Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

A Breast Cancer Stem Active Cobalt(III)-Cyclam Complex Containing Flufenamic Acid with Immunogenic Potential.

The cytotoxic and immunogenic-activating properties of a cobalt(III)-cyclam complex bearing the non-steroidal anti-inflammatory drug, flufenamic acid is reported within the context of anti-cancer stem cell (CSC) drug discovery. The cobalt(III)-cyclam complex 1 displays sub-micromolar potency towards breast CSCs grown in monolayers, 24-fold and 31-fold greater than salinomycin (an established anti-breast CSC agent) and cisplatin (an anticancer metallopharmaceutical), respectively. Strikingly, the cobalt(III)-cyclam complex 1 is 69-fold and 50-fold more potent than salinomycin and cisplatin towards three-dimensionally cultured breast CSC mammospheres. Mechanistic studies reveal that 1 induces DNA damage, inhibits cyclooxygenase-2 expression, and prompts caspase-dependent apoptosis. Breast CSCs treated with 1 exhibit damage-associated molecular patterns characteristic of immunogenic cell death and are phagocytosed by macrophages. As far as we are aware, 1 is the first cobalt complex of any oxidation state or geometry to display both cytotoxic and immunogenic-activating effects on breast CSCs.

Exploring TEAD2 as a drug target for therapeutic intervention of cancer: A multi-computational case study.

Transcriptional enhanced associate domain (TEAD) is a family of transcription factors that plays a significant role during embryonic developmental processes, and its dysregulation is responsible for tumour progression. TEAD is considered as druggable targets in various diseases, namely cancer, cardiovascular diseases and neurodegenerative disorders. Previous structural studies revealed the importance of the central hydrophobic pocket of TEAD as a potential target for small-molecule inhibitors and demonstrated flufenamic acid (FLU) (a COX-2 enzyme inhibitor) to bind and inhibit TEAD2 functions. However, to date, no drug candidates that bind specifically to TEAD2 with high selectivity and efficacy have been developed or proposed. Within this framework, we present here a case study where we have identified potential TEAD2 inhibitor candidates by integrating multiple computational approaches. Among the candidates, the top two ranked compounds ZINC95969481 (LG1) which is a fused pyrazole derivative and ZINC05203789 (LG2), a fluorene derivative resulted in much favourable binding energy scores than the reference ligand, FLU. The drug likeliness of the best compounds was also evaluated in silico to ensure the bioavailability of these compounds particularly LG1 as compared to FLU thus providing a strong rationale for their development as leads against TEAD. Molecular dynamics simulations results highlighted the role of key residues contributing to favourable interactions in TEAD2-LG1 complex with much favourable interaction and binding free energy values with respect to the reference compound. Altogether, this study provides a starting platform to be more exploited by future experimental research towards the development of inhibitors against TEAD, a persuasive strategy for therapeutic intervention in cancer treatment.

A Single High Dose of Flufenamic Acid in Rats does not Reduce the Damage Associated with the Rat Lithium-Pilocarpine Model of Status Epilepticus but Leads to Deleterious Outcomes.

BACKGROUND: Epilepsy is one of the most common neurologic diseases, and around 30% of all epilepsies, particularly the temporal lobe epilepsy (TLE), are highly refractory to current pharmacological treatments. Abnormal synchronic neuronal activity, brain glucose metabolism alterations, neurodegeneration and neuroinflammation are features of epilepsy. Further, neuroinflammation has been shown to contribute to dysregulation of neuronal excitability and the progression of epileptogenesis. Flufenamic acid (FLU), a non-steroidal anti-inflammatory drug, is also characterized by its wide properties as a dose-dependent ion channel modulator. In this context, in vitro studies have shown that it abolishes seizure-like events in neocortical slices stimulated with a gamma-aminobutyric acid A (GABAA) receptor blocker. However, little is known about its effects in animal models. Thus, our goal was to assess the efficacy and safety of a relatively high dose of FLU in the lithium-pilocarpine rat model of status epilepticus (SE). This animal model reproduces many behavioral and neurobiological features of TLE such as short-term brain hypometabolism, severe hippocampal neurodegeneration and inflammation reflected by a marked reactive astrogliosis. METHODS: FLU (100 mg/kg, i.p.) was administered to adult male rats, 150 min before SE induced by pilocarpine. Three days after the SE, brain glucose metabolism was assessed by 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) positron emission tomography (PET). Markers of hippocampal integrity, neurodegeneration and reactive astrogliosis were also evaluated. RESULTS: FLU neither prevented the occurrence of the SE nor affected brain glucose hypometabolism as assessed by [18F]FDG PET. Regarding the neurohistochemical studies, FLU neither prevented neuronal damage nor hippocampal reactive astrogliosis. On the contrary, FLU increased the mortality rate and negatively affected body weight in the rats that survived the SE. CONCLUSIONS: Our results do not support an acute anticonvulsant effect of a single dose of FLU. Besides, FLU did not show short-term neuroprotective or anti-inflammatory effects in the rat lithium-pilocarpine model of SE. Moreover, at the dose administered, FLU resulted in deleterious effects.

Influence of Characteristics of Oily Vehicle on Skin Penetration of Ufenamate.

Skin penetration amounts of a highly lipophilic drug, ufenamate, prepared in four oily vehicles, including white petrolatum (WP), liquid paraffin (LP), isopropyl myristate (IPM), and isocetyl stearate (ICS), were compared. Ufenamate was mixed in each vehicle at 5% and applied at a rate of 2 mg/cm2 to intact, stripped, and delipidized Yucatan micropig skin. The amounts of ufenamate and IPM in the stratum corneum (SC), epidermis, and dermis were determined. The skin penetration amounts of ufenamate from liquid oils were significantly higher than those from WP; the amounts of ufenamate were in the order WP

An immunogenic anti-cancer stem cell bi-nuclear copper(II)-flufenamic acid complex.

An asymmetric bi-nuclear copper(II) complex with both cytotoxic and immunogenic activity towards breast cancer stem cells (CSCs) is reported. The bi-nuclear copper(II) complex comprises of two copper(II) centres bound to flufenamic acid and 3,4,7,8-tetramethyl-1,10-phenanthroline. The bi-nuclear copper(II) complex exhibits sub-micromolar potency towards breast CSCs grown in monolayers and three-dimensional cultures. Remarkably, the bi-nuclear copper(II) complex is up to 25-fold more potent toward breast CSC mammospheres than salinomycin (a gold standard anti-breast CSC agent) and cisplatin (a clinically administered metallodrug). Mechanistic studies showed that the bi-nuclear copper(II) complex readily enters breast CSCs, elevates intracellular reactive oxygen species levels, induces apoptosis, and promotes damage-associated molecular pattern release. The latter triggers phagocytosis of breast CSCs by macrophages. As far as we are aware, this is the first report of a bi-nuclear copper(II) complex to induce engulfment of breast CSCs by immune cells.

Corneocyte quantification by NIR densitometry and UV/Vis spectroscopy for human and porcine skin and the role of skin cleaning procedures.

Optical methods of corneocyte quantification during tape stripping experiments on the skin are useful tools for the rapid evaluation of the skin penetration potential of dermally applied substances. However, a comparative investigation of the different methods proposed for this task, namely NIR densitometry and UV/Vis spectroscopy, is still missing. Thus, the aim of the present work was to employ these two techniques in comparative tape stripping experiments both in vivo on human forearm skin and in vitro on porcine ear skin. Standard tape stripping experiments were performed in the absence and presence of a marketed formulation containing flufenamic acid as a model drug. In the context of these methodological investigations, different methods of skin cleaning prior to the tape stripping procedure were evaluated to identify the most appropriate working protocol among the approaches proposed in the respective literature. The results showed that the investigated methods of NIR densitometry and UV/Vis spectroscopy deliver highly comparable results. Both optical methods are suitable to determine the skin penetration profiles of active substances during in vivo and in vitro tape stripping, especially if a simple working protocol without any cleaning procedures is maintained.

The role of corneocytes in skin transport revised--a combined computational and experimental approach.

PURPOSE: To investigate mechanisms of compound-corneocyte interactions in a combined experimental and theoretical approach. MATERIALS AND METHODS: Experimental methods are presented to investigate compound-corneocyte interactions in terms of dissolution within water of hydration and protein binding and to quantify the extent of the concurrent mechanisms. Results are presented for three compounds: caffeine, flufenamic acid, and testosterone. Two compartmental stratum corneum models M1 and M2 are formulated based on experimentally determined input parameters describing the affinity to lipid, proteins and water. M1 features a homogeneous protein compartment and considers protein interactions only via intra-corneocyte water. In M2 the protein compartment is sub-divided into a cornified envelope compartment interacting with inter-cellular lipids and a keratin compartment interacting with water. RESULTS: For the non-protein binding caffeine the impact of the aqueous compartment on stratum corneum partitioning is overestimated but is successfully modeled after introducing a bound water fraction that is non-accessible for compound dissolution. For lipophilic, keratin binding compounds (flufenamic acid, testosterone) only M2 correctly predicts a concentration dependence of stratum corneum partition coefficients. CONCLUSIONS: Lipophilic and hydrophilic compounds interact with corneocytes. Interactions of lipophilic compounds are probably confined to the corneocyte surface. Interactions with intracellular keratin may be limited by their low aqueous solubility.

Hair removal and bioavailability of chemicals: Effect of physicochemical properties of drugs and surfactants on skin permeation ex vivo.

Cosmetic hair removal procedures are everyday routines in our society. However, it is unclear if such routines lead to increased uptake of applied substances such as drugs or formulation compounds, potentially resulting in skin irritation or sensitization. The aim of this study was to elucidate the effect of common depilation and epilation methods on skin penetration of two surfactants and four model drugs of different physicochemical properties using the porcine ear model. It should be elucidated whether the substances' skin penetration behavior would be affected by hair removal procedures and if potential effects would be related to their polarity. Confocal Raman spectroscopy revealed no effect of hair removal on total penetration depths of SDS and sulfathiazole. Significantly higher relative penetrated amounts within 0-6 µm of stratum corneum depth were found for SDS after dry shaving, depilatory cream and waxing and for sulfathiazole after all depilation methods and partly after epilation. ATR-FTIR spectroscopy revealed no effect of hair removal on the penetration depth of lecithin LPC80, but higher relative amounts at the skin surface after wet shaving and electric epilation. Diffusion cell experiments using a lecithin-based microemulsion as carrier system for fluconazole, fludrocortisone acetate and flufenamic acid showed higher cumulative amounts, higher drug fluxes and shorter lag times for the more lipophilic drugs for some of the methods, but only shorter lag times in some cases for fluconazole. In summary, the observed effects appeared to depend on drug polarity and experimental setup.

Redox-cycling and intercalating properties of novel mixed copper(II) complexes with non-steroidal anti-inflammatory drugs tolfenamic, mefenamic and flufenamic acids and phenanthroline functionality: Structure, SOD-mimetic activity, interaction with albumin, DNA damage study and anticancer activity.

Copper(II) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) have been the subject of many research papers and reviews. Here we report the synthesis, spectroscopic study and biological activity of novel mixed copper(II) complexes with NSAIDs: tolfenamic (tolf), mefenamic (mef) and flufenamic (fluf) acids and phenanthroline (phen): [Cu(tolf-O,O')2(phen)] (1), [Cu(mef-O,O')2(phen)] (2), [Cu(fluf-O,O')2(phen)] (3). Complexes were characterized by X-ray analysis and EPR spectroscopy. Complexes 1-3 are monomeric, six-coordinate and crystallize in a monoclinic space group. Interaction of Cu(II) complexes with DNA was studied by means of absorption titrations, viscosity measurements and gel electrophoresis. The relative ability of the complexes to cleave DNA even in the absence of hydrogen peroxide is in the order 3 > 2 > 1. Application of the reactive oxygen species (ROS) scavengers, L-histidine, DMSO and SOD confirmed that singlet oxygen, hydroxyl radicals (Fenton reaction) and superoxide radical were formed, respectively. Thus, in addition to mechanism of intercalation, redox-cycling mechanism which in turn lead to the formation of ROS contribute to DNA damage. Cu(II) complexes exhibit excellent SOD-mimetic activity in the order 3~1 > 2. The fluorescence spectroscopy revealed that albumin may act as a targeted drug delivery vehicle for Cu(II) complexes (K~106). The anticancer activities of complexes 1-3 were investigated using an MTS assay (reduction of the tetrazolium compound) against three cancer cell lines (HT-29 human colon adenocarcinoma, HeLa and T-47D breast cancer cells) and mesenchymal stromal cells (MSC). The most promising compound, from the viewpoint of its NSAID biological activity is 3, due to the presence of the three fluorine atoms participating in the formation of weak hydrogen-bonds at the DNA surface.

Charge compensation for NADPH oxidase activity in microglia in rat brain slices does not involve a proton current.

The membrane properties of isolated cultured microglia have been extensively studied but it is important to understand their properties in situ, where they protect the brain against infection, but also contribute to neurodegenerative diseases. Microglia and macrophages attack bacteria by generating reactive oxygen species, a process which involves NADPH oxidase pumping electrons out across the cell membrane. The resulting inward current evokes a depolarization, which would inhibit the activity of the NADPH oxidase if there were no charge-compensating current which moves positive charge out across the membrane. The mechanism of this charge compensation is controversial. In neutrophils and in cultured microglia a depolarization-activated H(+) conductance has been proposed to provide charge compensation, and also to remove protons generated intracellularly by the NADPH oxidase. Alternatively, a depolarization-activated K(+) conductance has been proposed to mediate charge compensation. Here we show that in microglia, either in the resting state or when activated by the bacterial coat component lipopolysaccharide, both in acute and in cultured hippocampal slices, no significant H(+) current is detectable. This implies that the membrane properties of microglia in their normal cellular environment differ from those of cultured microglia (similarly, microglia generated a current in response to ATP but, unlike in culture, not to glutamate or GABA). Furthermore, the K(+) current (Kv1.3) that is activated by lipopolysaccharide is inactivated by depolarization, making it unsuitable for mediating charge compensation on a long time scale at positive voltages. Instead, charge compensation may be mediated by a previously undescribed non-selective cation current.

Glucuronidation of fenamates: kinetic studies using human kidney cortical microsomes and recombinant UDP-glucuronosyltransferase (UGT) 1A9 and 2B7.

Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis-Menten (MM) kinetics were observed for mefenamic (K(m)(app) 23 microM) and niflumic acid (K(m)(app) 123 microM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CL(int) 17+/-5.5 microL/minmg protein) was fifteen fold higher than that of niflumic acid (CL(int) 1.1+/-0.8 microL/minmg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic (K(m)(app) 48 microM) and niflumic acid (K(m)(app) 135 microM) glucuronidation and atypical kinetics with mefenamic acid. Similarity in K(m)(app) between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic (S(50) 449 microM, h 0.4) and niflumic acid (S(50) 7344 microM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate 'probe' 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney.

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs: flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid.

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H(2)O(2)/Cl(-) system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H(2)O(2)/Cl(-) system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC(50) = 1.1+/-0.3 microM) is the most efficient inhibitor of the MPO/H(2)O(2)/Cl(-) system and nimesulide (IC(50) = 2.1+/-0.3 microM) is more active than the other NSAIDs of the oxicam family (IC(50) = 8-12 microM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H(2)O(2)/Cl(-) system. Flufenamic acid has the best inhibiting activity towards the MPO/H(2)O(2)/Cl(-) system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.

Pharmacokinetics of Transdermal Etofenamate and Diclofenac in Healthy Volunteers.

Little is known about the course of the plasma concentration and the bioavailability of non-steroidal anti-inflammatory drugs (NSAIDs) contained in dermal patches. We compared an etofenamate prototype patch (patent EP 1833471) and a commercially available diclofenac epolamine patch regarding the bioavailability of the active ingredients relative to respective i.m. applications and regarding their plasma concentration-time course. Twenty-four healthy human volunteers were treated using a parallel group design (n = 12 per group) with a single dermal patch (removed after 12 hr) followed (after a latency of 48 hr) by eight consecutive dermal patches every 12 hr to reach steady-state conditions. The patches were generally well tolerated, but one volunteer treated with etofenamate developed an allergic contact dermatitis. After the first patch, Cmax was 0.81 ± 0.11 (mean ± S.E.M.) ng/mL (reached 12 hr after patch removal) for diclofenac and 31.3 ± 3.8 ng/mL for flufenamic acid (reached at patch removal), the main metabolite of etofenamate. Etofenamate was not detectable. After repetitive dosing, trough plasma concentrations after the eighth dose were 1.72 ± 0.32 ng/mL for diclofenac and 48.7 ± 6.6 ng/mL for flufenamic acid. Bioavailabilities (single dose) relative to i.m. applications were 0.22 ± 0.04% for diclofenac and 1.15 ± 0.06% for flufenamic acid. In conclusion, the relative bioavailability (compared to the respective i.m. application) of both drugs is low. The maximal plasma concentrations after topical administration of these drugs are well below the IC50 values for COX-1 and COX-2, explaining the absence of dose-dependent toxicities.

Flufenamic acid protects against intestinal fluid secretion and barrier leakage in a mouse model of Vibrio cholerae infection through NF-κB inhibition and AMPK activation.

Nuclear factor kappa B (NF-κB)-mediated inflammatory responses play crucial roles in the pathogenesis of diarrhea caused by the Vibrio cholerae El Tor variant (EL), which is a major bacterial strain causing recent cholera outbreaks. Flufenamic acid (FFA) has previously been demonstrated to be a potent activator of AMP-activated protein kinase (AMPK), which is a negative regulator of NF-κB signaling. This study aimed to investigate the anti-diarrheal efficacy of FFA in a mouse model of EL infection and to investigate the mechanisms by which FFA activates AMPK in intestinal epithelial cells (IEC). In a mouse closed loop model of EL infection, FFA treatment (20mg/kg) significantly abrogated EL-induced intestinal fluid secretion and barrier disruption. In addition, FFA suppressed NF-κB nuclear translocation and expression of proinflammatory mediators and promoted AMPK phosphorylation in the EL-infected mouse intestine. In T84 cells, FFA induced AMPK activation. Furthermore, FFA promoted tight junction assembly and prevented interferon gamma (IFN-γ)-induced barrier disruption in an AMPK-dependent manner. Biochemical and molecular docking analyses indicated that FFA activates AMPK via a direct stimulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß) activity. Collectively, our data indicate that FFA represents a class of existing drugs that may be of potential utility in the treatment of cholera caused by EL infection via AMPK-mediated suppression of NF-κB signaling in IEC.

TEWL measurements as a routine method for evaluating the integrity of epidermis sheets in static Franz type diffusion cells in vitro. Limitations shown by transport data testing.

The suitability of transepidermal water loss (TEWL) measurements in vitro as a barrier integrity test for human heat separated epidermis (HSE) was investigated. A model system consisting of a Teflon membrane mounted in Franz diffusion cells (FDC) filled with phosphate buffer saline (PBS) was set up. The membrane was used intact and punctured with a needle (up to five holes). After each puncturing the TEWL was measured. Only the TEWL of intact and punctured membrane differed significantly regardless of the number of holes. From three donors intact human HSE and punctured HSE were compared and no significant difference of the TEWL was found. Permeation experiments with flufenamic acid (FFA) showed a significantly higher diffusion rate through punctured HSE. TEWL and drug permeation were compared for skin stripped three, seven and 15 times prior to heat separation to an intact control group. Only the TEWL values of intact HSE and HSE stripped 15 times differed significantly. However, seven and 15 times stripping resulted in significantly higher diffusion rate. In conclusion, TEWL measurements can detect severe damage of the stratum corneum (SC) but not small changes, which nevertheless may already influence drug diffusion. Therefore, TEWL measurements appears to be of limited use as a barrier integrity test for human HSE in in vitro test systems.

Comparison of bovine udder skin with human and porcine skin in percutaneous permeation experiments.

Rat and pig animal skin has been the most common replacement material for human skin for use in in vitro permeability experiments. Unfortunately, the permeability barrier of skin from laboratory animals is known to be relatively weak, due to significant follicular transport. Pig skin has been shown to be a suitable model for human skin. Unfortunately, it cannot be gathered from the regular slaughtering process, which makes it unsuitable for permeation experiments. We therefore studied the suitability of bovine udder skin, an untreated waste material of the butchering process, as a possible replacement material for use in in vitro permeability tests. We investigated the barrier strength of bovine udder skin against four different substances, and its histology and lipid profile, in comparison with pig skin and heat separated human epidermis. Pig and human skin were found to be equally permeable, whilst bovine udder skin seemed to exhibit a weaker, but less variable, barrier against caffeine, benzoic acid, testosterone, and flufenamic acid. The skin of all three species contained variable contents of the major lipid classes: cholesterol, ceramides, cholesterol ester, fatty acids and triglycerides. Morphological differences mainly comprised variations in the density of hair follicles. Based on these results, the amount of free fatty acids and triglycerides and the density of hair follicles seem to be important factors in the differences between the skin barriers in the three species.

Crystal structures of prostaglandin D(2) 11-ketoreductase (AKR1C3) in complex with the nonsteroidal anti-inflammatory drugs flufenamic acid and indomethacin.

It is becoming increasingly well established that nonsteroidal anti-inflammatory drugs (NSAID) protect against tumors of the gastrointestinal tract and that they may also protect against a variety of other tumors. These activities have been widely attributed to the inhibition of cylooxygenases (COX) and, in particular, COX-2. However, several observations have indicated that other targets may be involved. Besides targeting COX, certain NSAID also inhibit enzymes belonging to the aldo-keto reductase (AKR) family, including AKR1C3. We have demonstrated previously that overexpression of AKR1C3 acts to suppress cell differentiation and promote proliferation in myeloid cells. However, this enzyme has a broad tissue distribution and therefore represents a novel candidate for the target of the COX-independent antineoplastic actions of NSAID. Here we report on the X-ray crystal structures of AKR1C3 complexed with the NSAID indomethacin (1.8 A resolution) or flufenamic acid (1.7 A resolution). One molecule of indomethacin is bound in the active site, whereas flufenamic acid binds to both the active site and the beta-hairpin loop, at the opposite end of the central beta-barrel. Two other crystal structures (1.20 and 2.1 A resolution) show acetate bound in the active site occupying the proposed oxyanion hole. The data underline AKR1C3 as a COX-independent target for NSAID and will provide a structural basis for the future development of new cancer therapies with reduced COX-dependent side effects.

Identification of Potential Therapeutics to Conquer Drug Resistance in Salmonella typhimurium: Drug Repurposing Strategy.

BACKGROUND: Salmonella typhimurium is the main cause of gastrointestinal illness in humans, and treatment options are decreasing because drug-resistant strains have emerged. OBJECTIVE: The objective of this study was to use computational drug repurposing to identify a novel candidate with an effective mechanism of action to circumvent the drug resistance. METHODS: We used the Mantra 2.0 database to initially screen drug candidates that share similar gene expression profiles to those of quinolones. Data were further reduced using pharmacophore mapping theory. Finally, we employed molecular-simulation studies to calculate the binding affinity of the screened candidates with DNA gyrase, alongside an analysis of side effects. RESULTS: A total of 16 drug candidates from the Mantra 2.0 database were screened. The pharmacophoric features of the screened candidates were examined and nalidixic acid features compared using the PharamGist program. A total of 11 compounds with the highest pharmacophore score were considered for binding energy calculation. Finally, we analysed the side effects of the eight drug candidates that showed significant binding affinity in the simulation study. CONCLUSION: Overall, flufenamic acid and sulconazole may be potential drug candidates that could be studied in vitro to assess their resistance profile against Salmonella enterica Typhimurium.

The asymmetry of chloride transport at 38 degrees C in human red blood cell membranes.

Band 3-mediated Cl- exchange in human red blood cells and resealed ghosts was measured at 38 degrees C by the continuous flow tube method. When external Cl- concentration, C(o), is varied with constant internal Cl- concentration, C(i), the flux fits a simple Michaelis-Menten saturation curve (MM fit), with K1/2o = 3.8 +/- 0.4 mM. When the Cl- concentration is varied simultaneously at both sides of the membrane in resealed ghosts (C(i) = C(o) = C(i = o)), the flux rises toward a flat maximum between 200 and 450 mM Cl-, and then decreases at very high C(i = o). An MM fit to the data with C(i = o) < 500 mM gives K1/2s of 106 +/- 13 mM; fits including modifier site inhibition (MS fit) give an over threefold higher K1/2s. Despite this uncertainty, the intrinsic asymmetry of unloaded transport sites, A (defined as E(o)/E(i) with C(i) = C(o), where E(i) is the fraction of unloaded inward-facing sites and E(o) is the fraction of unloaded outward-facing sites), calculated from K1/2s and K1/2o, ranges only from 0.046 to 0.107. A new method, which uses the initial slope of a plot of Cl- flux versus C(i = o), gives A values of 0.023 to 0.038. Flufenamic acid (FA) inhibits Cl- exchange by binding to an external site different from the transport site. At 38 degrees C, FA binds 24-36 times more tightly to E(o) than to E(i). Estimates of A from FA inhibitory potency range from 0.01 to 0.05. All methods, including bicarbonate data from the preceding paper, indicate that at 38 degrees C, like 0 degree C, far more band 3 molecules are in the E(i) than in the E(o) form. The agreement of various methods supports the ping-pong model for anion exchange, and demonstrates that the intrinsic asymmetry is very slightly, if at all, affected by temperature.