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Occupancy of prostate cancer (PCa) cell androgen receptors (AR) signals proliferation, therefore testosterone biosynthesis inhibitors and AR antagonists are important PCa treatments. Conversely, androgen mimics (e.g., prednisone) used in management of PCa might cause proliferation. The balance between PCa proliferation and inhibition predicts treatment success. We used in silico molecular modelling to explore interactions between ARs, androgens (testosterone, dihydrotestosterone (DHT)) and drugs used to treat (bicalutamide) and manage (dexamethasone, prednisone, hydrocortisone) PCa. We found that hydrogen (H-) bonds between testosterone, DHT and Arg752, Asn705 and Thr877 followed by ligand binding cleft hydrophobic interactions signal proliferation, whereas bicalutamide antagonism is via Phe764 interactions. Hydrocortisone, dexamethasone and prednisone H-bond Asn705 and Thr877, but not Arg752 in the absence of a water molecule. Studies with a bicalutamide agonist AR mutation showed different amino acid interactions, indicating testosterone and DHT would not promote proliferation as effectively as via the native receptor. However, hydrocortisone and bicalutamide form Arg752 and Asn705 H-bonds indicating agonism. Our results suggest that as PCa progresses the resulting mutations will change the proliferative response to androgens and their drug mimics, which have implications for the treatment of prostate cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The pharmaceutical industry has to tackle the explosion of high amounts of poorly soluble APIs. This phenomenon leads to numerous sophisticated solutions. These include the use of multifactorial data analysis identifying correlations between the components and dosage form properties, laboratory and production process parameters with respect to the API liberation Example of such API is bicalutamide. Improved liberation is achieved by particle size reduction. Laboratory batches, with different PSD of API, were filled into gelatinous capsules and consequently granulated for tablet compression. Comparative dissolution profiles with Casodex 150 mg (Astra Zeneca) were performed. The component analysis was used for the statistical evaluation of f 1 and f 2 factors and D(v,0.9) and D[4,3] parameters of PSD to identify optimal PSD values. Suitable PSD limits for API were statistically confirmed in laboratory and in commercial scale with respect to optimized tablet properties. The tablets were bioequivalent with originator (n = 20; 90% CI for ln AUC 0-120 : 99.8-111.9%; 90% CI for ln c max : 101.1-112.9%). In conclusion, the micronisation of the API is still an efficient and inexpensive method improving the bioavailability, although there are more complicated and expensive methods available. Statistical multifactorial methods improved the safety and reproducibility of production.

A library of thirty N-substituted tosyl N'-acryl-hydrazones was prepared with p-toluenesulfonyl hydrazide, methyl propiolate and different aldehydes in a one-pot synthesis via an aza-Michael reaction. The scope of the reaction was studied, including aliphatic, isoprenylic, aromatic and carbocyclic aldehydes. The prepared collection was tested against Mycobacterium tuberculosis H37Rv. Nine analogs of the collection showed Minimum Inhibitory Concentration ≤10 μM, of which the most active members (MIC of 1.25 μM) were exclusively E isomers. In order to validate the mechanism of action of the most active acrylates, we tested their activity on a M. tuberculosis InhA over-expressing strain obtaining MIC that consistently doubled those obtained on the wild type strain. Additionally, the binding mode of those analogs on M. tuberculosis InhA was investigated by docking simulations. The results displayed a hydrogen bond interaction between the sulfonamide and Ile194 and the carbonyl of the methyl ester with Tyr 158 (both critical residues in the interaction with the fatty acyl chain substrate), where the main differences on the binding mode relays on the hydrophobicity of the nitrogen substituent. Additionally, chemoinformatic analysis was performed to evaluate in silico possible cytotoxicity risk and ADME-Tox profile. Based on their simple preparation and interesting antimycobacterial activity profile, the newly prepared aza-acrylates are promising candidates for antitubercular drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

BACKGROUND Zafirlukast is an antagonist of cysteinyl leukotriene receptor 1 (CysLTR1). Advanced glycation end-products (AGEs) are formed by the glycation of lipids and proteins in hyperglycemia, including diabetes mellitus. Zafirlukast has not previously been studied in diabetic nephropathy. This study aimed to investigate the effects of zafirlukast on rat renal mesangial cells cultured with AGEs in vitro. MATERIAL AND METHODS Mesangial cells were cultured in AGEs (0, 20, 50, 100 μg/ml), and with AGEs (100 μg/ml) and zafirlukast (2.5 μm, 5 μm, and 100 μm). An enzyme-linked immunoassay (ELISA) was used to measure the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß (IL-1ß), IL-6, and monocyte chemoattractant protein-1 (MCP-1). Reactive oxygen species (ROS) were assessed by intracellular fluorescence measurement of 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and detection kits were used to measure malondialdehyde (MDA), lactate dehydrogenase (LDH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD). Cell apoptosis was assessed by flow cytometry, and Western blot was used to measure protein levels. RESULTS In mesangial cells cultured with AGEs, markers of inflammation, oxidative stress, and apoptosis and levels of CysLTR1 increased, and these effects were reduced by zafirlukast in a dose-dependent manner. The effects of zafirlukast as a CysLTR1 antagonist protected mesangial cells from the effects of AGE in vitro. CONCLUSIONS Zafirlukast, a CysLTR1 antagonist, reduced the levels of inflammatory cytokines, markers of oxidative stress, and cell apoptosis induced by AGE in mesangial cells in a dose-dependent way. Future in vivo studies are needed to investigate the potential role for zafirlukast in models of diabetic nephropathy.

The G protein-coupled cysteinyl leukotriene receptor CysLT 1 R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT 1 R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT 1 R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue-coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Targeted delivery of clinically approved anticancer drug to tumor sites is an effective way to achieve enhanced drug efficacy as well as reduced side effects and toxicity. Here bicalutamide is caged by the Ru(II) center through the nitrile group, and three photoactive Ru(II) complexes were designed and synthesized. Docking study showed that the ruthenium(II) fragments can effectively block the binding of complexes 1-3 with AR (androgen receptor) owing to the large steric structures, thus bicalutamide in complexes 1-3 could not interact with AR-LBD (ligand binding domain). Once irradiation with blue light (465nm), complexes 1-3 can release bicalutamide and anticancer Ru(II) fragments, which possesses dual-action of AR binding and DNA interaction simultaneously. In vitro cytotoxicity study on these complexes further confirmed that complexes 1-3 exhibited considerable cytotoxicity upon irradiation with blue light. Significantly, complex 3 could be activated at 660nm, which greatly increases the scope of complex 3 to treat deeper within tissue. Theoretical calculations showed that the lowest singlet excitation energy of complex 3 is lower than those of complexes 1-2, which explains the experimental results well. Moreover, the 3 MC (metal centered) states of these complexes are more stable than their 3 MLCT (metal to ligand charge transfer) states, indicating that the photoactive processes of these complexes are likely to result in ligand dissociation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Diabetes is a hyperglycemic condition characterized by pancreatic β-cell dysfunction and depletion. Whereas methods for monitoring β-cell function in vivo exist, methods to deliver therapeutics to β cells are lacking. We leveraged the rare ability of β cells to concentrate zinc to preferentially trap zinc-binding molecules within β cells, resulting in β-cell-targeted compound delivery. We determined that zinc-rich β cells and islets preferentially accumulated TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) in a zinc-dependent manner compared with exocrine pancreas. Next, we asked whether appending a zinc-chelating moiety onto a β-cell replication-inducing compound was sufficient to confer preferential β-cell accumulation and activity. Indeed, the hybrid compound preferentially accumulated within rodent and human islets in a zinc-dependent manner and increased the selectivity of replication-promoting activity toward β cells. These data resolve the fundamental question of whether intracellular accumulation of zinc-chelating compounds is influenced by zinc content. Furthermore, application of this principle yielded a proof-of-concept method for β-cell-targeted drug delivery and bioactivity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hydrogen peroxide (H 2 O 2 ) is one of the reactive oxygen species (ROS), endogenously produced during metabolism, which acts as a second messenger. In skeletal muscles, hypoxia- or hyperthermia-induced increase in H 2 O 2 might affect synaptic transmission by targeting the most redox-sensitive presynaptic compartment (Giniatullin et al., 2006). However, the effects of H 2 O 2 as a signal molecule have not previously been studied in different patterns of the synaptic activity. Here, using optical and microelectrode recording of synaptic vesicle exocytosis, we studied the use-dependent action of low concentrations of H 2 O 2 and other oxidants in the mouse neuromuscular junction. We found that: (i) H 2 O 2 at low micromole concentrations inhibited both spontaneous and evoked transmitter releases from the motor nerve terminals in a use-dependent manner, (ii) the antioxidant N-acetylcysteine (NAC) eliminated these depressant effects, (iii) the influence of H 2 O 2 was not associated with lipid oxidation suggesting a pure signaling action, (iv) the intracellular oxidant Chloramine-T or (v) the glutathione depletion produced similar to H 2 O 2 depressant effects. Taken together, our data revealed the effective inhibition of neurotransmitter release by ROS, which was proportional to the intensity of synaptic activity at the neuromuscular junction. The combination of various oxidants suggested an intracellular location for redox-sensitive sites responsible for modulation of the synaptic transmission in the skeletal muscle., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Bicalutamide (BCT), a drug used in the treatment of prostate cancer, antagonises the actions of androgens, at the receptor level, thereby inhibiting the growth of prostate tumours. Alpha-2-macroglobulin (α 2 M), a pan-proteinase inhibitor, inhibits proteinase, regardless of specificity and catalytic mechanism. α 2 M is deficient in patients of advanced prostate cancer with bone metastases. Our studies explored the interaction of BCT with α 2 M and analysed the BCT induced structural alteration to the α 2 M. The result suggests that BCT decreases the antiproteolytic potential and causes structural and functional change in human α 2 M. UV-visible absorption spectroscopy confirms the formation of α 2 M-BCT complex. Fluorescence analysis shows significant quenching in fluorescence intensity of α 2 M upon binding with BCT. Synchronous fluorescence result suggests the interaction of BCT with α 2 M changed the microenvironment around tyrosine residues. Secondary structure of α 2 M also undergoes a slight change upon complexation with the drug as evident by shift in negative ellipticity in far UV CD spectroscopy. FTIR results confirm the alteration in secondary structure of α 2 M upon drug interaction. Molecular docking studies show that BCT bind to a monomer of α 2 M primarily through hydrophobic force. Thermodynamics parameters were determined by isothermal titration calorimetry found that the binding was exothermic in nature., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Advanced technologies for controlled cell adhesion and detachment in novel biointerface designs profit from stimuli-responsive systems that are able to react to their environment. Here, a multilayer system made of thiolated chitosan and thiolated chondroitin sulfate was constructed, with the potential of switchable inter- and intramolecular thiol/disulfide interactions representing a redox-sensitive nanoplatform. Owing to the formation and cleavage of inherent disulfide bonds by oxidation and reduction, surface properties of the multilayer can be controlled toward protein adsorption/desorption and cell adhesion in a reversible manner. Oxidation of thiols by chloramine-T promotes fibronectin (FN) adsorption and fibroblast cell adhesion, whereas the reduction by tris(2-carboxyethyl)phosphine reverses these effects, leading to low FN adsorption and little cell adhesion and spreading. These effects on the biological systems are related to significant changes of wetting properties, zeta potential, and mechanical properties of these multilayer films. The system presented may be useful for biomedical applications as responsive and obedient surfaces in medical implants and support tissue regeneration.

Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents.

The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5-10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.

Besides their development for therapeutic purposes, non-steroidal selective androgen receptor modulators (non-steroidal SARMs) are also known to impact growth-associated pathways as ligands of androgenic receptors (AR). They present a potential for abuse in sports and food-producing animals as an interesting alternative to anabolic androgenic steroids (AAS). These compounds are easily available and could therefore be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies should be developed for specific and sensitive detection of these compounds in biological matrices. The present study focused on Bicalutamide, a non-steroidal SARM used in human treatment of non-metastatic prostate cancer because of its anti-androgenic activity exhibiting no anti-anabolic effects. To select the most appropriate matrix to be used for control purposes, different animal matrices (urine and faeces) have been investigated and SARM metabolism studied to highlight relevant metabolites of such treatments and establish associated detection time windows. The aim of this work was thus to compare the urinary and faecal eliminations of bicalutamide in a calf, and investigate phase I and II metabolites. The results in both matrices showed that bicalutamide was very rapidly and mainly excreted under its free form. The concentration levels were observed as higher in faeces (ppm) than urine (ppb); although both matrices were assessed as suitable for residue control. The metabolites found were consistent with hydroxylation (phase I reaction) combined or not with glucuronidation and sulfation (phase II reactions). Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Nitric oxide (NO) is both an important regulatory molecule in biological systems and a toxic xenobiotic. Its oxidation products react with sulfhydryl groups and either nitrosylate or oxidize them. The aerobic reaction of NO supplied by diethylamine NONOate (DEA-NO) with pig kidney LLC-PK 1 cells and Zn-proteins within the isolated proteome was examined with three fluorescent zinc sensors, zinquin (ZQ), TSQ, and FluoZin-3 (FZ-3). Observations of Zn 2+ labilization from Zn-proteins depended on the specific sensor used. Upon cellular exposure to DEA-NO, ZQ sequestered about 13% of the proteomic Zn 2+ as Zn(ZQ) 2 and additional Zn 2+ as proteome·Zn-ZQ ternary complexes. TSQ, a sensor structurally related to ZQ with lower affinity for Zn 2+ , did not form Zn(TSQ) 2 . Instead, Zn 2+ mobilized by DEA-NO was exclusively bound as proteome·Zn-TSQ adducts. Analogous reactions of proteome with ZQ or TSQ in vitro displayed qualitatively similar products. Titration of native proteome with Zn 2+ in the presence of ZQ resulted in the sole formation of proteome·Zn-ZQ species. This result suggested that sulfhydryl groups are involved in non-specific proteomic binding of mobile Zn 2+ and that the appearance of Zn(ZQ) 2 after exposure of cells and proteome to DEA-NO resulted from a reduction in proteomic sulfhydryl ligands, favoring the formation of Zn(ZQ) 2 instead of proteome·Zn-ZQ. With the third sensor, FluoZin-3, neither Zn-FZ-3 nor proteome·Zn-FZ-3 was detected during the reaction of proteome with DEA-NO. Instead, it reacted independently with DEA-NO with a modest enhancement of fluorescence.

Prostate cancer (PC) is one of the major causes of male death worldwide and the development of new and more potent anti-PC compounds is a constant requirement. Among the current treatments, (R)-bicalutamide and enzalutamide are non-steroidal androgen receptor antagonist drugs approved also in the case of castration-resistant forms. Both these drugs present a moderate antiproliferative activity and their use is limited due to the development of resistant mutants of their biological target. Insertion of fluorinated and perfluorinated groups in biologically active compounds is a current trend in medicinal chemistry, applied to improve their efficacy and stability profiles. As a means to obtain such effects, different modifications with perfluoro groups were rationally designed on the bicalutamide and enzalutamide structures, leading to the synthesis of a series of new antiproliferative compounds. Several new analogues displayed improved in vitro activity towards four different prostate cancer cell lines, while maintaining full AR antagonism and therefore representing promising leads for further development. Furthermore, a series of molecular modelling studies were performed on the AR antagonist conformation, providing useful insights on potential protein-ligand interactions., (Copyright © 2016 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186.

Inhibition of drug metabolizing enzymes is a major mechanism in drug-drug interactions (DDIs). A number of cases of DDIs via inhibition of UDP-glucuronosyltranseferases (UGTs) have been reported, although the changes in pharmacokinetics are relatively small in comparison with drugs that are metabolized by cytochrome P450s. Most of the past studies have investigated hepatic UGTs, although recent studies have revealed a significant contribution of UGTs in the small intestine to drug clearance. To evaluate potential DDIs caused by inhibition of intestinal UGTs, we assessed inhibitory effects of 578 compounds, including drugs, xenobiotics, and endobiotics, on human UGT1A8 and UGT1A10, which are major contributors to intestinal glucuronidation. We identified 29 inhibitors by monitoring raloxifene glucuronidation with recombinant UGTs. All of the inhibitors potently inhibited UGT1A1 activity, as well. We found that zafirlukast is a potent general inhibitor of UGT1As and a moderate inhibitor of UGT2Bs because it monitors 4-methylumbelliferone glucuronidation by recombinant UGTs. However, zafirlukast did not potently inhibit diclofenac glucuronidation, suggesting that the inhibitory effects might be substrate specific. Inhibitory effects of zafirlukast on some UGT substrates were further investigated in human liver and human small intestine microsomes in order to evaluate potential DDIs. The R values (the ratios of intrinsic clearance with and without an inhibitor) revealed that zafirlukast has potential to cause clinical DDIs in the small intestine. Although we could not identify specific UGT1A8 and UGT1A10 inhibitors, zafirlukast was identified as a general inhibitor for UGTs in vitro. The present study suggests that the inhibition of UGT in the small intestine would be an underlying mechanism for DDIs., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Bicalutamide (BCM) is an anti-androgen drug used to treat prostate cancer. In this study, nanostructured lipid carriers (NLCs) were chosen as a carrier for delivery of BCM using Box-Behnken (BB) design for optimizing various quality attributes such as particle size and entrapment efficiency which is very critical for efficient drug delivery and high therapeutic efficacy. Stability of formulated NLCs was assessed with respect to storage stability, pH stability, hemolysis, protein stability, serum protein stability and accelerated stability. Hot high-pressure homogenizer was utilized for formulation of BCM-loaded NLCs. In BB response surface methodology, total lipid, % liquid lipid and % soya lecithin was selected as independent variable and particle size and %EE as dependent variables. Scanning electron microscopy (SEM) was done for morphological study of NLCs. Differential scanning calorimeter and X-ray diffraction study were used to study crystalline and amorphous behavior. Analysis of design space showed that process was robust with the particle size less than 200 nm and EE up to 78%. Results of stability studies showed stability of carrier in various storage conditions and in different pH condition. From all the above study, it can be concluded that NLCs may be suitable carrier for the delivery of BCM with respect to stability and quality attributes.

Introducing nongenetically encoded, synthetic probes into specific proteins is now recognized as a key component in chemical biology. In particular, the ability to chemically modify specific "native" proteins in various contexts from in vitro to cellular systems is of fundamental importance to study biological systems. We developed a protein-labeling technique termed ligand-directed tosyl (LDT) chemistry for this purpose. This method is capable of labeling specific native proteins with diverse synthetic probes with high site specificity and target selectivity without compromising protein function. Here we describe the principle of the LDT chemistry and the protocol for selective chemical labeling of native carbonic anhydrase in vitro, in blood cells (ex vivo), and in living mice (in vivo).

The binding of some novel bicalutamide analogues to human serum albumin (HSA) and rat serum albumin (RSA) was investigated by surface plasmon resonance (SPR) based optical biosensor technique. The serum protein binding of the bicalutamide analogues was determined and compared to that of the parent compound. Furthermore, HSA and RSA were used as target plasma proteins, in order to highlight possible differences among species when performing pharmacokinetic studies. HSA and RSA were covalently immobilized on carboxymethyl dextran matrixes, using an amine coupling procedure. The anchoring method was validated by determining the dissociation constant (KD) of a standard analyte to confirm that the binding properties of the proteins were maintained. The ranking of the bicalutamide analogues for their HSA and RSA bound fractions was used to compare the behaviour of the two albumins. Most of the bicalutamide analogues showed higher binding levels with respect to the lead compound, (R)-bicalutamide. Further, meaningful differences in the binding level to the two serum proteins were obtained. The dissociation constants (KD) of the interaction between the lead compound, (R)-bicalutamide, and the two proteins were calculated. As a result, the KD obtained with HSA was one order of magnitude higher than that obtained with RSA. The observed differences in the HSA and RSA bonding of the bicalutamide analogues increase the knowledge on the possible low reliability in extrapolating the distribution data obtained on animals to humans. This work demonstrates that SPR based optical biosensor technique is well suited for the medium-high throughput screening of compounds' ligand binding to serum albumins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bicalutamide (Casodex®) is a non-steroidal anti-androgen drug used in the treatment of prostate cancer, which represents the second most common malignancy diagnosed in men worldwide. In this work, we analyze the ability of some novel bicalutamide analogs to bind the androgen receptor, by using an optical biosensor. Androgen receptor was covalently immobilized on a carboxy methyl dextran matrix. The immobilized receptor chip was then used for the binding experiments of the bicalutamide analogs. The (R)-bicalutamide dissociation constant was in good agreement to the value reported in literature obtained by using radiolabeled targets. Most of the new synthesized compounds showed higher androgen receptor binding level, when compared to the reference. Our results clearly indicate that the surface plasmon resonance (SPR) technique offers many advantages with respect to other available technologies in terms of studying biomolecular interactions. Moreover, this study provides an effective methodology for determining the binding affinity of novel chemical entities for the isolated androgen receptor, thus excluding possible off-target interactions occurring in conventional cell-based techniques., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Prostate cancer growth depends on androgens. Synthetic antiandrogens are used in the cancer treatment. However, antiandrogens, such as bicalutamide (BIC), have a mixed agonist/antagonist activity. Here we compare the antiandrogenic capacity of BIC to a new antiandrogen, MDV3100 (MDV) or Enzalutamide™. By reconstitution of a hormone-regulated enhancer in Xenopus oocytes we show that both antagonists trigger the androgen receptor (AR) translocation to the nucleus, albeit with a reduced efficiency for MDV. Once in the nucleus, both AR-antagonist complexes can bind sequence specifically to DNA in vivo. The forkhead box transcription factor A (FoxA1) is a negative prognostic indicator for prostate cancer disease. FoxA1 expression presets the enhancer chromatin and makes the DNA more accessible for AR binding. In this context the BIC-AR antiandrogenic effect is seriously compromised as demonstrated by a significant chromatin remodeling and induction of a robust MMTV transcription whereas the MDV-AR complex displays a more persistent antagonistic character., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry (ToF-MS) and triple quadrupole mass spectrometry (QqQ-MS). Several metabolites were identified and results were compared with literature data on metabolism using a human cell line. Monohydroxylation, nitro-reduction, dephenylation and demethylation were the main S9 in vitro metabolic routes established. Next, an in vivo study was performed by oral administration of the arylpropionamide ostarine to a male calf and urine samples were analysed with UPLC-QToF-MS. Apart from two metabolites resulting from hydroxylation and dephenylation that were also observed in the in vitro study, the bovine in vivo metabolites of ostarine resulted in glucuronidation, sulfation and carboxylation, combined with either a hydroxylation or a dephenylation step. As the intact mother compounds of all SARMs tested are the main compounds present after in vitro incubations, and ostarine is still clearly present in the urine after the in vivo metabolism study in veal calves, the intact mother molecules were selected as the indicator to reveal treatment. The analytical UPLC-QqQ-MS/MS procedure was validated for three commercially available arylpropionamides according to European Union criteria (Commission Decision 2002/657/EC), and resulted in decision limits ranging from 0.025 to 0.05 µg l⁻¹ and a detection capability of 0.025 µg l⁻¹ in all cases. Adequate precision and intra-laboratory reproducibility (relative standard deviation below 20%) were obtained for all SARMs and the linearity was 0.999 for all compounds. This newly developed method is sensitive and robust, and therefore useful for confirmation and quantification of SARMs in bovine urine samples for residue control programmes and research purposes.

KFase (kynurenine formamidase), also known as arylformamidase and formylkynurenine formamidase, efficiently catalyses the hydrolysis of NFK (N-formyl-L-kynurenine) to kynurenine. KFase is the second enzyme in the kynurenine pathway of tryptophan metabolism. A number of intermediates formed in the kynurenine pathway are biologically active and implicated in an assortment of medical conditions, including cancer, schizophrenia and neurodegenerative diseases. Consequently, enzymes involved in the kynurenine pathway have been considered potential regulatory targets. In the present study, we report, for the first time, the biochemical characterization and crystal structures of Drosophila melanogaster KFase conjugated with an inhibitor, PMSF. The protein architecture of KFase reveals that it belongs to the α/β hydrolase fold family. The PMSF-binding information of the solved conjugated crystal structure was used to obtain a KFase and NFK complex using molecular docking. The complex is useful for understanding the catalytic mechanism of KFase. The present study provides a molecular basis for future efforts in maintaining or regulating kynurenine metabolism through the molecular and biochemical regulation of KFase.

Autoimmune synaptic encephalitides are recently described human brain diseases leading to psychiatric and neurological syndromes through inappropriate brain-autoantibody interactions. The most frequent synaptic autoimmune encephalitis is associated with autoantibodies against extracellular domains of the glutamatergic N-methyl-d-aspartate receptor, with patients developing psychotic and neurological symptoms in an autoantibody titre-dependent manner. Although N-methyl-d-aspartate receptors are the primary target of these antibodies, the cellular and molecular pathway(s) that rapidly lead to N-methyl-d-aspartate receptor dysfunction remain poorly understood. In this report, we used a unique combination of high-resolution nanoparticle and bulk live imaging approaches to demonstrate that anti-N-methyl-d-aspartate receptor autoantibodies from patients with encephalitis strongly alter, in a time-dependent manner, the surface content and trafficking of GluN2-NMDA receptor subtypes. Autoantibodies laterally displaced surface GluN2A-NMDA receptors out of synapses and completely blocked synaptic plasticity. This loss of extrasynaptic and synaptic N-methyl-d-aspartate receptor is prevented both in vitro and in vivo, by the activation of EPHB2 receptors. Indeed, the anti-N-methyl-d-aspartate receptor autoantibodies weaken the interaction between the extracellular domains of the N-methyl-d-aspartate and Ephrin-B2 receptors. Together, we demonstrate that the anti-N-methyl-d-aspartate receptor autoantibodies from patients with encephalitis alter the dynamic retention of synaptic N-methyl-d-aspartate receptor through extracellular domain-dependent mechanism(s), shedding new light on the pathology of the neurological and psychiatric disorders observed in these patients and opening possible new therapeutic strategies.

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 μM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 μM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.

In the brain, free zinc levels are under the exquisite control of a variety of zinc-regulating systems, in which zinc transporter (ZnT) proteins play a central role. ZnT3, which is prominently expressed in the brain, facilitates the concentration of free zinc in pre-synaptic vesicles. In addition to histochemical staining methods, a variety of zinc-specific fluorescence dyes has been developed to image or analyze zinc in brain tissue. In this study, we demonstrate the close correlations between histofluorescently reactive zinc and ZnT3. We examined the overlapping distribution of the zinc-specific fluorescent dye, N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ)-, and ZnT3-immunoreactive fluorescence throughout the normal brain. TSQ and ZnT3-antibody intensely stained the hippocampus, cortex and amygdala, highlighting the characteristic laminar organization of these regions by variably staining the different layers. TSQ fluorescence and ZnT3 immunoreactivity were roughly co-localized with synaptophysin along the neuropil, but were absent in the neuronal soma. However, albeit relatively faint, TSQ fluorescence was also found throughout the brains of ZnT3-knockout mice. Although these results may indicate the presence of very small cerebral free zinc pools distinct from synaptic vesicle zinc, the synaptic vesicle zinc pool is predominant, accounting for more than 95% of the entire histofluorescently reactive zinc pool in the hippocampus and cortex. Thus, the physiological activity of free zinc in the normal brain might largely depend on the pool of synaptic vesicle zinc that is determined by ZnT3., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Zinquin (ZQ) is a commonly used sensor for cellular Zn(2+) status. It has been assumed that it measures accessible Zn(2+) concentrations in the nanomolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn(2+) bound as Zn-proteins. The predominant class of products were ZQ-Zn-protein adducts that were characterized in vivo and in vitro by a fluorescence emission spectrum centered at about 470 nm, by their migration over Sephadex G-75 as protein not low molecular weight species, by the exclusion of reaction with lipid vesicles, and by their large aggregate concentration. In addition, variable, minor formation of Zn(ZQ)(2) with a fluorescence band at about 490 nm was observed in vivo in each case. Because incubation of isolated Zn-proteome with ZQ also generated similar amounts of Zn(ZQ)(2), it was concluded that this species had formed through direct ligand substitution in which ZQ had successfully competed for protein-bound Zn(2+). Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH), and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQ(ACID) (Zinquin acid, (2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate)) able to bind to one Zn(2+) and extract the other in Zn(2)-ADH, whereas it removed one Zn(2+) from Zn(2)-AP and did not bind to the other. Zinquin ethyl ester (ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate); ZQ(EE)) bound to both proteins without sequestering Zn(2+) from either one. In contrast to a closely related sensor, 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ), neither ZQ(ACID) nor ZQ(EE) associated with Zn-carbonic anhydrase. A survey of reactivity of these sensors with partially fractionated Zn-proteome confirmed that ZQ and TSQ bind to distinct, overlapping subsets of the Zn-proteome.

Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery. We have developed a computational drug repositioning pipeline to perform large-scale molecular docking of small molecule drugs against protein drug targets, in order to map the drug-target interaction space and find novel interactions. Our method emphasizes removing false positive interaction predictions using criteria from known interaction docking, consensus scoring, and specificity. In all, our database contains 252 human protein drug targets that we classify as reliable-for-docking as well as 4621 approved and experimental small molecule drugs from DrugBank. These were cross-docked, then filtered through stringent scoring criteria to select top drug-target interactions. In particular, we used MAPK14 and the kinase inhibitor BIM-8 as examples where our stringent thresholds enriched the predicted drug-target interactions with known interactions up to 20 times compared to standard score thresholds. We validated nilotinib as a potent MAPK14 inhibitor in vitro (IC50 40 nM), suggesting a potential use for this drug in treating inflammatory diseases. The published literature indicated experimental evidence for 31 of the top predicted interactions, highlighting the promising nature of our approach. Novel interactions discovered may lead to the drug being repositioned as a therapeutic treatment for its off-target's associated disease, added insight into the drug's mechanism of action, and added insight into the drug's side effects.

The androgen receptor (AR) plays a key role in regulating gene expression in a variety of tissues, including the prostate. In that role, it is one of the primary targets in the development of new chemotherapeutics for treatment of prostate cancer and the target of the most widely prescribed current drug, bicalutamide (Bcu), for this disease. In view of its importance, and the absence of a crystal structure for any antagonist--AR complex, we have conducted a series of molecular dynamics-based simulations of the AR--Bcu complex and quantum mechanical (QM) calculations of Bcu, to elucidate the structural basis for antagonism of this key target. The structures that emerge show that bicalutamide antagonizes AR by accessing an additional binding pocket (B-site) adjacent to the hormone binding site (HBS), induced by displacing helix 12. This distorts the coactivator binding site and results in the inactivation of transcription. An alternative equienergetic conformational state of bicalutamide was found to bind in an expanded hormone pocket without materially perturbing either helix 12 or the coactivator binding site. Thus, both the structural basis of antagonism and the mechanism underlying agonist properties displayed by bicalutamide in different environments may be rationalized in terms of these structures. In addition, the antagonist structure and especially the induced second site (B-site) provide a structural framework for the design of novel antiandrogens.

Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.

Semisynthetic fluorescent biosensors consisting of a protein framework and a synthetic fluorophore are powerful analytical tools for specific detection of biologically relevant molecules. We report herein a novel method that allows for the construction of turn-on fluorescent semisynthetic biosensors in a one-step manner. The strategy is based on the ligand-directed tosyl (LDT) chemistry, a new type of affinity-guided protein labeling scheme which can site-specifically introduce synthetic probes to the surface of proteins with concomitant release of the affinity ligands. Novel quenched ligand-directed tosylate (Q-LDT) reagents were designed by connecting an organic dye to a conjugate of a protein ligand and a fluorescence quencher through a tosyl linker. The Q-LDT-mediated labeling directly converts a natural protein to a fluorescently labeled protein that remains noncovalently complexed with the cleaved ligand-tethered quencher. The fluorescence of this labeled protein is initially quenched and only in the presence of specific analytes is the fluorescence enhanced (turned on) due to the expulsion of the ligand-quencher fragment. Using a single labeling step, this approach was successfully applied to carbonic anhydrase II (CAII) and a Src homology 2 (SH2) domain to generate turn-on fluorescent biosensors toward CAII inhibitors and phosphotyrosine peptides, respectively. Detailed investigations revealed that the obtained biosensors exhibit their natural ligand selectivity. The high target-specificity of the LDT chemistry also allowed us to prepare the SH2 domain-based biosensor not only in a purified form but also in a bacterial cell lysate. These results demonstrate the utility of the Q-LDT-based approach to expand the applications of semisynthetic biosensors.

We report on the synthesis and investigation of two diastereomers (5a and 5b) of a new bicalutamide analog with an asymmetric carbon atom and a chiral sulfoxide group. These bicalutamide analogs are novel androgen receptor antagonists with biological activities that depend significantly on the configuration of their stereogenic centers. We determined the absolute configuration at the SO center by combining X-ray and NMR measurements with quantum chemical calculations. Since 5a and 5b failed to yield satisfactory crystals for X-ray crystal structure determination, analogs 6a and 6b differing in only one remote functional group relative to the chiral sulfoxide were synthesized, which yielded satisfactory crystals. X-ray structure determination of 6a and 6b provided the absolute configuration at the chiral sulfoxide. Since the structural difference between 5 and 6 is remote from the chiral sulfoxide, the structural assignment was extended from the diastereomers of 6 to those of 5 provisionally. These assignments were verified with the help of measured and DFT-calculated proton and carbon NMR chemical shifts., ((c) 2009 Wiley-Liss, Inc.)

The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo.

Metastatic prostate cancer is treated with drugs that antagonize androgen action, but most patients progress to a more aggressive form of the disease called castration-resistant prostate cancer, driven by elevated expression of the androgen receptor. Here we characterize the diarylthiohydantoins RD162 and MDV3100, two compounds optimized from a screen for nonsteroidal antiandrogens that retain activity in the setting of increased androgen receptor expression. Both compounds bind to the androgen receptor with greater relative affinity than the clinically used antiandrogen bicalutamide, reduce the efficiency of its nuclear translocation, and impair both DNA binding to androgen response elements and recruitment of coactivators. RD162 and MDV3100 are orally available and induce tumor regression in mouse models of castration-resistant human prostate cancer. Of the first 30 patients treated with MDV3100 in a Phase I/II clinical trial, 13 of 30 (43%) showed sustained declines (by >50%) in serum concentrations of prostate-specific antigen, a biomarker of prostate cancer. These compounds thus appear to be promising candidates for treatment of advanced prostate cancer.

A CE method using CDs as chiral selectors was developed and validated to achieve the separation of glycidyl tosylate enantiomers originated by in situ derivatization of glycidol enantiomers obtained in asymmetric epoxidation of allyl alcohol with chiral titanium-tartrate complexes as catalysts. The effects of the nature, pH and concentration of the buffer, the nature and concentration of chiral selector, the addition of SDS, methanol, ethanol or 2-propanol, the capillary temperature, the effective capillary length and the applied voltage on the chiral resolution of glycidyl tosylate enantiomers were investigated. The best separation conditions were achieved using a Tris-borate buffer mixture (50 and 25 mM, respectively) at pH=9.3 with a dual CD system consisting of 2.5% succinyl-beta-CD and 1.0% beta-CD w/v at 15 degrees C. A baseline separation (resolution approximately 2.0) of the glycidyl tosylate enantiomers was obtained in a relatively short time (less than 12 min). Satisfactory results were obtained in terms of linearity (r>0.99) and intermediate precision (RSD below 8.5%). The LOD and LOQ were 3.0 and 10.0 mg/L, respectively, and the recoveries ranged from 99.8 to 108.8%. Finally, the method was applied to the determination of the enantiomeric excess and the yield obtained in the asymmetric epoxidation of allyl alcohol employing chiral titanium-tartrate complexes as catalysts after an in situ derivatization of glycidol enantiomers to glycidyl tosylate.

Purpose: Zinc is an essential cofactor for normal cell function. Altered expression and function of zinc transporters may contribute to the pathogenesis of neurodegenerative disorders including macular degeneration. The expression and regulation of zinc transporters in the RPE and the toxicity of zinc to these cells were examined., Methods: Zinc transporters were identified in a human RPE cell line, ARPE19, using a 28K human array, and their expression was confirmed by PCR, immunocytochemistry, and Western blot analysis in primary human RPE cultures and ARPE19. Zinc toxicity to ARPE19 was determined using monotetrazolium, propidium iodide, and TUNEL assays, and Zn(2+) uptake was visualized with Zinquin ethyl ester. The effect of various growth factors on zinc transporter expression also was examined., Results: Transcripts for 20 of 23 zinc transporters are expressed in fetal human RPE, 16 of 23 in adult human RPE, and 21 of 23 in ARPE19. Zn transporter proteins were also detected in ARPE19. ZnT5 expression was not observed, whereas ZnT6, ZIP1, and ZIP13 were the most abundantly expressed in all RPE samples. The addition of low concentrations of Zn(2+) to cultures resulted in a dose-dependent increase in intracellular Zn(2+) content in ARPE19, and >30 nM Zn(2+) induced necrosis with an LC(50) of 117.4 nM. Brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), and pigment epithelial-derived neurotrophic factor (PEDF) increased ZIP2 expression, GDNF and PEDF increased ZnT2 expression, and PEDF increased ZnT3 and ZnT8 expression. These neurotrophic factors also promoted Zn(2+) uptake in the RPE., Conclusions: The array of zinc transporters expressed by the RPE may play a key role in zinc homeostasis in the retina and in ocular health and diseases.

The aim of this study was to investigate the direct iodination of the recently discovered peptide obestatin by LC-UV/ESI ion trap MS analysis. The influence of selected reaction parameters on obestatin iodination by chloramine-T, Iodo-Gen((R)) and lactoperoxidase was investigated by experimental design. Different responses, i.e. species percentage and yield, peptide recovery and iodination yield were evaluated. Mono-up till tetra-iodinated species are possible depending on the reaction conditions with electrophilic substitutions occurring at Tyr(16) and His(19) as confirmed by LC/MS/MS. The two possible mono-iodinated obestatin isomers, i.e. [I(1)-Tyr(16)]-obestatin and [I(1)-His(19)]-obestatin, could be chromatographically separated. Several significant main and quadratic effects, and interaction of factors were observed from which optimum conditions for a specific response could be derived. The highest impact on the response surface diagrams was overall attributed to the amount of iodide added. Synthesis methods were compared relative to the different response factors: lactoperoxidase was found to be the overall most robust iodination technique, and also gave the highest mono-iodinated species yield. The applicability of our research was demonstrated by non-carrier-added (125)I-radioiodination. To our knowledge, this is the first time an LC separation of mono-iodinated peptide isomers has been reported.

Cell permeable carrier peptides are currently of interest for their potential to improve the delivery of bioactive molecules into cells and to specific cellular compartments. We have investigated the activity of a derivative of the antiandrogen drug, bicalutamide, attached to the cell-permeable carrier peptide penetratin(R). We have used both disulfide (labile) and thioether (nonlabile) linkages to attach the bicalutamide derivative to the peptide in order to assess whether one type of chemistry has advantages over the other. In addition we have added a nuclear localization sequence (NLS) to the carrier peptide to investigate whether localization of the drug to the nucleus of the cell affects the activity of the drug. Biotin-labeled peptides were used to demonstrate that the carrier peptide is rapidly accumulated inside cultured cells, and that the incorporation of an NLS in the sequence results in its nuclear targeting. The bicalutamide derivative linked to carrier peptides via a disulfide-linkage exerted no greater antiproliferative effect in LNCaP cells, than the bicalutamide derivative alone. The bicalutamide derivative linked to the carrier peptide by a non-labile thioether linkage showed a similar activity profile. When the construct includes a nuclear targeting sequence, however, a markedly increased antiproliferative effect was observed. This study has thus shown that the activity of bicalutamide may be enhanced by the nonlabile attachment of a cell-permeable and nuclear-targeted peptide, which has implications for the development of novel antiandrogens for the treatment of prostate cancer., ((c) 2008 Wiley Periodicals, Inc.)

Disifin has emerged as a unique and very effective agent used in disinfection of wounds, disinfection of surfaces, materials and water, and other substances contaminated with almost every type of pathogenic microorganism ranging from viruses, bacteria, fungi and yeast, and, very possibly, protozoan parasites, as well. The major active component of Disifin is tosylchloramide sodium (chloramine T). However, the mechanism by which Disifin suppresses the activities of pathogenic microbial agents remains enigmatic. The molecular mechanisms, and the receptors and the signal transducing pathways responsible for the biological effects of Disifin are largely unknown. Despite considerable advances, enormous investigative efforts and large resources invested in the research on infectious diseases, microbial infection still remains a public health problem in many parts of the world. The exact nature of the pathogenic agents responsible for many infectious diseases, and the nature of the receptors mediating the associated inflammatory events are incompletely understood. Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns (PAMPs) by a family of transmembrane pattern-recognizing and signal transducing receptor proteins called Toll-like receptors (TLRs). The TLR family plays an instructive role in innate immune responses against microbial pathogens, as well as the subsequent induction of adaptive immune responses. TLRs mediate recognition and inflammatory responses to a wide range of microbial products and are crucial for effective host defense by eradication of the invading pathogens. Now, recent updates demonstrated the ability of Disifin-derived products, Disifin-Animal and Disifin-Pressant to effectively suppress the progression and activities of Chikungunya fever and that of avian influenza A virus [A/cardialis/Germany/72, H7N1: the agent of a highly pathogenic avian influenza (HPAI)] infection, respectively. Overall, the above findings led me to suggest that Disifin and TLRs may mechanistically overlap in the processes of executing their functions against pathogenic microbial organisms. Thus, elucidating and better understanding of the molecular underpinnings responsible for the biochemical effects of Disifin-products, and the nature and mode of the interaction(s) of Disifin with TLRs in the process of exerting their biological effects may open a novel dimension in the research of infectious diseases, which may provide novel therapeutic targets for the prevention and treatment of a wide range of infectious diseases.

Objectives: This study aims to investigate the importance of ISO11405 recommended storage regime for extracted teeth in surface disinfectant chloramine T (chlT) prior to use in biofilm or in vitro caries studies involving microorganisms. ChlT may be absorbed into dentin and undergoes breakdown with organic material., Methods: Extracted roots were stored in chlT (2 days), rinsed and transferred to distilled deionised water. HPLC at regular intervals determined chlT elution. At 4 weeks roots were boiled in water and eluent assessed with HPLC. ChlT breakdown (+/-organic material) over time was monitored with HPLC. ChlT minimum inhibitory/bactericidal concentration (MIC/MBC) against Lactobacillus acidophilus was evaluated using L. acidophilus broth and chlT serial dilutions., Results: No significant increase in chlT elution was detected between 2h and 4 weeks (ANOVA, Tukeys, p>0.05), although levels tended to increase with time. ChlT detected in water was 0.005%, corresponding to 0.05% in dentin. After boiling (4 weeks) chlT breakdown products in water corresponded to 0.015% in dentin. MIC/MBC of chlT against L. acidophilus was 0.031%., Significance: ChlT breakdown is accelerated by organic material. L. acidophilus is highly sensitive to chlT. ChlT readily leaches from dentin but rinsing does not reduce chlT concentration below MIC/MBC. Low levels of chlT may remain but will probably be in a less active form. Teeth disinfected in chlT for use in research involving bacteria must be stored in distilled water for at least 2h to reduce chlT concentration below MBC, although longer will give greater elution and breakdown.

Zinquin (Zn(2+) selective fluorophore), when used to visualize intracellular Zn(2+), typically shows brightly fluorescent perinuclear endosome-like structures, presumably identifying Zn(2+) containing organelles. In this study, zinquin identified numerous and widespread sites of Zn(2+) compartmentalization in primary cultures of embryonic rat cortical neurons. Nuclear fluorescence, however, was absent. We labeled neuronal mitochondria with MitoTracker Green in the presence of zinquin and show that the fluorescent patterns of MitoTracker Green and zinquin were distinct and clearly different in both the perinuclear region and in processes. The mitochondrial compartment was much larger than the sum of the areas of zinquin fluorescence, as indicated by the small amount (<10% MitoTracker Green over zinquin) of overlap of MitoTracker Green on zinquin. Zinquin fluorescence was unaffected by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) treatment. The zinquin fluorescent objects were generally spherical in shape with a average diameter of about 0.6 mum. Most fluorescent objects, nearly two thirds on average, appeared to be docked, but both anterograde and retrograde movements were observed by time lapse image analysis. Although some fluorescent objects moved as much as 1 mum in 5 min, typical movements were smaller, usually 0.5 mum or less. Colchicine treatment caused striking aggregation of MitoTracker Green most noticeable in the perinuclear region. Zinquin fluorescence similarly showed reduced distribution throughout the cytoplasm, suggesting that zinquin fluorescent structures were associated with microtubules. Treatment with cytochalasin D had little noticeable effect on either the pattern of zinquin and MitoTracker Green fluorescence or their coincidence. Thus, numerous Zn(2+) sequestering organelles/structures are present in perinuclear regions and processes of cultured neurons and are sometimes found coincident with mitochondria. We demonstrated real time trafficking of sequestered Zn(2+), using zinquin fluorescence, apparently associated with an endosome-like compartment or protein complexes in the cytosol.

The trace element zinc is essential for the survival and function of all cells. Zinc deficiency, whether nutritional or genetic, is fatal if left untreated. The effects of zinc deficiency are particularly obvious in the skin, seen as an erythematous rash, scaly plaques, and ulcers. Electron microscopy reveals degenerative changes within keratinocytes. Despite the well-documented association between zinc deficiency and skin pathology, it is not clear which cellular processes are most sensitive to zinc deficiency and could account for the typical pathological features. We used the cultured HaCaT keratinocyte line to obtain insight into the cellular effects of zinc deficiency, as these cells show many characteristics of normal skin keratinocytes. Zinc deficiency was induced by growing cells in the presence of the zinc chelator, TPEN, or by growth in zinc-deficient medium. Growth of cells in zinc-deficient medium resulted in a 44% reduction of intracellular zinc levels and a 75% reduction in the activity of the zinc-dependent enzyme, 5'-nucleotidase, relative to the control cells. Over a period of 7 days of exposure to zinc-deficient conditions, no changes in cell viability and growth, or in the cytoskeletal and cell adhesion systems, were found in HaCaT cells. At 7 days, however, induction of apoptosis was indicated by the presence of DNA fragmentation and expression of active caspase-3 in cells. These results demonstrate that apoptosis is the earliest detectable cellular change induced by zinc deficiency in HaCaT keratinocytes. Our observations account for many of the features of zinc deficiency, including the presence of degenerate nuclei, chromatin aggregates and abnormal organization of keratin, that may represent the later stages of apoptosis. In summary, a major causal role for apoptosis in the pathology of zinc deficiency in the skin is proposed. This role is consistent with the previously unexplained diverse range of degenerative cellular changes seen at the ultrastructural level in zinc-deficient keratinocytes.

Zinc chelation and non-steroidal anti-inflammatory drugs (NSAIDs) have been explored as potential neuroprotective agents. However, it remains unknown whether NSAIDs and zinc chelation may converge on a similar cellular process. Using two-photon microscopy to observe hippocampal neurons labeled with a zinc-sensitive dye, we provide evidence that three chemically unrelated NSAIDs, niflumic acid, ibuprofen, and naproxen, acutely increase intracellular zinc stores from extracellular metal pools. Phospholipase A2 inhibitors triggered similar responses, suggesting that NSAIDs likely control zinc stores by their activity as cyclooxygenase inhibitors. These results provide evidence for a new link between cyclooxygenase metabolites and the mechanisms controlling neuronal zinc pools.

The scope of photobiological processes that involve absorbers within a protein matrix may be limited by the vulnerability of the peptide group to attack by highly reactive redox centers consequent upon electronic excitation. We have explored the nature of this vulnerability by undertaking comprehensive product analyses of aqueous photolysates of 12 N-p-toluenesulfonyl peptides with systematically selected structures. The results indicate that degradation includes a major pathway that is initiated by intramolecular electron transfer in which the peptide bond serves as electron donor, and the data support the likelihood of a relay process in dipeptide derivatives.

Zafirlukast is a leukotriene antagonist indicated for the treatment of mild to moderate asthma, but the drug has been associated with occasional idiosyncratic hepatotoxicity. Structurally, zafirlukast is similar to 3-methylindole because it contains an N-methylindole moiety that has a 3-alkyl substituent on the indole ring. The results presented here describe the metabolic activation of zafirlukast via a similar mechanism to that described for 3-methylindole. NADP(H)-dependent biotransformation of zafirlukast by hepatic microsomes from rats and humans afforded a reactive metabolite, which was detected as its GSH adduct. Mass spectrometry and NMR data indicated that the GSH adduct was formed by the addition of GSH to the methylene carbon between the indole- and methoxy-substituted phenyl rings of zafirlukast. The formation of this reactive metabolite in human liver microsomes was shown to be exclusively catalyzed by CYP3A enzymes. Evidence for in vivo metabolic activation of zafirlukast was obtained when the same GSH adduct was detected in bile of rats given an iv or oral dose of the drug. On the basis of results with model peroxidases and of the structures of product alcohols from incubations containing H2(18)O, it appeared that zafirlukast underwent dehydrogenation by two sequential one-electron oxidations. In addition, zafirlukast proved to be a mechanism-based inhibitor of CYP3A4 activity in human liver microsomes and in microsomes containing cDNA-expressed CYP3A4. The enzyme inhibitory property of zafirlukast was selective for this enzyme among all of the P450 enzymes that were tested in human liver microsomes. The inactivation was characterized by a K(I) of 13.4 microM and k(inact) of 0.026 min(-1). In summary, zafirlukast dehydrogenation to an electrophilic alpha,beta-unsaturated iminium intermediate may be associated with idiosyncratic hepatotoxicity and/or cause drug-drug interactions through inactivation of CYP3A4.

Digestive enzymes of Catla catla were studied during ontogenic development. Specific amylase activity was 0.12+/-0.01 mg maltose mg protein(-1) h(-1) in fish 4 days after hatching (DAH) and reached a maximum on (0.41+/-0.12 mg maltose mg protein(-1) h(-1)) 34 DAH. Total protease activity was minimum (123.2+/-16.5 mU mg protein(-1) min(-1)) on day-8 and reached its highest level (2713+/-147.2 mU mg protein(-1) min(-1)) on day-32. Trypsin activity showed constant increasing trend from day-16 onwards and was maximum on day-34 (118.1+/-7.09 mU mg protein(-1) min(-1)). Highest chymotrypsin activity was found on day-32 (1789.0+/-111.7 mU mg protein(-1) min(-1)). Lipase activity was detected in 4 DAH catla. Lipase activity increased steadily from day-22 onwards. SDS-PAGE of crude enzyme extracts showed that high molecular mass bands (41.8-127.8 kDa) appeared during the early stages followed by low molecular mass bands (17.8-37.2 kDa). The number of protease activity bands in substrate SDS-PAGE increased with age of fish. During ontogenesis of carp, soybean trypsin inhibitor (SBTI), PMSF and TLCK inhibited 75.5+/-1.19% to 92.8+/-0.85%, 53.3+/-9.47% to 90.5+/-2.6% and 39.8+/-3.8% to 84.7+/-1.54% of total protease activity, respectively. There was only 2.58+/-0.66% to 10.21+/-0.09% inhibition of protease activity with EDTA. SBTI and PMSF inhibited 8 and 4 activity bands, respectively. TLCK, a specific trypsin inhibitor, inhibited four trypsin-like enzymes in carp during ontogenesis.

Crystal structure of ubiquitous toxin from barley alpha-hordothionin (alpha-HT) has been determined at 1.9A resolution by X-ray crystallography. The primary sequence as well as the NMR solution structure of alpha-HT firmly established that alpha-HT belongs to a family of membrane active plant toxins-thionins. Since alpha-HT crystallized in a space group (P4(1)2(1)2) that is different from the space group (I422) of previously determined alpha(1)- and beta-purothionins, and visocotoxin A3, therefore, it provided independent information on protein-protein interactions that may be relevant to the toxin mechanism. The structure of alpha-HT not only confirms overall architectural features (crambin fold) but also provides an additional confirmation of the role for crucial solute molecules, that were postulated to be directly involved in the mechanism of toxicity for thionins.

Extracellular zinc ions are effectors of many signaling pathways in mammalian cells, including the insulin/IGF-1 pathway. Molecular targets of zinc are intracellular, however, because otherwise ineffective zinc concentrations alter the extent of protein phosphorylation only in the presence of the ionophore pyrithione. The tight inhibition of protein tyrosine phosphatases by zinc (nanomolar inhibition constants) is likely responsible for the known insulinomimetic effects of zinc ions, which increase net phosphorylation of the insulin/IGF-1-receptors and activate their signaling cascades. More importantly, not only do extracellular zinc ions affect signal transduction, but growth factors induce cellular zinc fluctuations that are of sufficient magnitude to inhibit protein tyrosine phosphatases. In conclusion, a pool of cellular, available zinc participates in phosphorylation/dephosphorylation cascades, suggesting the existence of a cellular signaling system based on zinc as a second messenger.