Testosterone/bicalutamide antagonism at the predicted extracellular androgen binding site of ZIP9.

ZIP9 is a Zn2+ transporter, testosterone receptor, and mediator of signaling events through G-proteins. Despite these pivotal properties, however, its physiological and pathophysiological significance has not yet been comprehensively addressed. Using a cell line that lacks the classical androgen receptor we show that ZIP9-mediated phosphorylation of Erk1/2, CREB, or ATF-1 and expression of claudin-5 and zonula occludens-1 by testosterone can be completely antagonized by bicalutamide (Casodex), an anti-androgen of significant clinical impact. Computational modeling and docking experiments with ZIP9 reveal typical characteristics of ZIP transporters and an extracellular binding site for testosterone capable of accommodating bicalutamide. The presence of this site is verified by our demonstration that the membrane-impermeable testosterone analogue T-BSA-FITC labels the membrane only when ZIP9 is expressed and that this labeling is completely prevented by bicalutamide. The study connects structural features of ZIP9 to its functions and indicates a possible relevance of ZIP9 as a pharmacological target.

Pinpoint chemical modification of Asp160 in the 49 kDa subunit of bovine mitochondrial complex I via a combination of ligand-directed tosyl chemistry and click chemistry.

Through a ligand-directed tosyl (LDT) chemistry strategy using the synthetic acetogenin ligand AL1, we succeeded in the pinpoint alkynylation (-C≡CH) of Asp160 in the 49 kDa subunit of bovine complex I, which may be located in the inner part of the putative quinone binding cavity of the enzyme [Masuya, T., et al. (2014) Biochemistry, 53, 2307-2317]. This study provided a promising technique for diverse chemical modifications of complex I. To further improve this technique for its adaptation to intact complex I, we here synthesized the new acetogenin ligand AL2, possessing an azido (-N3) group in place of the terminal alkyne in AL1, and attempted the pinpoint azidation of complex I in bovine heart submitochondrial particles. Careful proteomic analyses revealed that, just as in the case of AL1, azidation occurred at 49 kDa Asp160 with a reaction yield of ∼50%, verifying the high site specificity of our LDT chemistry using acetogenin ligands. This finding prompted us to speculate that a reactivity of the azido group incorporated into Asp160 (Asp160-N3) against externally added chemicals can be employed to characterize the structural features of the quinone/inhibitor binding cavity. Consequently, we used a ring-strained cycloalkyne possessing a rhodamine fluorophore (TAMRA-DIBO), which can covalently attach to an azido group via so-called click chemistry without Cu¹âº catalysis, as the reaction partner of Asp160-N3. We found that bulky TAMRA-DIBO is capable of reacting directly with Asp160-N3 in intact complex I. Unexpectedly, the presence of an excess amount of short-chain ubiquinones as well as some strong inhibitors (e.g., quinazoline and fenpyroximate) did not interfere with the reaction between TAMRA-DIBO and Asp160-N3; nevertheless, bullatacin, a member of the natural acetogenins, markedly interfered with this reaction. Taking the marked bulkiness of TAMRA-DIBO into consideration, it appears to be difficult to reconcile these results with the proposal that only a narrow entry point accessing to the quinone/inhibitor binding cavity exists in complex I [Baradaran, R., et al. (2013) Nature, 494, 443-448]; rather, they suggest that there may be another access path for TAMRA-DIBO to the cavity.

Bicalutamide 80 mg combined with a luteinizing hormone-releasing hormone agonist (LHRH-A) versus LHRH-A monotherapy in advanced prostate cancer: findings from a phase III randomized, double-blind, multicenter trial in Japanese patients.

To compare combination therapy with bicalutamide 80 mg and a luteinizing hormone-releasing hormone agonist (LHRH-A) versus LHRH-A alone in Japanese men with untreated advanced prostate cancer. A total of 205 patients with stage C/D prostate cancer were randomized to either LHRH-A+once-daily oral bicalutamide 80 mg or placebo. Primary study variables have been reported previously. Secondary variables included: time to achieve prostate-specific antigen < or = 4 ng/ml, time-to-treatment failure (TTTF), time-to-disease progression (TTP), overall survival (OS), adverse events and adverse drug reactions. Following combination therapy with bicalutamide 80 mg, there were significant (P<0.001) advantages over LHRH-A alone in terms of TTTF and TTP, but the difference in the interim OS was not statistically significant. First-line combination therapy with bicalutamide 80 mg in Japanese patients with advanced prostate cancer offers significant benefits over LHRH-A alone, with respect to TTTF and TTP. Follow-up for OS continues.

Tetrapentylammonium block of chloramine-T and veratridine modified rat brain type IIA sodium channels.

Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel alpha subunit was studied using whole cell patch clamp. Results indicate that TPeA blocks the inactivating brain sodium channel in a potential and use-dependent manner similar to that of the cardiac sodium channel. Removal of inactivation using chloramine-T (CT) unmasks a time-dependent block by TPeA consistent with slow blocking kinetics. On the other hand, no time dependence is observed when inactivation is abolished by modification with veratridine. TPeA does not bind in a potential-dependent fashion to veratridine-modified channels and does not significantly affect gating of veratridine-modified channels suggesting that high affinity binding of TPeA to the brain sodium channel is lost after veratridine modification.

Interaction between AR signalling and CRKL bypasses casodex inhibition in prostate cancer.

The underlying mechanism of failed androgen ablation therapy is unknown. It is recognised that under therapeutic conditions the androgen receptor (AR) remains functionally active independent of hormone stimulation and may function through an alternative pathway. We report a novel cooperative interaction between CRKL (an intracellular signalling adaptor protein) and the AR. We demonstrate by biochemical and genetic approaches that CRKL is associated with the AR complex and is localised in the nucleus of prostate cancer cells and patient tissue biopsies. The interaction between CRKL and the AR is functionally relevant as demonstrated by its presence on the enhancer region of an androgen regulated gene (human Kallikrein-2), its upregulation of PSA, and reduction in AR transactivation following its disruption by siRNA knockdown. In the presence of the AR inhibitor casodex, the expression of CRKL co-stimulated by growth factors is able to rescue AR activity independent of hormone. Our data provides insight on how a non-nuclear factor such as CRKL may interact with the AR complex to bypass hormone dependency by using an alternative growth factor signalling pathway in advanced prostate cancer.

Diazald, a newly recognized antimicrobial agent and its spectrophotometric determination.

Diazald, a chemical intermediate for the synthesis of biologically active compounds, was found to be a potent in vitro antimicrobial agent against yeasts, yeast-like and filamentous fungi as well as Gram-positive and Gram-negative bacterial strains. Its activity is not inhibited by either para-aminobenzoic acid (PABA) or the nitroso group-specific 2-aminothiazole-methoxyimino acetic acid (ATMAA). This suggests that the molecule as such is responsible for the antimicrobial activity. For its quick measurement a sensitive spectrophotometric method has been developed.

Comparison of the bronchodilating effect of salmeterol and zafirlukast in combination with that of their use as single treatments in asthma and chronic obstructive pulmonary disease.

BACKGROUND: It has been suggested that the effect of a beta2-agonist is additive with that of a cysteinyl leukotriene 1 receptor antagonist. OBJECTIVES: The present study was designed to answer the question of whether combined administration of inhaled salmeterol and oral zafirlukast at the standard doses would result in greater bronchodilation in patients with chronic airway obstruction than the use of either drug alone. METHODS: The study was performed using a double-blind, double-dummy, crossover, randomised design, and was conducted on 4 non-consecutive days. Sixteen patients with moderate to severe chronic obstructive pulmonary disease (COPD) and 10 non-smoker asthmatic patients received 40 mg of oral zafirlukast, 50 microg of inhaled salmeterol, 50 microg of inhaled salmeterol plus 40 mg of oral zafirlukast of placebo. Lung function was assessed before drug administration and 30, 60, 120, 180 and 240 min thereafter. At the end of the 4-hour period, each patient received 400 microg of inhaled salbutamol and spirometric testing was performed 30 min later. RESULTS: In both asthmatic and COPD patients, the overall effect of salmeterol and zafirlukast on the forced expiratory volume in 1 s (FEV1) was considered extremely significant (p < 0.0001), with a maximum bronchodilation above baseline after 180 min (20.7 and 11.0%, respectively) in asthmatics and after 2 h (21.7 and 11.2%, respectively) in COPD subjects. Zafirlukast did not produce any further significant acute bronchodilation in addition to that achieved with salmeterol alone in either asthmatic or COPD patients. Nevertheless, 7 out of 16 COPD patients and 7 out of 10 asthmatic patients had a further improvement after the combination of salmeterol and zafirlukast. The mean difference in pre- and post-salbutamol FEV1 values in both asthmatic and COPD patients after zafirlukast was significant (p < 0.05), but that after salmeterol and the combination of the two drugs was not significant (p > 0.05). The difference between placebo and zafirlukast was not significant following inhaled salbutamol given 4 h after each treatment. CONCLUSIONS: Both salmeterol and zafirlukast induced a significant increase in FEV1, although salmeterol elicited a greater improvement in both asthmatic and COPD patients. Apparently, zafirlukast at the clinically recommended dose did not produce any further significant acute bronchodilation in addition to that achieved with salmeterol alone, either in asthma or COPD. In any case, evaluation of the effect of the combination over a 12-hour period is mandatory.