Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms

The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.     

An ivermectin-sensitive glutamate-gated chloride channel subunit from Dirofilaria immitis

Dirofilaria immitis is a filarial nematode that infects dogs and causes cardiopulmonary disease. The most effective way of controlling the infection is by chemoprophylaxis, using members of the avermectin/milbemycin (A/M) class of anthelmintics, which includes ivermectin; these drugs act at invertebrate glutamate-gated chloride channels (GluCl). We have cloned two cDNAs encoding D. immitis GluCl subunits and demonstrated that at least one may be an important molecular target for the A/Ms in vivo. The subunits are orthologues of the alternatively spliced GluClalpha3A and alpha3B subunits (encoded by the avr-14 gene) previously identified in Caenorhabditis elegans and in Haemonchus contortus. Although the alternative splicing of avr-14 is conserved across the species, the processing of the mature GluClalpha3A mRNA differs in D. immitis compared to C. elegans and H. contortus. Two-electrode voltage clamp recordings were made from Xenopus oocytes injected with subunit-specific cRNAs. The DiGluClalpha3B subunit formed channels that were gated by L-glutamate (1-100 mM) and ivermectin (1 microM). Oocytes injected with DiGluClalpha3A cRNA failed to respond to L-glutamate. The qualitative responses obtained were consistent with the pharmacology observed for the GluClalpha3 subunits from C. elegans and H. contortus.     

The molecular targets of ivermectin and lotilaner in the human louse Pediculus humanus humanus: New prospects for the treatment of pediculosis

Control of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of parasites resistant to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanisms of action remain largely unknown. The macrocyclic lactone ivermectin is active against a broad range of insects including human lice. Isoxazolines are a new chemical class exhibiting a strong insecticidal potential. They preferentially act on the γ-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and, to a lesser extent on glutamate-gated chloride channels (GluCls) in some species. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Using toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and GluCl (Phh-GluCl) subunits were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC₅₀ values of 16.0 μM and 9.3 μM. Importantly, ivermectin displayed a super agonist action on Phh-GluCl, whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC₅₀ value of 40.7 nM, whereas it had no effect on Phh-GluCl. We report here for the first time the insecticidal activity of isoxazolines on human ectoparasites and reveal the mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize an expected extension of the use of the isoxazoline drug class as new pediculicidal agents to tackle resistant-louse infestations in humans.     

Mutations in the extracellular domains of glutamate-gated chloride channel alpha3 and beta subunits from ivermectin-resistant Cooperia oncophora affect agonist sensitivity

Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluClalpha3 subunits differ at three amino acid positions, while the IVS and IVR GluClbeta subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluClalpha3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluClalpha3 receptor. Mutations in the IVR GluClbeta subunit abolished glutamate sensitivity. Co-expressing the IVS GluClalpha3 and GluClbeta subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluClalpha3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluClalpha3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluClalpha3 homomeric channels.     

Molecular Cloning and Characterization of Novel Glutamate-Gated Chloride Channel Subunits from Schistosoma mansoni

Cys-loop ligand-gated ion channels (LGICs) mediate fast ionotropic neurotransmission. They are proven drug targets in nematodes and arthropods, but are poorly characterized in flatworms. In this study, we characterized the anion-selective, non-acetylcholine-gated Cys-loop LGICs from Schistosoma mansoni. Full-length cDNAs were obtained for SmGluCl-1 (Smp_096480), SmGluCl-2 (Smp_015630) and SmGluCl-3 (Smp_104890). A partial cDNA was retrieved for SmGluCl-4 (Smp_099500/Smp_176730). Phylogenetic analyses suggest that SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 belong to a novel clade of flatworm glutamate-gated chloride channels (GluCl) that includes putative genes from trematodes and cestodes. The flatworm GluCl clade was distinct from the nematode-arthropod and mollusc GluCl clades, and from all GABA receptors. We found no evidence of GABA receptors in S. mansoni. SmGluCl-1, SmGluCl-2 and SmGluCl-3 subunits were characterized by two-electrode voltage clamp (TEVC) in Xenopus oocytes, and shown to encode Cl⁻-permeable channels gated by glutamate. SmGluCl-2 and SmGluCl-3 produced functional homomers, while SmGluCl-1 formed heteromers with SmGluCl-2. Concentration-response relationships revealed that the sensitivity of SmGluCl receptors to L-glutamate is among the highest reported for GluCl receptors, with EC₅₀ values of 7–26 µM. Chloride selectivity was confirmed by current-voltage (I/V) relationships. SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group. SmGluCl receptors are also insensitive to 10 µM meclonazepam, a schistosomicidal benzodiazepine. These results provide the first molecular evidence showing the contribution of GluCl receptors to L-glutamate signaling in S. mansoni, an unprecedented finding in parasitic flatworms. Further work is needed to elucidate the roles of GluCl receptors in schistosomes and to explore their potential as drug targets.     

Picrotoxin Blockade of Invertebrate Glutamate-Gated Chloride Channels: Subunit Dependence and Evidence for Binding Within the Pore

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClα and GIuClβ, were cloned from the free living nematode Caenorhabditls elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClα and GluClβ RNAs were injected individually or coinjected into Xenopus oocytes. The EC₅₀ values for picrotoxin block of homomeric GluClα and GluClβ were 59 μM and 77 nM, respectively. Picrotoxin block of homomeric GluClβ channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClα and the C-terminal membrane-spanning domain of GIuClβ localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClβ. A point mutation within the M2 membrane-spanning domain of GluClβ reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.     

Identification and Functional Expression of a Glutamate- and Avermectin-Gated Chloride Channel from Caligus rogercresseyi,a Southern Hemisphere Sea Louse Affecting Farmed Fish

Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC₅₀ value of around 200 nM, being cooperative (n_H = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy.     

A Predicted Binding Site for Cholesterol on the GABAA Receptor

Modulation of the GABA type A receptor (GABA_AR) function by cholesterol and other steroids is documented at the functional level, yet its structural basis is largely unknown. Current data on structurally related modulators suggest that cholesterol binds to subunit interfaces between transmembrane domains of the GABA_AR. We construct homology models of a human GABA_AR based on the structure of the glutamate-gated chloride channel GluCl of Caenorhabditis elegans. The models show the possibility of previously unreported disulfide bridges linking the M1 and M3 transmembrane helices in the α and γ subunits. We discuss the biological relevance of such disulfide bridges. Using our models, we investigate cholesterol binding to intersubunit cavities of the GABA_AR transmembrane domain. We find that very similar binding modes are predicted independently by three approaches: analogy with ivermectin in the GluCl crystal structure, automated docking by AutoDock, and spontaneous rebinding events in unbiased molecular dynamics simulations. Taken together, the models and atomistic simulations suggest a somewhat flexible binding mode, with several possible orientations. Finally, we explore the possibility that cholesterol promotes pore opening through a wedge mechanism.     

Synthesis,biological evaluation and molecular docking studies of chromone hydrazone derivatives as α-glucosidase inhibitors

A series of chromone hydrazone derivatives 4a–4p have been synthesized, characterized by ¹H NMR and ¹³C NMR and evaluated for their in vitro α-glucosidase inhibitory activity. Out of these tested compounds, six (4a, 4b, 4d, 4j, 4o and 4p) displayed potent α-glucosidase inhibitory activity with IC₅₀ values in the range of 20.1 ± 0.19 μM to 45.7 ± 0.23 μM, as compared to the standard drug acarbose (IC₅₀ = 817.38 ± 6.27 μM). Among this series, compound 4d (IC₅₀ = 20.1 ± 0.19 μM) with 4-sulfonamide substitution at phenyl part of hydrazide was found to be the most active compound. Lineweaver-Burk plot analysis indicated that compound 4d is a non-competitive inhibitor of α-glucosidase. The binding interactions of the most active analogs were confirmed through molecular docking studies. Docking studies showed 4d are interacting with the residues Glu-276, Asp-214, Asp-349 and Arg-439 through hydrogen bonds, arene-anion and arene-cation interactions. In summary, our studies shown that these chromone hydrazone derivatives are a new class of α-glucosidase inhibitors.     

Expression and localization of glutamate-gated chloride channel variants in honeybee brain (Apis mellifera)

Due to its specificity to invertebrate species, glutamate-gated chloride channels (GluCls) are the target sites of antiparasitic agents and insecticides, e.g. ivermectin and fipronil, respectively. In nematodes and insects, the GluCls diversity is broadened by alternative splicing. GluCl subunits have been characterized according to their sensitivity to drugs, and to their anatomical localization. In the honeybee, the GluCl gene can encode different alpha subunits due to alternative splicing of exon 3. We examined mRNA expression in brain parts and we confirmed the existence of two GluCl variants with RT-PCR, Amel_GluCl A and Amel_GluCl B. Surprisingly, a mixed isoform not yet described in insect was obtained, we called it Amel_GluCl C. We determined precise immunolocalization of peptide sequence corresponding to Amel_GluCl A and Amel_GluCl B in the honeybee brain. Amel_GluCl A is mainly located in neuropils, whereas Amel_GluCl B is mostly expressed in cell bodies. Both proteins can also be co-localized. According to their anatomical localization, different GluCl variants might be involved in olfactory and visual modalities and in learning and memory.     

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.     

New antiprotozoal agents: Synthesis and biological evaluation of different 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives

In an endeavor to develop efficacious antiprotozoal agents 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives (5–14) were synthesized, characterized and biologically evaluated for antiprotozoal activity. The compounds were screened in vitro against the HM1: IMSS strain of Entamoeba histolytica and NF54 chloroquine-sensitive strain of Plasmodium falciparum. Among the synthesized compounds six exhibited promising antiamoebic activity with IC₅₀ values (0.14–1.26 μM) lower than the standard drug metronidazole (IC₅₀ 1.80 μM). All nine compounds exhibited antimalarial activity (IC₅₀ range: 1.42–19.62 μM), while maintaining a favorable safety profile to host red blood cells. All the compounds were less effective as an antimalarial and more toxic (IC₅₀ range: 14.67–81.24 μM) than quinine (IC₅₀: 275.6 ± 16.46 μM) against the human kidney epithelial cells. None of the compounds exhibited any inhibitory effect on the viability of Anopheles arabiensis mosquito larvae.     

Chromium(III) and iron(III) inhibits replication of DNA and RNA viruses

The aim of this study was to examine the effect of treating of chromium(III) and iron(III) and their combinations on Herpes Simplex Virus type 1 (HSV-1) and Bovine Viral Diarrhoea virus (BVDV) replication. The antiviral efficacies of chromium(III) and iron(III) on HSV-1 and BVDV were evaluated using Real Time PCR method. Moreover, the cytotoxicity of these microelements was examined using the MTT reduction assay. The IC₅₀ (50% inhibiotory concentration) for the chromium chloride was 1100 μM for Hep-2 cells and 1400 μM for BT cells. The IC₅₀ for the iron chloride was 1200 μM for Hep-2 cells and more than1400 μM for BT cells. The concentration-dependent antiviral activity of chromium chloride and iron chloride against HSV-1 and BVDV viruses was observed. In cultures simultaneously treated with (1) 200 μM of CrCl₃ and 1000 μM of FeCl₃, (2) 1000 μM of CrCl₃ and 200 μM of FeCl₃, (3) 400 μM of CrCl₃ and 800 μM of FeCl₃, (4) 800 μM of CrCl₃ and 400 μM of FeCl₃ a decrease in number of DNA or RNA copies was observed compared with control cells and cells incubated with chromium(III) and iron(III) used separately. The synergistic antiviral effects were observed for chromium(III) and iron(III) against HSV-1 and BVDV.     

Anti-diabetic xanthones from the bark of Garcinia xanthochymus

An ethyl acetate extract the bark of Garcinia xanthochymus exhibited strong inhibition towards α-glucosidase and PTP1B with IC₅₀ values of 0.3 ± 0.1 μg/mL and 2.3 ± 0.4 μg/mL, respectively. Chemical constituents of the extract were therefore examined, and two new compounds, xanthochymusxanthones A (1) and B (2), along with ten known xanthones (3–12), were isolated. Their structures were determined using spectroscopic methods, mainly 1D and 2D NMR. Inhibitory activity of the isolated compounds was then tested, and subelliptenone F (12) showed significant effect towards α-glucosidase with IC₅₀ value of 4.1 ± 0.3 μM (compared with acarbose, IC₅₀ = 900.0 ± 3.0 μM) whilst xanthochymusxanthone B (2) exhibited remarkable activity towards PTP1B with IC₅₀ value of 8.0 ± 0.6 μM (compared with RK682, IC₅₀ = 4.4 ± 0.3 μM).     

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.     

Establishment of dedifferentiated callus of haploid origin from unfertilized ovaries of tea (Camellia sinensis (L.) O. Kuntze) as a potential source of total phenolics and antioxidant activity

This is the first report of induction of haploid callus with significant antioxidant activity from unpollinated ovary cultures of tea. Out of the five cultivars tested, TV18 gave the highest percentage of callus induction. Within 1 wk of induction, ovules swelled to almost double their original size, and white, friable callus emerged. A high cytokinin/auxin ratio, provided by 8.5 μM benzyl adenine and 4.5 μM 2,4-dichlorophenxyacetic acid, and high-temperature treatment (33°C) for 10 d in the dark promoted maximum callus induction. Callus was maintained on MS medium containing 22.2 μM benzyl adenine and 9.8 μM indolebutyric acid (callus line RM 1) in the light at 25°C. Well-developed tracheids were formed within 4 wk in callus subcultured on MS medium containing 1.8 μM thidiazuron and 5.0 μM 2,3,5-triiodobenzoic acid (line RM 2). Flow cytometric analysis revealed that most cells were haploid. Both RM 1 and RM 2 produced phenolic compounds with significant antioxidant capacity. Phenolic content showed a positive linear correlation with antioxidant activity. The total phenolic content of RM 1 was 3.47?±?0.21 gallic acid equivalents (GAE) mg/g dry weight and that of RM 2 was 2.39?±?0.12 GAE mg/g dry weight. Antioxidant activity was measured using IC₅₀, a measure of inhibitory concentration; a lower IC₅₀ value reflects greater antioxidant activity. The IC₅₀ value of RM 1 was 2,530 μg/ml and that of RM 2 was 3,170 μg/ml. The results suggested that the phenolic compounds contributed significantly to the antioxidant capacity of the in vitro cell lines.     

avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans.

Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectin's effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr-15, a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr-15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate-gated chloride channel subunits. An avr-15-encoded subunit forms a homomeric channel that is ivermectin-sensitive and glutamate-gated. These results indicate that: (i) an ivermectin-sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate-gated chloride channels in essential excitable cells such as those of the pharynx.     

Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights

Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC_{50 L. braziliensis} 30.47 ± 6.35 μM; IC_{50 L. amazonensis} 40.46 ± 14.21 μM; IC_{50 L. infantum} 65.93 ± 15.12 μM) and amastigotes (IC_{50 L. braziliensis} 68.75 ± 16.55 μM; IC_{50 L. amazonensis} 38.45 ± 12.05 μM; IC_{50 L. infantum} 64.08 ± 23.52 μM), with low cytotoxicity against mouse peritoneal macrophages (CC₅₀ 235.80 ± 36.95 μM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment.     

Synthesis of novel hybrids of pyrazole and coumarin as dual inhibitors of COX-2 and 5-LOX

In our previous study, we designed a series of pyrazole derivatives as novel COX-2 inhibitors. In order to obtain novel dual inhibitors of COX-2 and 5-LOX, herein we designed and synthesized 20 compounds by hybridizing pyrazole with substituted coumarin who was reported to exhibit 5-LOX inhibition to select potent compounds using adequate biological trials sequentially including selective inhibition of COX-2 and 5-LOX, anti-proliferation in vitro, cells apoptosis and cell cycle. Among them, the most potent compound 11g (IC₅₀ = 0.23 ± 0.16 μM for COX-2, IC₅₀ = 0.87 ± 0.07 μM for 5-LOX, IC₅₀ = 4.48 ± 0.57 μM against A549) showed preliminary superiority compared with the positive controls Celecoxib (IC₅₀ = 0.41 ± 0.28 μM for COX-2, IC₅₀ = 7.68 ± 0.55 μM against A549) and Zileuton (IC₅₀ = 1.35 ± 0.24 μM for 5-LOX). Further investigation confirmed that 11g could induce human non-small cell lung cancer A549 cells apoptosis and arrest the cell cycle at G2 phase in a dose-dependent manner. Our study might contribute to COX-2, 5-LOX dual inhibitors thus exploit promising novel cancer prevention agents.