitor, Attenuates Haemolysate-Induced BV-2 Cell Activation by Suppressing the MyD88/NF-κB Pathway.

M1 microglia induce neuroinflammation-related neuronal death in animal models of spontaneous subarachnoid haemorrhage. Zileuton is a 5-lipoxygenase inhibitor that reduces the levels of downstream pro-inflammatory cytokines. This study aimed to investigate whether zileuton inhibits microglial activation and describe its underlying mechanisms. BV-2 cells were exposed to 1 mg/mL haemolysate for 30 min, followed by treatment with different concentrations (5, 10, 15, or 20 µM) of zileuton for 24 h. The cells were then assessed for viability, polarisation, and protein expression levels. Haemolysate increases the viability of BV-2 cells and induces M1 polarisation. Subsequent exposure to high concentrations of zileuton decreased the viability of BV-2 cells, shifted the polarisation to the M2 phenotype, suppressed the expression of 5-lipoxygenase, decreased tumour necrosis factor α levels, and increased interleukin-10 levels. Furthermore, high concentrations of zileuton suppressed the expression of myeloid differentiation primary response protein 88 and reduced the phosphorylated-nuclear factor-kappa B (NF-kB)/NF-kB ratio. Therefore, phenotype reversal from M1 to M2 is a possible mechanism by which zileuton attenuates haemolysate-induced neuroinflammation after spontaneous subarachnoid haemorrhage.

5-Lipoxygenase Inhibition Protects Retinal Pigment Epithelium from Sodium Iodate-Induced Ferroptosis and Prevents Retinal Degeneration.

Excessive reactive oxygen species (ROS) contribute to damage of retinal cells and the development of retinal diseases including age-related macular degeneration (AMD). ROS result in increased metabolites of lipoxygenases (LOXs), which react with ROS to induce lipid peroxidation and may lead to ferroptosis. In this study, the effect of 5-LOX inhibition on alleviating ROS-induced cell death was evaluated using sodium iodate (NaIO3) in the retinal pigment epithelium (RPE) cell line ARPE-19 and a mouse model investigating oxidative stress in AMD. We demonstrated that NaIO3 induced cell death in the RPE cells through mechanisms including ferroptosis. Inhibition of 5-LOX with specific inhibitor, Zileuton, or siRNA knockdown of ALXO5 mitigated NaIO3-induced lipid peroxidation, mitochondrial damage, DNA impairment, and cell death in ARPE-19 cells. Additionally, in the mouse model, pretreatment with Zileuton reduced the NaIO3-induced lipid peroxidation of RPE cells, cell death in the photoreceptor layer of the retina, inflammatory responses, and degeneration of both the neuroretina and RPE monolayer cells. Our results suggest that 5-LOX plays a crucial role in ROS-induced cell death in the RPE and that regulating 5-LOX activity could be a useful approach to control ROS and ferroptosis-induced damage, which promote degeneration in retinal diseases.

Abnormally decreased renal Klotho is linked to endoplasmic reticulum-associated degradation in mice.

Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded proteins retained in the ER, is implicated in various diseases including chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial fibrosis (RIF). Methods: Following establishment of a mouse RIF model by unilateral ureteral obstruction (UUO), a specific ERAD inhibitor, Eeyarestatin I (EerI), was administered to experimental animals by intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by enzyme-linked immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1, glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and protein disulfide isomerase (PDI). HK-2 cells were exposed to transforming growth factor (TGF)-ß1 with or without EerI, and expressions of related proteins including Klotho, Derlin-1, GRP78, ATF4 and PDI were determined by western blotting analyses. Results: UUO induced severe kidney injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-ß1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded proteins, subsequently contributing to the additional production of mature Klotho protein. Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.

Long-term role of zileuton in the treatment of chronic rhinosinusitis in aspirin exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis (CRS) in the setting of Aspirin Exacerbated Respiratory Disease (AERD) have high rate of treatment failure and disease recurrence. OBJECTIVE: Evaluate the long-term effect of zileuton on sinonasal outcomes in patients with AERD. METHODS: AERD patients were reviewed and divided into two cohorts, depending if they were treated with zileuton during their clinical course. Demographic data, 22-item sinonasal outcome test (SNOT-22), Lund-Kennedy (LK) endoscopy score, duration of treatment, and number of sinus surgeries performed were collected. RESULTS: 40 AERD patients were included, with follow-up duration up to 10 years (avg of 5.2 years). All patients were treated with topical saline and budesonide irrigations, intranasal steroid spray, and montelukast. 19 patients had uncontrolled sinus disease requiring multiple steroid tapers and were switched from montelukast to zileuton (cohort 1, 47.5%) at some point in their treatment. 21 patients (cohort 2, 52.5%) never needed zileuton. The average duration of treatment with zileuton was 6 years. Patients who required zileuton had a worse SNOT-22 (32.1 vs 19, p = 0.117), worse LK score (8.1 vs 7.5, p = 0.504), and higher average number of surgeries (1.9 vs 1.6, p = 0.343). The outcomes in the zileuton cohort trended toward improvement, however these did not reach statistical significance with an improved SNOT-22 from 32.1 to 27.4 (p = 0.617) and LK score from 7.9 to 6.2 (p = 0.092); The addition of zileuton significantly lowered the number of surgeries needed to an average of 0.5 (p < 0.0001). CONCLUSION: Zileuton may help decrease the number of sinus surgeries needed in AERD.

Zileuton inhibits arachidonate-5-lipoxygenase to exert antitumor effects in preclinical cervical cancer models.

BACKGROUND: Inhibitors of arachidonate lipoxygenase 5 (ALOX5) exhibit anticancer activity. Zileuton is an FDA-approved drug for treating asthma and an ALOX5 inhibitor. This study evaluated the efficacy of zileuton in cervical cancer, determined the molecular mechanism of action, and assessed ALOX5 expression in cervical cancer patients. METHODS: The effects of zileuton were evaluated using cervical cancer cell lines and xenograft mouse models. Loss-of-function analysis of ALOX5 was performed using siRNA. The levels of ALOX5 and 5-HETE were determined using immunohistochemistry and ELISA. RESULTS: Zileuton resulted in cell proliferation inhibition and apoptosis induction in a dose-dependent manner, regardless of cellular origin or HPV infection. In two independent cervical cancer xenograft mouse models, zileuton at nontoxic doses significantly prevented tumor formation and decreased tumor growth. Zileuton acts on cervical cancer cells by inhibiting the ALOX5-5-HETE axis. Of note, ALOX5-5-HETE was significantly upregulated in cervical cancer compared with normal tissue. Inhibition of ALOX5 via the siRNA approach mimics the inhibitory effects of zileuton and confirms the roles of ALOX5 in cervical cancer. CONCLUSIONS: Our work demonstrates that the ALOX5-5-HETE axis is activated in cervical cancer, with important roles in growth and survival, and this can be therapeutically targeted by zileuton. Our findings also provide preclinical evidence to assess the efficacy of zileuton in cervical cancer in clinical settings.

Inhibition of Lipoxygenases Showed No Benefit for the Musculoskeletal System in Estrogen Deficient Rats.

Background: In previous studies, we reported the beneficial impact of two lipoxygenase-inhibitors, Baicalein and Zileuton, on osteoporotic bone in a postmenopausal rat model. Whereas subcutaneous Baicalein predominantly improved cortical bone, Zileuton enhanced vertebral and femoral trabecular bone. In this study, we aimed to reveal whether the oral administration of Baicalein caused similar effects on bone and whether a combined administration of Baicalein and Zileuton could act synergistically to ameliorate the formerly reported effects in the musculoskeletal system. Methods: We treated ovariectomized (OVX) female Sprague-Dawley rats either with Baicalein (10mg/kg BW), Zileuton (10mg/kg BW) or a combination of both (each 10mg/kg BW) for 13 weeks and compared with untreated OVX and NON-OVX groups (n=12-16 rats per group). Lumbar vertebral bodies and femora were analyzed. Tibiae were osteotomized, plate-stabilized (at week 8 after OVX) and likewise analyzed by biomechanical, histological, micro-computed tomographical and ashing tests. The skeletal muscle structure was analyzed. Results: Oral administration of Baicalein did not confirm the reported favorable cortical effects in neither vertebra nor femur. Zileuton showed a beneficial effect on trabecular vertebra, while the femur was negatively affected. Callus formation was enhanced by all treatments; however, its density and biomechanical properties were unaltered. Lipoxygenase inhibition did not show a beneficial effect on skeletal muscle. The combination therapy did not ameliorate OVX-induced osteoporosis but induced even more bone loss. Conclusions: The preventive anti-osteoporotic treatments with two lipoxygenase inhibitors applied either alone or in combination showed no benefit for the musculoskeletal system in estrogen deficient rats.

Development of polymer-based nanoparticles for zileuton delivery to the lung: PMeOx and PMeOzi surface chemistry reduces interactions with mucins.

In this paper, two amphiphilic graft copolymers were synthesized by grafting polylactic acid (PLA) as hydrophobic chain and poly(2-methyl-2-oxazoline) (PMeOx) or poly(2-methyl-2-oxazine) (PMeOzi) as hydrophilic chain, respectively, to a backbone of α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA). These original graft copolymers were used to prepare nanoparticles delivering Zileuton in inhalation therapy. Among various tested methods, direct nanoprecipitation proved to be the best technique to prepare nanoparticles with the smallest dimensions, the narrowest dimensional distribution and a spherical shape. To overcome the size limitations for administration by inhalation, the nano-into-micro strategy was applied, encapsulating the nanoparticles in water-soluble mannitol-based microparticles by spray-drying. This process has allowed to produce spherical microparticles with the proper size for optimal lung deposition, and, once in contact with fluids mimicking the lung district, able to dissolve and release non-aggregated nanoparticles, potentially able to spread through the mucus, releasing about 70% of the drug payload in 24 h.

ALOX5-5-HETE promotes gastric cancer growth and alleviates chemotherapy toxicity via MEK/ERK activation.

BACKGROUND: Recent studies highlight the regulatory role of arachidonate lipoxygenase5 (Alox5) and its metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) in cancer tumorigenesis and progression. In this study, we analyzed the expression, biological function and the downstream signaling of Alox5 in gastric cancer. METHODS: Alox5 protein levels were measured using IHC and ELISA. Growth, migration and survival assays were performed. Phosphorylation of molecules involved in growth and survival signaling were analyzed by WB. Analysis of variance and t-test were used for statistic analysis. RESULTS: Alox5 and 5-HETE levels were upregulated in gastric cancer patients. ALOX5 overexpression or 5-HETE addition activates gastric cancer cells and reduces chemotherapy's efficacy. In contrast, ALOX5 inhibition via genetic and pharmacological approaches suppresses gastric cancer cells and enhances chemotherapy's efficacy. In addition, Alox5 inhibition led to suppression of ERK-mediated signaling pathways whereas ALOX5-5-HETE activates ERK-mediated signaling in gastric cancer cells. CONCLUSIONS: Our work demonstrates the critical role of ALOX5-5-HETE in gastric cancer and provides pre-clinical evidence to initialize clinical trial using zileuton in combination with chemotherapy for treating gastric cancer.

5-LO-derived LTB4 plays a key role in MCP-1 expression in HMGB1-exposed VSMCs via a BLTR1 signaling axis.

Monocyte chemoattractant protein-1 (MCP-1) plays an important role in initiating vascular inflammation; however, its cellular source in the injured vasculatures is unclear. Given the importance of high mobility group box 1 (HMGB1) in tissue injury, we investigated the role of vascular smooth muscle cells (VSMCs) in MCP-1 production in response to HMGB1. In primary cultured rat aortic VSMCs stimulated with HMGB1, the expression of MCP-1 and 5-lipoxygenase (LO) was increased. The increased MCP-1 expression in HMGB1 (30 ng/ml)-stimulated cells was significantly attenuated in 5-LO-deficient cells as well as in cells treated with zileuton, a 5-LO inhibitor. Likewise, MCP-1 expression and production were also increased in cells stimulated with exogenous leukotriene B4 (LTB4), but not exogenous LTC4. LTB4-induced MCP-1 expression was attenuated in cells treated with U75302, a LTB4 receptor 1 (BLTR1) inhibitor as well as in BLTR1-deficient cells, but not in 5-LO-deficient cells. Moreover, HMGB1-induced MCP-1 expression was attenuated in BLTR1-deficient cells or by treatment with a BLTR1 inhibitor, but not other leukotriene receptor inhibitors. In contrast to MCP-1 expression in response to LTB4, the increased MCP-1 production in HMGB1-stimulated VSMC was markedly attenuated in 5-LO-deficient cells, indicating a pivotal role of LTB4-BLTR1 signaling in MCP-1 expression in VSMCs. Taken together, 5-LO-derived LTB4 plays a key role in MCP-1 expression in HMGB1-exposed VSMCs via BLTR1 signaling, suggesting the LTB4-BLTR1 signaling axis as a potential therapeutic target for vascular inflammation in the injured vasculatures.

5-Lipoxygenase inhibition reduces inflammation and neuronal apoptosis via AKT signaling after subarachnoid hemorrhage in rats.

Early brain injury (EBI) is a major contributor to the high mortality and morbidity after subarachnoid hemorrhage (SAH). Inflammatory responses and neuronal apoptosis are important causes of EBI. Because 5- lipoxygenase (5-LOX) is known to be involved various central nervous system diseases, we investigated the effects of 5-LOX inhibition during EBI after SAH. Zileuton and LY294002 were used to inhibit expression of 5-LOX and Akt, respectively. We found that 5-LOX expression was significantly increased in the cytoplasm of cortical neurons after SAH and was accompanied by upregulated expression of the inflammatory factors LTB4, TNF-α, IL-1ß and IL-6; upregulation of the pro-apoptotic factor Bax; downregulation of the anti-apoptotic factor Bcl-2; and an increased apoptosis rate. Gastric Zileuton administration significantly suppressed all of those effects and improved neurological function. Zileuton also upregulated activated (phosphorylated) AKT levels, and these beneficial effects of Zileuton were abolished by intracerebroventricular infusion of the PI3K inhibitor LY294002. Taken together, these findings indicate that 5-LOX mediates pro-inflammatory and pro-apoptotic effects that contribute to EBI after SAH and that those effects are suppressed by activation of PI3K/Akt signaling. This suggests targeting 5-LOX may be an effective approach to treating EBI after SAH.

Inhibition of ERAD synergizes with FTS to eradicate pancreatic cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers, is driven by oncogenic KRAS mutations. Farnesyl thiosalicylic acid (FTS), also known as salirasib, is a RAS inhibitor that selectively dislodges active RAS proteins from cell membrane, inhibiting downstream signaling. FTS has demonstrated limited therapeutic efficacy in PDAC patients despite being well tolerated. METHODS: To improve the efficacy of FTS in PDAC, we performed a genome-wide CRISPR synthetic lethality screen to identify genetic targets that synergize with FTS treatment. Among the top candidates, multiple genes in the endoplasmic reticulum-associated protein degradation (ERAD) pathway were identified. The role of ERAD inhibition in enhancing the therapeutic efficacy of FTS was further investigated in pancreatic cancer cells using pharmaceutical and genetic approaches. RESULTS: In murine and human PDAC cells, FTS induced unfolded protein response (UPR), which was further augmented upon treatment with a chemical inhibitor of ERAD, Eeyarestatin I (EerI). Combined treatment with FTS and EerI significantly upregulated the expression of UPR marker genes and induced apoptosis in pancreatic cancer cells. Furthermore, CRISPR-based genetic ablation of the key ERAD components, HRD1 and SEL1L, sensitized PDAC cells to FTS treatment. CONCLUSION: Our study reveals a critical role for ERAD in therapeutic response of FTS and points to the modulation of UPR as a novel approach to improve the efficacy of FTS in PDAC treatment.

Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens.

Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) associated protein degradation, Sec61-dependent Ca2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61-translocon, and captures it in an open conformation that is translocation-incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG-translocon. Transcriptomic analysis suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clinically relevant strains. Furthermore, the antibacterial activity of ES24 is equivalent to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG-dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo.

New Zileuton-Hydroxycinnamic Acid Hybrids: Synthesis and Structure-Activity Relationship towards 5-Lipoxygenase Inhibition.

A novel series of zileuton-hydroxycinnamic acid hybrids were synthesized and screened as 5-lipoxygenase (5-LO) inhibitors in stimulated HEK293 cells and polymorphonuclear leukocytes (PMNL). Zileuton's (1) benzo[b]thiophene and hydroxyurea subunits combined with hydroxycinnamic acid esters' ester linkage and phenolic acid moieties were investigated. Compound 28, bearing zileuton's (1) benzo[b]thiophene and sinapic acid phenethyl ester's (2) α,ß-unsaturated phenolic acid moiety 28, was shown to be equipotent to zileuton (1), the only clinically approved 5-LO inhibitor, in stimulated HEK293 cells. Compound 28 was three times as active as zileuton (1) for the inhibition of 5-LO in PMNL. Compound 37, bearing the same sinapic acid (3,5-dimethoxy-4-hydroxy substitution) moiety as 28, combined with zileuton's (1) hydroxyurea subunit was inactive. This result shows that the zileuton's (1) benzo[b]thiophene moiety is essential for the inhibition of 5-LO product biosynthesis with our hydrids. Unlike zileuton (1), Compound 28 formed two π-π interactions with Phe177 and Phe421 as predicted when docked into 5-LO. Compound 28 was the only docked ligand that showed a π-π interaction with Phe177 which may play a part in product specificity as reported.

High ability of zileuton ((±)-1-(1-benzo[b]thien-2-ylethyl)-1-hydroxyurea) to stimulate IK(Ca) but suppress IK(DR) and IK(M) independently of 5-lipoxygenase inhibition.

Zileuton (Zyflo®) is regarded to be an inhibitor of 5-lipoxygenase. Although its effect on Ca2+-activated K+ currents has been reported, its overall ionic effects on neurons are uncertain. In whole-cell current recordings, zileuton increased the amplitude of Ca2+-activated K+ currents with an EC50 of 3.2 µM in pituitary GH3 lactotrophs. Furthermore, zileuton decreased the amplitudes of both delayed-rectifier K+ current (IK(DR)) and M-type K+ current (IK(M)). Conversely, no modification of hyperpolarization-activated cation current (Ih) was demonstrated in its presence of zileuton, although the subsequent addition of cilobradine effectively suppressed the current. In inside-out current recordings, the addition of zileuton to the bath increased the probability of large-conductance Ca2+-activated K+ (BKCa) channels; however, the subsequent addition of GAL-021 effectively reversed the stimulation of channel activity. The kinetic analyses showed an evident shortening in the slow component of mean closed time of BKCa channels in the presence of zileuton, with minimal change in mean open time or that in the fast component of mean closed time. The elevation of BKCa channels caused by zileuton was also observed in hippocampal mHippoE-14 neurons, without any modification of single-channel amplitude. In conclusion, except for its suppression of 5-lipoxygenase, our results indicate that zileuton does not exclusively act on BKCa channels, and its inhibitory effects on IK(DR) and IK(M) may combine to exert strong influence on the functional activities of electrically excitable cells in vivo.

Targeting Neutrophils for Enhanced Cancer Theranostics.

Improving tumor accumulation and delivery efficiency is an important goal of nanomedicine. Neutrophils play a vital role in both chemically mediating inflammatory response through myeloperoxidase (MPO) and biologically promoting metastasis during inflammation triggered by the primary tumor or environmental stimuli. Herein, a novel theranostic nanomedicine that targets both the chemical and biological functions of neutrophils in tumor is designed, facilitating the enhanced retention and sustained release of drug cargos for improved cancer theranostics. 5-hydroxytryptamine (5-HT) is equipped onto nanoparticles (NPs) loaded with photosensitizers and Zileuton (a leukotriene inhibitor) to obtain MPO and neutrophil targeting NPs, denoted as HZ-5 NPs. The MPO targeting property of 5-HT modified NPs is confirmed by noninvasive positron emission tomography imaging studies. Furthermore, photodynamic therapy is used to initiate the inflammatory response which further mediated the accumulation and retention of neutrophil targeting NPs in a breast cancer model. This design renders a greatly improved theranostic nanomedicine for efficient tumor suppression, and more importantly, inhibition of neutrophil-mediated lung metastasis via the sustained release of Zileuton. This work presents a novel strategy of targeting neutrophils for improved tumor theranostics, which may open up new avenues in designing nanomedicine through exploiting the tumor microenvironment.