Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

Dual Farnesoid X Receptor/Soluble Epoxide Hydrolase Modulators Derived from Zafirlukast.

The nuclear farnesoid X receptor (FXR) and the enzyme soluble epoxide hydrolase (sEH) are validated molecular targets to treat metabolic disorders such as non-alcoholic steatohepatitis (NASH). Their simultaneous modulation in vivo has demonstrated a triad of anti-NASH effects and thus may generate synergistic efficacy. Here we report dual FXR activators/sEH inhibitors derived from the anti-asthma drug Zafirlukast. Systematic structural optimization of the scaffold has produced favorable dual potency on FXR and sEH while depleting the original cysteinyl leukotriene receptor antagonism of the lead drug. The resulting polypharmacological activity profile holds promise in the treatment of liver-related metabolic diseases.

The underlying factors that explain why nucleophilic reagents rarely co-elute with test chemicals in the ADRA.

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for skin sensitization potential that uses two different nucleophilic reagents and it is known that ADRA hardly exhibts co-elution compared with the Direct Peptide Reactivity Assay (DPRA) based on the same scientific principles. In this study, we have analyzed the factors underlying why co-elution, which is sometimes an issue during DPRA testing, virtually never occurs during ADRA testing. Chloramine T and dimethyl isophthalate both exhibited co-elution during DPRA testing, but when quantified at both DPRA's 220 nm and ADRA's 281 nm, we found that when the later detection wavelength was used, these test chemicals produced extremely small peaks that did not interfere with quantification of the peptides. And although both salicylic acid and penicillin G exhibited co-elution during DPRA testing, when tested at a concentration just 1% of that used in DPRA, the very broad peak produced at the higher concentration was reduced significantly. However, both these test chemicals exhibited very sharp peaks when the pH of the injection sample was adjusted to be acidic. Based on these results, we were able to clarify that the reasons why nucleophlic reagents hardly co-elute with test chemicals during ADRA testing are depend on the following three major reasons: (1)differences in the detection wavelength, (2)differences in test chemical concentrations in the injection sample, (3)differences in composition of the injection solvent.

Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor.

The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC50 value of 32 µM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC50 of 22 µM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition.

p-Toluenesulfonic Acid-based Deep Eutectic Solvent as Transesterification Catalyst for Biodiesel Production.

In this study, acidic deep eutectic solvents (DESs) have been studied as catalyst in transesterification of soybean oil with methanol to produce biodiesel. p-Toluenesulfonic acid-based deep eutectic solvent (P-DES), composed of p-toluenesulfonic acid and choline chloride (1:3, mol/mol), was found to be an efficient transesterification catalyst for biodiesel production. The optimum conditions for the transesterification process were as follows: 8% P-DES catalyst, 8:1 molar ratio of methanol to oil, reaction temperature of 110°C and 2 h reaction time. The transesterification yield could reach 98.66±0.17%. And the P-DES exhibited as a heterogeneous catalyst after the reaction, which could be separated easily. And P-DES could remove the trace amount of by-product (glycerol) in the final biodiesel product, making this process more simple and cleaner.

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy.

Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.

A cellulose-based bioassay for the colorimetric detection of pathogen DNA.

Cellulose-paper-based colorimetric bioassays may be used at the point of sampling without sophisticated equipment. This study reports the development of a colorimetric bioassay based on cellulose that can detect pathogen DNA. The detection was based on covalently attached single-stranded DNA probes and visual analysis. A cellulose surface functionalized with tosyl groups was prepared by the N,N-dimethylacetamide-lithium chloride method. Tosylation of cellulose was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and elemental analysis. Sulfhydryl-modified oligonucleotide probes complementary to a segment of the DNA sequence IS6110 of Mycobacterium tuberculosis were covalently immobilized on the tosylated cellulose. On hybridization of biotin-labelled DNA oligonucleotides with these probes, a colorimetric signal was obtained with streptavidin-conjugated horseradish peroxidase catalysing the oxidation of tetramethylbenzamidine by H2O2. The colour intensity was significantly reduced when the bioassay was subjected to DNA oligonucleotide of randomized base composition. Initial experiments have shown a sensitivity of 0.1 µM. A high probe immobilization efficiency (more than 90 %) was observed with a detection limit of 0.1 µM, corresponding to an absolute amount of 10 pmol. The detection of M. tuberculosis DNA was demonstrated using this technique coupled with PCR for biotinylation of the DNA. This work shows the potential use of tosylated cellulose as the basis for point-of-sampling bioassays.

In situ dimerization of multiple wild type and mutant zinc transporters in live cells using bimolecular fluorescence complementation.

Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells.

Development and validation of an HPLC-DAD method for quantification of bornesitol in extracts from Hancornia speciosa leaves after derivatization with p-toluenesulfonyl chloride.

Cylitol L-(+)-bornesitol is regarded as a bioactive constituent from Hancornia speciosa leaves, a plant species traditionally used in Brazil to treat diabetes and hypertension. We report a simple HPLC-DAD method for the quantification of bornesitol in extracts from H. speciosa leaves, after derivatization with p-toluenesulfonyl chloride, using pentaerythritol as internal standard. A gradient of methanol, acetonitrile and water was employed for elution on an ODS column and detection was set up at a wavelength of 230 nm. The method was selective and linear over the range 60.4-302.0 µg/ml with r² of 0.9981, and showed satisfactory precision for intra-day (RSD=2.37%) and inter-day (RSD=3.17%) assays. The recovery varied between 92.3% and 99.9% and the limits of quantification and detection were respectively 5.00 and 1.67 µg/ml. The method was applied to quantify bornesitol in extracts from H. speciosa leaves of different specimens.

Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons and their pharmacologies. Part 2: Synthesis of novel triplet drugs with the epoxymethano structure (capped homotriplet).

An improved synthetic method for triplet drugs with the 1,3,5-trioxazatriquinane skeleton was developed that used p-toluenesulfonylmethyl isocyanide (TosMIC) instead of 1,3-dithiane. Using the improved method, we synthesized compounds with two identical pharmacophore units and an epoxymethano group, that is, capped homotriplets. Among the synthesized capped homotriplets, KNT-123 showed high selectivity for the µ receptor over the κ receptor, and the µ selectivity was the highest among the reported µ selective nonpeptide ligands. KNT-123 administered subcutaneously induced a dose-dependent analgesic effect in the acetic acid writhing assay, and its potency was 11-fold more potent than that of morphine. KNT-123 may serve as a useful tool for the study of the pharmacological actions mediated specifically via the µ receptor.

Sorafenib and its tosylate salt: a multikinase inhibitor for treating cancer.

Sorafenib, a drug that targets malignant cancer cells and cuts off the blood supply feeding the tumour, has been crystallized as the free base, 4-(4-{3-[4-chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N-methylpyridine-2-carboxamide, C(21)H(16)ClF(3)N(4)O(3), (I), and as a tosylate salt, 4-(4-{3-[4-chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-2-(N-methylcarbamoyl)pyridinium 4-methylbenzenesulfonate, C(21)H(17)ClF(3)N(4)O(3)(+)·C(7)H(7)O(3)S(-), (II). In both structures, the sorafenib molecule is in an extended conformation. The pyridine-2-carboxamide group exhibits a syn conformation of the N atoms in (I), whereas an almost anti orientation is present in (II). In both crystal structures, the two terminal groups, viz. pyridine-2-carboxamide and the trifluorophenyl ring, are oriented differently to the conformations found in enzyme-bound sorafenib. The sorafenib molecules in (I) are linked into zigzag chains by N-H···O hydrogen bonds, whereas in (II) the presence of the additional tosylate anion results in the formation of chains of fused hydrogen-bonded rings. This study reveals the variations in the solid-state conformation of the sorafenib molecule in different crystalline environments.

Polyphenol-rich beverages enhance zinc uptake and metallothionein expression in Caco-2 cells.

The effect of red wine (RW), red grape juice (RGJ), green tea (GT), and representative polyphenols on Caco-2 cell (65)Zn uptake was explored. RW, RGJ, and GT enhanced the uptake of zinc from rice matrix. Fractionation of RW revealed that enhancing activity of zinc uptake was exclusively resided in the polyphenol fraction. Among the polyphenols tested, only tannic acid and quercitin stimulated the uptake of zinc while others did not influence the uptake. In tune with these results, only tannic acid and quercitin competed with zinquin (a zinc selective fluorophore) for zinc in vitro. Although all the polyphenols tested appear to enhance the expression of metallothionein (MT), the induction was higher with tannic acid, quercitin, and RW extract. Furthermore, phytic acid abrogated the tannic acid-induced MT expression. These results suggest that polyphenol-rich beverages, tannic acid, and quercitin bind and stimulate the zinc uptake and MT expression in Caco-2 cells.

Al(OTf)(3)-mediated epoxide ring-opening reactions: toward piperazine-derived physiologically active products.

Al(OTf)(3) is a good catalyst for the ring opening of epoxides, forming beta-amino alcohols bearing the piperazine motif. Two different strategies were examined, where the glycidyl ether resided on one-half of the molecule or the other, allowing insight into a best-case approach for the ring-opening step. Each half of the molecule contained an heteroatom that could be used either to attach the glycidyl moiety or as the nucleophile in the ring-opening reaction, for the same set of reagents, allowing this approach.

Computationally identified novel diphenyl- and phenylpyridine androgen receptor antagonist structures.

We have identified and profiled a set of androgen receptor (AR) binding compounds representing two nonsteroidal scaffolds from a public chemical database supplied by Asinex with virtual screening procedure incorporating our recently published 3D QSAR model of AR ligands. The diphenyl- and phenylpyridine-based compounds act as antagonists in wild-type AR in CV1 cells and also retain this antagonistic character in CV1 cells expressing T877A mutant receptor. This mutation is frequently associated with prostate cancer. Two of the compounds repress the androgen-dependent cell growth of LNCaP prostate cancer cells expressing the T877A AR mutant. Molecular modeling of the observed in vitro antagonism with induced fit docking suggests that W741 and M895 could be mechanistically involved in the initiation of the antagonism. The results indicate finding of nonsteroidal AR antagonist compounds from a public chemical database with computational methods. Compounds could serve as a novel platform to develop more potent AR antagonists with inhibitory activity in both wild-type and T877A mutant AR.

Determination of the global fibrinolytic state.

Fibrinolysis consists of a plasmatic part and a cellular part. A rapid global assay for plasmatic fibrinolysis is the fibrinolysis parameters assay (FIPA). Cellular fibrinolysis is measured by testing the clot lysis capacity using the microtitre plate clot lysis assay with polymorphonuclear neutrophils (CLA-PMN). Individual citrated plasma or pooled normal plasma (50 microl) of 232 patients was recalcified, incubated for 90 min at 37 degrees C, oxidized with 0 or 1.5 mmol/l (final concentration) chloramine-T, and supplemented with 50 microl respective polymorphonuclear neutrophil plasma. The turbidity of the clots was measured at 405 nm after 12 h and 60 h (37 degrees C). Plasma (50 microl) was also incubated with 5 microl of 100 IU/ml urokinase, 6 mmol/l tranexamic acid, 6% human albumin for 10 min (37 degrees C). Then 100 microl of 0.5 mmol/l Val-Leu-Lys-pNA in 2.45 mol/l arginine, pH 8.6, was added and the increase in absorbance with time was measured. The different CLA-PMN assay versions correlated with each other with r = 0.543-0.782. Cellular fibrinolysis (34 +/- 30% lysis; normal: 25 +/- 10%) did not correlate with the FIPA (72 +/- 27%; normal: 100 +/- 15%), prothrombin time, activated partial thromboplastin time, fibrinogen, C-reactive protein, or the blood counts of thrombocytes, leukocytes, or polymorphonuclear neutrophils. Chloramine (1.5 mmol/l) oxidation of the microclots favours their fibrinolytic breakdown, especially if lysis-resistant microclots are oxidized. The FIPA and CLA-PMN are new economical tests for the fibrinolytic state in patient blood.

Bactericidal and cytotoxic effects of chloramine-T on wound pathogens and human fibroblasts in vitro.

OBJECTIVES: To evaluate cytotoxicity and bactericidal effects of chloramine-T. METHODS: In vitro study of various concentrations and exposure times to preparations containing human fibroblasts or 1.5 x 10 colony forming units per milliliter (CFU/mL) of 3 gram-positive bacteria-Staphylococcus aureus, methicillin-resistant S aureus, and vancomycin-resistant Enterococcus faecalis-and 2 gram-negative bacteria-Escherichia coli and Pseudomonas aeruginosa-with and without fetal bovine serum present. MAIN OUTCOME MEASURES: Percentage reduction of bacterial growth and percentage of viable fibroblasts 48 hours after exposure. RESULTS: All gram-positive growth was reduced by 95% to 100%, regardless of dose, with or without serum. E coli (gram-negative; with/without serum) was reduced 94% to 100% at antiseptic concentrations of 300 and 400 ppm. At 200 ppm, E coli growth was fully inhibited without serum present and by 50% with serum. P aeruginosa (gram-negative) was not significantly affected under any conditions. At 100 and 200 ppm, cell viability remained greater than 90% under all experimental conditions. A 300-ppm, 3-minute exposure to chloramine-T resulted in cell viability of up to 70%, with longer exposures producing lower viabilities. Serum did not affect cell viability in any condition. CONCLUSIONS: In vitro, chloramine-T at 200 ppm for 5 to 20 minutes was effective against 3 virulent gram-positive bacteria without fibroblast damage. At 300 ppm and 3 and 5 minutes, 30% of fibroblasts were damaged and 95% to 100 % of E coli were inhibited, respectively.

The ZIP7 gene (Slc39a7) encodes a zinc transporter involved in zinc homeostasis of the Golgi apparatus.

It has been suggested that ZIP7 (Ke4, Slc39a7) belongs to the ZIP family of zinc transporters. Transient expression of the V5-tagged human ZIP7 fusion protein in CHO cells led to elevation of the cytoplasmic zinc level. However, the precise function of ZIP7 in cellular zinc homeostasis is not clear. Here we report that the ZIP7 gene is ubiquitously expressed in human and mouse tissues. The endogenous ZIP7 was associated with the Golgi apparatus and was capable of transporting zinc from the Golgi apparatus into the cytoplasm of the cell. Moreover, by using the yeast mutant strain Deltazrt3 that was defective in release of stored zinc from vacuoles, we found that ZIP7 was able to decrease the level of accumulated zinc and in the meantime to increase the nuclear/cytoplasmic labile zinc level in the ZIP7-expressing zrt3 mutant. We showed that the protein expression of ZIP7 was repressed under zinc-rich condition, whereas there were no effects of zinc on ZIP7 gene expression and intracellular localization. Neither did zinc deficiency affect the intracellular distribution of ZIP7 in mammalian cells. Our study demonstrates that ZIP7 is a functional zinc transporter that acts by transporting zinc from the Golgi apparatus to the cytoplasm of the cell.

Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution.

Leukotriene modifiers: novel therapeutic opportunities in asthma.

Cysteinyl leukotrienes (Cys-LT) are powerful proinflammatory autacoids that cause long-lasting bronchoconstriction, plasma leakage, increased mucus production; their biological activity suggests a prominent role in the etiopathology of asthma and several Cys-LT receptor antagonists and synthetase inhibitors have been developed as new antiasthmatic drugs. Zafirlukast was discovered by a mechanism-based approach to drug discovery; early structure-activity relationship analyses of the prototype SRS-A antagonist FPL-55712, lead to the identification of an indole-containing lead compound that was more specific than FPL-55712. Modifications were made on the lipid-like tail, indole backbone and acidic head region of this lead compound, resulting in potent and selective leukotriene receptor antagonists such as ICI-198615 and 204219 (zafirlukast). On the basis of successful results in preclinical asthma models, zafirlukast was recommended for clinical development and became the first leukotriene-modifier to be approved for the treatment of asthma. Leukotriene biosynthesis inhibitors (LSI) also represent a promising approach to the treatment of asthma and may theoretically provide a broader protection than Cys-LT receptor antagonists by inhibition of the synthesis of the two major leukotrienes, the Cys-LT and the chemotactic LTB4. The LSI BAY X-1005 is the result of a broad chemistry program that identified 15-HETE as an endogenous inhibitor of leukotriene synthesis and REV 5901 as a lead prototypic quinoline-based 5-lipoxygenase (5-LO) inhibitor. Clinical studies demonstrated the effectiveness of BAY X-1005 in experimental conditions such as allergen provocation and cold-air induced asthma. However, no consistent treatment effect in the overall asthma population (mild to moderately severe asthmatics) lead to discontinuation of its development.