Synthesis of Hydantoin Androgen Receptor Antagonists and Study on Their Antagonistic Activity

Hydroxymethylthiohydantoin, hydroxymethylthiohydantoin, and hydantoin, containing a pyridine group, were synthesized to study their androgen receptor antagonistic activities. Among them, compounds 6a/6c/7g/19a/19b exhibited excellent androgen receptor antagonistic activity, which was consistent with or even superior to enzalutamide. In addition, compounds 19a and 19b exhibited better antiproliferative activity than enzalutamide in prostate cancer cells. The results show that compound 19a has great potential as a new AR antagonist.     

Effects of Heat Shock on Glucocorticoid Receptor

The changes of glucocorticoid receptor (GR) during the heat shock response have been studied using a human osteosarcoma cell line (HOS-8603) as the model. The expression of the heat shock protein 70 (hsp70) mRNA in HOS-8603 cells has been enhanced markedly after a heat treatment at 43 ℃ for 30 min. A mild thermal pretreatment (42℃ for 1 h) protects the HOS-8603 cells against a subsequent heat challenge (46℃). This induced thermotolerance is reflected by the increase of cell viability of HOS-8603 cells. The GR binding activity in HOS-8603 cells decreased rapidly after the heat treatment at 43℃; only 42. 61% of controls were detected 60 min after the heat treatment. However, there was no significant change in the dissociation constant value (Kd). These results indicate that the heat shock induce not only the heat shock mRNA expression, but also the rapid reduction in GR binding activity, suggesting that there might be a functional relationship between GR action and the heat shock response.     

The Glucocorticoid Receptor of the Neural Retina of the Chick Embryo: Gradual Changes in Charge of the Glucocorticoid Receptor Complex During Transformation,Detected by Electrofocusing

Abstract

Two main difficulties impede the monitoring of the changes in the surface charge of the triamcinolone acetonide-glucocorticoid-receptor (TA-GR) complex concomitant with GR-transformation by electrofocusing (EF). One difficulty relates to the high sensitivity of the nonactivated [³H]TA-GR complex to temperature. The second difficulty relates to the transforming effects of basic carrier ampholytes, pI′ range 8-10 (basic SCAMs) on GR. Performing EF at strictly controlled cold temperature, and omitting the use of basic SCAMS revealed that the nontransformed molybdate-stabilized TA-GR complexes in the cytosol of the neural retina consist of a homogeneously charged GR population exhibiting an apparent pI′ (pI) value of 5.0 ± 0.2 (contrary to the previously reported heterogeneity of GR in ‘nonactivated’ state¹). Exposure of the [³H]TA-GR to activation by physiological temperatures, or to activation by 0.4M KC1 in the cold, (in the presence of PMSF, which inhibits proteolytic degradation of GR in the neural retina²) transformed the acidic nonactivated GR complex (designated TA-GR I) to more basic GR species: TA-GR II with a pI′ of 5.6 ± 0.2 and TA-GR III with a pI′ of 6.2 ± 0.1. TA-GR II appeared as a minor component, and TA-GR III appeared as the major form in the activated state. Molybdate inhibited the formation of TA-GR III induced by 0.4M KC1, while it augmented TA-GR II. The formation of TA-GR II from molybdate-stabilized TA-GR I was enhanced by cytosol dilution. The increase in the amount of TA-GR II in molybdate-stabilized cytosol correlated with the decrease in the amount of TA-GR I and the absence of III, suggesting that TA-GR II represents an intermediate state of GR transformation. The suppression of TA-GR III by molybdate is reversible at 30°C. Inhibitors of proteases, leupeptin, antipain and PMSF, caused a slight decrease in the pI′ value of TA-GR II found in the activated cytosol, (5.4 instead of 5.7), but did not change the heterogeneity of GR species found in the activated state. The omission of basic SCAMs in this study reduced the previously reported pI′ values¹ of TA-GR species which had been artifactually increased by basic SCAMs. The pI′ values reported in this study were confirmed by replacing SCAMs with simple buffers as carrier constituents, and appear to reflect the authentic surface net charge of TA-GR. EF analysis of [³H]TA-GR complexes that were formed in the intact tissue at 0°C (cell-bound [³H]TA-GR) confirmed the charge homogeneity of the nonactivated GR complexes in the nonactivated cytosol but showed that the cell-bound nonactivated [³H]TA-GR is more acidic than the TA-GR I formed under cell-free conditions, exhibiting a pI′ value of 4.2 ± 0.1. These results are consistent with the hypothesis that GR transformation is a multistage process characterized by a progressive decrease in the negative net charge of the GR complex. The nature of the changes in negative net charge is discussed elsewhere² in relation to the changes in size of the GR complex occurring in the process of GR transformation.     

Combination of Heme Oxygenase-1 Inhibition and Sigma Receptor Modulation for Anticancer Activity

Cancer is a multifactorial disease that may be tackled by targeting different signaling pathways. Heme oxygenase-1 (HO-1) and sigma receptors (σRs) are both overexpressed in different human cancers, including prostate and brain, contributing to the cancer spreading. In the present study, we investigated whether HO-1 inhibitors and σR ligands, as well a combination of the two, may influence DU145 human prostate and U87MG human glioblastoma cancer cells proliferation. In addition, we synthesized, characterized, and tested a small series of novel hybrid compounds (HO-1/σRs) 1–4 containing the chemical features needed for HO-1 inhibition and σR modulation. Herein, we report for the first time that targeting simultaneously HO-1 and σR proteins may be a good strategy to achieve increased antiproliferative activity against DU145 and U87MG cells, with respect to the mono administration of the parent compounds. The obtained outcomes provide an initial proof of concept useful to further optimize the structure of HO-1/σRs hybrids to develop novel potential anticancer agents.     

Computational Investigation on the Allosteric Modulation of Androgen Receptor

Androgens have similar structures with different biological activities. To identify molecular determinants responsible for the activity difference, we have docked six steroidal androgens to the binding site or the surface of androgen receptor by using molecular docking with computational investigation. The energy was calculated respectively based on the QM (quantum mechanics) and MM (molecular mechanics) methods. The result shows that the allosteric modulation of androgen receptor plays an important role in the binding process between androgens and receptor. The open state receptor is less stable than the close state one, but the latter is more favorable for binding with androgens. It is worthy of note that when the androgen receptors binding or without binding with androgen are in close state, they are difficult to return to their open state. This phenomenon is an exception of the well known two-state model theory in which the two states are reversible. Whether the internal of close state androgen receptor has a combination of androgen or not, the androgen receptor surface can be combined with another androgen, and their surface binding energies could be very close. The result is consistent with the experimental observations, but this phenomenon of continuous combination from open state is also an exception of the two-state model theory.     

Photocytotoxicity of the Fluorescent Nonsteroidal Androgen Receptor Ligand TDPQ†

1,2,3,4‐tetrahydro‐2,2‐dimethyl‐6‐(trifluoromethyl)‐8‐pyridono[5,6‐g]quinoline (TDPQ), a selective nonsteroidal androgen receptor (AR) ligand, is a fluorescent compound. We characterized its spectral properties in comparison with the structural precursor carbostyril 151 (C151) and with its racemic structural isomer 4‐ethyl‐1,2,3,4‐tetrahydro‐6‐(trifluoromethyl)‐8‐pyridino[5,6‐g]quinoline (ETPQ). The absorption maximum in CH₃CN of either TDPQ or ETPQ is 400 nm whereas that of C151 is 350 nm. The fluorescence lifetimes (τ) and quantum yields (?_f) in CH₃CN are typical of fluorescent dyes: TDPQ (4.2 ns, 0.8) and ETPQ (4.6 ns, 0.76). C151 showed lower τ and ?_f of 0.2 ns and 0.02, respectively. TDPQ can function as a fluorescent label at (sub)micromolar concentrations. We detected TDPQ fluorescence in human breast tumor cells using confocal microscopy. While the fluorescence maxima of the compounds were solvent insensitive, the ?_f for ETPQ decreased in aqueous solutions regardless of the presence of albumin or DNA. The ?_f of TDPQ was less affected. The quantum yield of singlet oxygen (¹O₂) photosensitization (?_so) by TDPQ and ETPQ was about 7% in CH₃CN, sufficient to induce photocytotoxicity. TDPQ was photocytotoxic in AR‐positive MDA‐MB‐453 breast cancer cells but not in AR‐negative MDA‐MB‐231 cells. The combination of AR selectivity with photocytotoxicity makes TDPQ a promising candidate for selective targeting of AR‐positive cells during photodynamic therapy.     

Investigation of Coculture of Human Adipose-Derived Stem Cells and Mature Adipocytes

The purpose of this study was to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into adipocytes by coculturing them with human mature adipocytes. The transwell culture system was utilized for indirect coculture of hADSCs and human mature adipocytes at four different hADSCs-to-mature adipocytes ratios, i.e., 1:5, 1:1, 2:1, and 5:1. After 8?days of coculture, the Oil Red O and Trypan Blue stainings were performed for the evaluation of adipogenic differentiation of hADSCs. In addition, flow cytometric analysis and Hoechst 33342/PI double staining were performed after 20?days of coculture. The Oil Red O and Trypan Blue stainings showed that hADSCs with high viability could not differentiate into mature adipocytes after 8 or 20?days of coculture. However, flow cytometric analysis indicated that CD105 expression of hADSCs decreased after 20?days of coculture. These results indicated that hADSCs cocultured with human adult adipocytes could not successfully differentiate into adipocytes.     

木犀草素抑制酪氨酸酶活性的分子机制

通过酶抑制动力学实验、荧光猝灭实验以及分子对接技术研究了木犀草素对酪氨酸酶活性的抑制作用。酶抑制动力学实验结果表明:木犀草素是酪氨酸酶的非竞争性抑制剂,抑制常数KI与IC50分别为86mmol/L和778.2μmol/L;荧光猝灭实验结果表明:木犀草素对酪氨酸酶产生静态荧光猝灭作用,疏水作用与氢键作用共同稳定其复合物结构,结合位点数为1;分子对接结果表明:木犀草素在酪氨酸酶疏水口袋边缘与其相互作用,相互作用力包括疏水作用力与氢键。     

Flow-Microcalorimetric Determination of Cellobiase Activity and Its Inhibition by Glucose

Abstract

A method for measuring cellobiase activity of the Trichoderma reesei CCF 1853 cellulase complex using a Thermal Activity Monitor and a flow - mix mode is described. The kinetic constant K_M and the linear dependence of dQ_max/dt (the maximum heat flow at the total saturation of enzyme with substrate) on the enzyme concentration were determined. The process of the end product inhibition of cellobiase activity by glucose has been observed too. The obtained results allow to determine the mechanism of the inhibition and an inhibition constant for glucose.

The procedure is completely general in nature and is applicable to other enzymatic systems.     

Stabilization of Human Telomeric G‐Quadruplex and Inhibition of Telomerase Activity by Propeller‐Shaped Trinuclear PtII Complexes

Two novel propeller‐shaped, trigeminal‐ligand‐containing, flexible trinuclear Pt^II complexes, {[Pt(dien)]₃(ptp)}(NO₃)₆ ( 1 ) and {[Pt(dpa)]₃(ptp)}(NO₃)₆ ( 2 ) (dien: diethylenetriamine; dpa: bis‐(2‐pyridylmethyl)amine; ptp: 6′‐(pyridin‐3‐yl)‐3,2′:4′,3′′‐terpyridine), have been designed and synthesized, and their interactions with G‐quadruplex (G4) sequences are characterized. A combination of biophysical and biochemical assays reveals that both Pt^II complexes exhibit higher affinity for human telomeric (hTel) and c‐myc promoter G4 sequences than duplex DNA. Complex 1 binds and stabilizes hTel G4 sequence more effectively than complex 2 . Both complexes are found to induce and stabilize either antiparallel or parallel conformation of G4 structures. Molecular docking studies indicate that complex 1 binds into the large groove of the antiparallel hTel G4 structure (PDB ID: 143D) and complex 2 stacks onto the exposed G‐quartet of the parallel hTel G4 structure (PDB ID: 1KF1). Telomeric repeat amplification protocol assays demonstrate that both complexes are good telomerase inhibitors, with IC₅₀ values of (16.0±0.4) μM and (4.20±0.25) μM for 1 and 2 , respectively. Collectively, the results suggest that these propeller‐shaped flexible trinuclear Pt^II complexes are effective and selective G4 binders and good telomerase inhibitors. This work provides valuable information for the interaction between multinuclear metal complexes with G4 DNA.     

电致化学发光抑制效应测定硫脲

在自制的ECL-1型电致化学发光仪上,用电化学发光法研究了硫脲.发现硫脲对新的酰肼类试剂6-[2,4-二羟基苯基偶氮]-2,3-二氢-1,4-酞嗪二酮(DHDHD)在Na₂CO₃-NaHCO₃-KCl体系中的电化学发光有较强的抑制效应.在0.05～5mg/L范围内,硫脲浓度的对数与发光强度有良好的线性关系.0.2mg/L的硫脲,相对标准偏差为2%.并探讨了发光机理.     

Directed Evolution of a Soluble Human IL-17A Receptor for the Inhibition of Psoriasis Plaque Formation in a Mouse Model

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Highlights? IL-17 is involved in many inflammatory diseases ? Directed evolution of IL-17A receptor results in improved IL-17A affinity ? The engineered receptors efficiently inhibit IL-17A induced cytokine secretion ? Engineered receptor promotes the recovery of psoriasis plaques in mouse model     

Antitumor Activity and Mechanism of Action of the Antimicrobial Peptide AMP-17 on Human Leukemia K562 Cells

Cancer is one of the most common malignant diseases in the world. Hence, there is an urgent need to search for novel drugs with antitumor activity against cancer cells. AMP-17, a natural antimicrobial peptide derived from Musca domestica, has antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. However, its antitumor activity and potential mechanism of action in cancer cells remain unclear. In this study, we focused on evaluating the in vitro antitumor activity and mechanism of AMP-17 on leukemic K562 cells. The results showed that AMP-17 exhibited anti-proliferative activity on K562 cells with an IC₅₀ value of 58.91 ± 3.57 μg/mL. The membrane integrity of K562 was disrupted and membrane permeability was increased after AMP-17 action. Further observation using SEM and TEM images showed that the cell structure of AMP-17-treated cells was disrupted, with depressions and pore-like breaks on the cell surface, and vacuolated vesicles in the cytoplasm. Furthermore, further mechanistic studies indicated that AMP-17 induced excessive production of reactive oxygen species and calcium ions release in K562 cells, which led to disturbance of mitochondrial membrane potential and blocked ATP synthesis, followed by activation of Caspase-3 to induce apoptosis. In conclusion, these results suggest that the antitumor activity of AMP-17 may be achieved by disrupting cell structure and inducing apoptosis. Therefore, AMP-17 is expected to be a novel potential agent candidate for leukemia treatment.     

Inhibition of Lipid Accumulation and Adipokine Levels in Maturing Adipocytes by Bauhinia rufescens (Lam.) Stem Bark Extract Loaded Titanium Oxide Nanoparticles

The present study reports a cost-effective, environmentally friendly method to increase the bioavailability and bio-efficacy of B. rufescens stem bark extract in the biological system via functional modification as B. rufescens stem bark nanoparticles (BR-TO₂-NPs). The biosynthesis of BR- -NPs was confirmed by UV-visible (UV-vis) and Fourier-transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction analyses. The shifts in FT-IR stretching vibrations of carboxylic and nitro groups (1615 cm⁻¹), the O–H of phenolics or carboxylic acids (3405 cm⁻¹), alkanes, and alkyne groups (2925 and 2224 cm⁻¹) of the plant extract and lattice (455) indicated successful biosynthesis of BR- -NPs. Compared with the stem bark extract, 40 ng/dL dose of BR- -NPs led to a reduction in adipogenesis and an increase in mitochondrial biogenesis-related gene expressions, adiponectin-R1, PPARγC1α, UCP-1, and PRDM16, in maturing-adipocytes. This confirmed the intracellular uptake, bioavailability, and bio-efficiency of BR-TiO₂-NPs. The lipid-lowering capacity of BR-TiO₂-NPs effectively inhibited the metabolic inflammation-related gene markers, IL-6, TNF-α, LTB4-R, and Nf-κb. Further, BR-TiO₂-NPs stimulating mitochondrial thermogenesis capacity was proven by the significantly enhanced CREB-1 and AMPK protein levels in adipocytes. In conclusion, BR-TiO₂-NPs effectively inhibited lipid accumulation and proinflammatory adipokine levels in maturing adipocytes; it may help to overcome obesity-associated comorbidities.     

Effects of Chemical Structures Interacting with Amine Oxidases on Glucose,Lipid and Hydrogen Peroxide Handling by Human Adipocytes

Benzylamine is a natural molecule present in food and edible plants, capable of activating hexose uptake and inhibiting lipolysis in human fat cells. These effects are dependent on its oxidation by amine oxidases present in adipocytes, and on the subsequent hydrogen peroxide production, known to exhibit insulin-like actions. Virtually, other substrates interacting with such hydrogen peroxide-releasing enzymes potentially can modulate lipid accumulation in adipose tissue. Inhibition of such enzymes has also been reported to influence lipid deposition. We have therefore studied in human adipocytes the lipolytic and lipogenic activities of pharmacological entities designed to interact with amine oxidases highly expressed in this cell type: the semicarbazide-sensitive amine oxidase (SSAO also known as PrAO or VAP-1) and the monoamine oxidases (MAO). The results showed that SZV-2016 and SZV-2017 behaved as better substrates than benzylamine, releasing hydrogen peroxide once oxidized, and reproduced or even exceeded its insulin-like metabolic effects in fat cells. Additionally, several novel SSAO inhibitors, such as SZV-2007 and SZV-1398, have been evidenced and shown to inhibit benzylamine metabolic actions. Taken as a whole, our findings reinforce the list of molecules that influence the regulation of triacylglycerol assembly/breakdown, at least in vitro in human adipocytes. The novel compounds deserve deeper investigation of their mechanisms of interaction with SSAO or MAO, and constitute potential candidates for therapeutic use in obesity and diabetes.     

Inhibition of Metalloproteinase Activity by Chinese Formulations Used to Treat Inflammatory Diseases

Introduction Matrixmetalloproteinases(MMPs)areaclassof zinc requiringextracellularendopeptidasesthatcanto getherdegradeallcomponentsoftheextracellularma trixandbasementmembranes[1—3].Theyplayimpor tantrolesinconnectivetissueremodeling,occurringin normalb…     

高效液相色谱法测定人体红细胞中儿茶酚氧位甲基转移酶的活性

 采用高效液相色谱紫外检测法测定人体红细胞中儿茶酚氧位甲基转移酶 (COMT)的活性。以 3,4 二羟基苯甲酸 (DBA)作为酶反应底物 ,S 腺苷甲硫氨酸 (SAM)作为甲基供体 ,在镁离子的存在下 ,将SAM上的甲基转移到DBA 3位的氧上。色谱法测定反应产物 4 羟基 3 甲氧基苯甲酸 (4 OH 3 MBA)的生成量。人体红细胞中COMT活性的线性范围在 1U/mL～ 6 0U/mL ,最低检测限为 0 5U/mL(S/N≥ 5 ) ,方法的精密度良好 (平均RSD <10 % )。