Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na ϩ -K ϩ -2Cl Ϫ cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (Ն100 M). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC 50 ϳ20 M, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC 50 ϳ8 M), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 M had no effect, and 20 M caused 20% block of human Na ϩ -K ϩ -2Cl Ϫ cotransporter activity, in contrast to Ͼ90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl Ϫ currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.
In order to improve the discovery and development of new drugs, a broad effort is being made to assess the ‘drug-like’ properties of molecules in early stages of the discovery-research process. Although there are numerous approaches to this problem, perhaps the simplest and most widespread one is that developed by Chris Lipinski and his co-workers at Pfizer, which is generally referred either as the Lipinski Rules or the Rule of Five (ROF). The ROF is based on four properties of molecules, namely, molecular weight (MW), logP, number of hydrogen bond donors (HBD), and the number of hydrogen bond acceptors (HBA). A ‘flag’ is set if the value of a given property exceeds the chosen threshold value for that property—MW 500 Da, logP 5, the number of HBDs 5, and the number of HBAs 10. Each flag corresponds to an ROF violation. The total number of violations is the ROF-Score, which lies between ‘0’ and ‘4’. Molecules with ROF-Scores greater than one are considered to be marginal for further development. The difficulty with this approach is that two molecules with nearly identical property values can, nonetheless, possess ROF-Scores that can differ by two or more. Thus, one molecule could be considered for further studies while the other, nearly identical molecule (in terms of its four ROF properties), would most likely not be. This problem arises because of the sharp thresholds imposed by the present formulation of the ROF, which is based upon classical sets. In the current work an alternative approach based on the use of utility functions, within the framework of the analytic hierarchy process (AHP), are employed to ‘soften’ the sharp boundaries inherent in classical sets. This provides a more realistic assessment of compounds in terms of their potential suitability in drug-discovery research programs.
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a “proof-of-principle” that enzymatic inhibition of QSOX1 may have clinical relevancy.
Abnormally low plasma concentrations of thyroid hormones during sepsis often occur in the absence of thyroidal illness; however, the mechanisms involved in the "euthyroid sick syndrome" remain poorly understood. Here, we describe a previously unrecognized interaction between the thyroid hormone thyroxine (T 4 ) and the proinflammatory cytokine macrophage migration inhibitory factor (MIF), together with its clinical relevance in sepsis. We found that in both patients with severe sepsis, and our rodent model, low plasma T 4 concentrations were inversely correlated with plasma MIF concentrations. The MIF molecule contains a hydrophobic pocket that is important for many of its proinflammatory activities. Binding of L-T 4 (or its hormonally inert isomer D-T 4 ) significantly, and dose-dependently, inhibited the catalytic activity of this pocket. Moreover, administration of exogenous D-T 4 significantly improved survival in mice with severe sepsis. To examine the specificity of the MIF∶T 4 interaction, wild-type and MIF knockout mice were subjected to the carrageenan-air pouch model of inflammation and then treated with D-T 4 or vehicle. D-T 4 significantly inhibited leukocyte infiltration in wild-type mice but not in MIF knockout mice, providing evidence that in vivo T 4 may influence MIF-mediated inflammatory responses via inhibition of its hydrophobic proinflammatory pocket. These findings demonstrate a new physiological role for T 4 as a natural inhibitor of MIF proinflammatory activity. The data may also, in part, explain the low plasma T 4 concentrations in critically ill, euthyroid patients and suggest that targeting the imbalance between MIF and T 4 may be beneficial in improving outcome from sepsis. S epsis is a critical illness with an important inflammatory component that occurs in millions of individuals each year and results in high mortality and morbidity. Low circulating thyroid hormone levels are common in critically ill patients with severe infections and sepsis. This phenomenon of low plasma thyroid hormone levels, occurring in the absence of thyroid illness, is often referred to as "euthyroid sick syndrome." Approximately 60% of critically ill patients have abnormally low plasma T 4 levels, with the lowest levels being observed in patients with sepsis. These low thyroid hormone levels are good indicators of disease severity and predictors of mortality (1, 2). Although the etiology of the euthyroid sick syndrome has been linked to stress responses, impaired tissue function, and altered peripheral thyroid hormone metabolism during sepsis (2-4), the mechanisms involved in its pathophysiology remain poorly understood.Migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a critical role in the pathogenesis of sepsis (5-7). Plasma MIF levels are significantly elevated in nonsurvivors, compared with survivors, of severe sepsis (8), and administration of antibodies against MIF improves survival in experimental sepsis (5). During sepsis, plasma MIF levels can be higher than 180 ng∕mL (6...
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