Are hERG channel blockers also phospholipidosis inducers?

Both pharmacophore models of the human ether-à-go-go-related gene (hERG) channel blockers and phospholipidosis (PLD) inducers contain a hydrophobic moiety and a hydrophilic motif/positively charged center, so it is interesting to investigate the overlap between the ligand chemical spaces of both targets. We have assayed over 4000 non-redundant drug-like compounds for both their hERG inhibitory activity and PLD inducing potential in a quantitative high throughput screening (qHTS) format. Seventy-seven percent of PLD inducing compounds identified from the screening were also found to be hERG channel blockers, and 96.9% of the dually active compounds were positively charged. Among the 48 compounds that induced PLD without inhibiting hERG channel, 24 compounds (50.0%) carried steroidal structures. According to our results, hERG channel blockers and PLD inducers share a large chemical space. In addition, a positively charged hERG channel blocker will most likely induce PLD, while a steroid PLD inducer is less likely a hERG channel blocker.     

低自旋氰配位紫色震颤菌血红蛋白血红素及其周围质子的核磁共振研究

报道了低自旋氰配位紫色震颤菌血红蛋白 (VtHb CN)血红素及其周围质子的核磁共振谱 .通过MCOSY ,NOESY ,1DNOE和TOE ,以及SUPERWEFT实验对VtHb -CN的¹H核磁共振谱进行识别 .已识别了血红素和配位组氨酸的几乎所有共振峰 ,获得了这些共振峰的自旋 晶格弛豫时间和它们的化学位移随温度的变化关系 ,由此可分别获得这些质子到血红素Fe³⁺ 的距离和抗磁化学位移 .通过化学位移的pH滴定 ,得到了配位组氨酸 (His 85 )的咪唑环的电离平衡常数pK_a( =4.95 ) .这一较低的pK_a 值 ,归因于带正电的Fe³⁺ 以及His 85与血红素Fe³⁺   的配位 .     

The Unstirrable Water Layer Is Unstirrable because It Does Not Exist

A number of membrane‐permeation models require the incorporation of an unstirred or unstirrable water layer (UWL). An example occurs in PAMPA models when the effective permeation rate of lipophilic acids and bases, P_e, falls behind the expected permeation rate, P_m, at pH values providing a high concentration of unionized species in the donor phase. In such cases, the compound has an apparent pK_a of a weaker acid or base. The explanation is that an UWL adjacent to the membrane provides a rate‐limiting diffusion barrier for such compounds. The thickness of the UWL is correlated with the difference between the aqueous pK_a and the apparent pK_a (pK ). Here, we provide an explanation for the pK term that requires no UWL. It comes from the fact that, in the process of passing into a membrane, an ionizable compound undergoes a change in pK_a. At some point along its path into the membrane, the compound attains a maximum free energy, at which point it is as likely to continue into the membrane, as it is to return to the donor phase. This is the transition state for absorption. The pK is the pK_a of the compound at the transition state. This is a testable hypothesis (see text). The relevance of absorption to permeation depends on the rate‐limiting step of permeation.     

Probing the mechanism of drug/lipid membrane interactions using Biacore

Assay conditions were established to screen a panel of drugs for binding to liposome surfaces using a surface plasmon resonance (SPR) biosensor. Drugs were found to bind negligibly or reversibly or were retained on the liposome surface. Cationic amphiphilic drugs fell into the last class and correlated with drugs that induce phospholipidosis in vivo. To a first approximation, a single-site model yielded apparent binding affinities that adequately described a drug's dose-dependent binding to liposome surfaces. Affinities ranged at least 1000-fold within the drug panel. A liposome's drug-binding capacity and affinity depended on both the lipid headgroup and the drug's structure. Although a drug's charge state generally dominated whether or not it remained bound to the liposome, subtle structural differences between members of certain drug families led to them having widely differing binding affinities. A comparison between the dissociation of drugs from liposome surfaces by Biacore and the lipid retention measurements determined by a parallel artificial membrane permeability assay was drawn. The results from this study demonstrate the potential of using SPR-based assays to characterize drug/liposome-binding interactions.     

A metabonomic approach to the investigation of drug-induced phospholipidosis: an NMR spectroscopy and pattern recognition study

¹H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. ¹H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.     

Sexual dimorphism in inorganic mercury toxicokinetics and the attendant lipotoxic and non-lipotoxic dyslipidemia in the rat

The influence of variability in the biology of living organisms is poorly appreciated in toxicology. However, multiple lines of evidence indicate that sex-differences modulate toxicokinetics and toxicodynamics from cellular/molecular to whole animal levels resulting in different toxic responses of living organisms to xenobiotics exposure. In order to investigate the influence of sex in inorganic mercury (Hg) exposure, male and female Wistar rats were exposed to 0.5, 1.0 and 1.5 mg Hg/kg body weight orally as HgCl₂ twice a week for 12 weeks. Higher Hg levels in the females (except heart) as compared to males were observed in the animals. At the highest dose of inorganic Hg, female renal Hg content was 3.3 times higher than that of the males. Mixed sexual dimorphism characterised circulating-lipid- and organ-lipid lipotoxic and non-lipotoxic dyslipidemia. The highest dose of inorganic Hg, induced hypercholesterolemia in the males as opposed to hypocholesterolemia in the female. Plasma and erythrocyte free fatty acids increased in both sexes, although the increase was more pronounced in the male. Reverse cholesterol transport was inhibited in the male at the highest dose of Hg, whereas female HDL became enriched with cholesterol. Female erythrocytes had all their lipids increased, whereas only male erythrocyte triglyceride increased. Brain cholesterol and phospholipids, and splenic phospholipids were depleted in both sexes. Our findings indicate that inorganic Hg exposure appears to affect Hg and lipid kinetics differently in both sexes, thus underscoring the need to develop sex-tailored approaches in the treatment of metal toxicosis and its metabolic outcomes.     

Endolysosomal phospholipidosis and cytosolic lipid droplet storage and release in macrophages

This review summarizes the current knowledge of endolysosomal and cytoplasmic lipid storage in macrophages induced by oxidized LDL (Ox-LDL), enzymatically degraded LDL (E-LDL) and other atherogenic lipoprotein modifications, and their relation to the adapter protein 3 (AP-3) dependent ABCA1 and ABCG1 cellular lipid efflux pathways. We compare endolysosomal lipid storage caused either through drug induced phospholipidosis, inheritable endolysosomal and cytosolic lipid storage disorders and Ox-LDL or E-LDL induced phagosomal uptake and cytosolic lipid droplet storage in macrophages. Ox-LDL is resistant to rapid endolysosomal hydrolysis and is trapped within the endolysosomal compartment generating lamellar bodies which resemble the characteristics of phospholipidosis. Various inherited lysosomal storage diseases including sphingolipidosis, glycosphingolipidosis and cholesterylester storage diseases also present a phospholipidosis phenotype. In contrast E-LDL resembling coreless unesterified cholesterol enriched LDL-particles, with a multilamellar, liposome-like structure, lead to rapid phagosomal degradation and cytosolic lipid droplet accumulation. As a consequence the uptake of E-LDL through type I and type II phagocytosis leads to increased lipid droplet formation and moderate upregulation of ABCA1 and ABCG1 while uptake of Ox-LDL leads to a rapid expansion of the lysosomal compartment and a pronounced upregulation of the ABCA1/ABCG1/AP-3 lipid efflux pathway.     

A Fast Method for pKa Determination by Capillary Electrophoresis

In this article, we describe a recently developed capillary‐electrophoresis method for the determination of acidity constants and compare it with other existing methods. The new method is based on the use of an internal standard (compound similar in nature and pK_a value to the analyte), and offers several benefits, since it has all the advantages of capillary electrophoresis. In addition, it is very fast, because the exact measure of the pH of the separation electrolytes is not needed, and only a few electrophoretic runs are required to perform a pK_a determination. The acidity constants of some monoprotic weak acids and bases were determined by this fast method, yielding a very good agreement with literature values.     

创伤后血二胺氧化酶的变化与肠粘膜损伤

探讨创伤感染对肠道屏障功能的影响。以山羊、大鼠手术+失血再灌注+内毒素(LPS),大鼠肠缺血再灌注和犬低温枪伤多种创伤动物为模型,测定血浆二胺氧化酶(DAO)活性,并测定血乳酶、TNF和LPS含量。观察小肠病理形态改变。结果:失血再灌注后血浆DAO水平显著升高,给予内毒素后山羊血DAO水平再度升高。血浆DAO的变化与血乳酸,TNF和LPS变化呈高度相关(r=0.872,0.842和0.817,p<0.01)。光、电镜检查表明肠粘膜损伤,失血再灌注损伤可致肠粘膜屏障功能损伤,测定血浆DAO活性变化对判断小肠粘膜损伤有帮助     

Solution Structure and Acid-Base Properties of Reduced α-Conotoxin MI

The reduced derivative of α-conotoxin MI, a 14 amino acid peptide is characterized by NMR-pH titrations and molecular dynamics simulations to determine the protonation constants of the nine basic moieties, including four cysteine thiolates, and the charge-dependent structural properties. The peptide conformation at various protonation states was determined. The results show that the disulfide motifs in the native globular α-conotoxin MI occur between those cysteine moieties that exhibit the most similar thiolate basicities. Since the basicity of thiolates correlates to its redox potential, this phenomenon can be explained by the higher reactivity of the two thiolates with higher basicities. The folding of the oxidized peptide is further facilitated by the loop-like structure of the reduced form, which brings the thiolate groups into sufficient proximity. The 9 group-specific protonation constants and the related, charge-dependent, species-specific peptide structures are presented.     

l-2,4-diaminobutyric acid decarboxylase activity responsible for the formation of 1,3-diaminopropane in Enterobacter aerogenes

High content of 1,3-diaminopropane (DAP), a normally minor derivative of polyamine metabolism, have been observed in cells of Enterobacter aerogenes. Supplementation of the growth medium with L-2,4-diaminobutyric acid (L-DABA) resulted in increased production of DAP, but not if supplemented with spermidine. On the basis of these observations, the biosynthetic route for DAP was evaluated. It has appeared that this bacterium possesses a novel enzyme activity catalysing the formation of DAP from L-DABA. Lack of the activity for oxidative cleavage of spermidine yielding DAP suggests that the enzyme termed DABA decarboxylase is responsible for the formation of DAP in this bacterium. The enzyme was partially purified 360-fold and some properties were examined. The pH optimum for the activity was 7.75-8.0, and the enzyme showed an absolute requirement for pyridoxal 5'-phosphate with the Km value of 41 microM. The Km value for L-DABA was 0.32 mM, and neither L-2,3-diaminopropionic acid, L-ornithine nor L-lysine showed detectable substrate activity towards the partially purified enzyme. Mg2+ and dithiothreitol greatly activated the enzyme.     

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

Reductive methylation of lysine residues in proteins offers a way to introduce ¹³C methyl groups into otherwise unlabeled molecules. The ¹³C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of ¹³C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca²⁺-bound Calmodulin, Lysozyme, Ca²⁺-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of ¹³C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Equilibrium and structure of thallium(III)–ethylenediamine complexes in pyridine solution and in solid

The formation of three [Tl(en)_n]³⁺ complexes (n=1–3) in a pyridine solvent has been established by means of ²⁰⁵Tl and ¹H NMR. Their stepwise stability constants based on concentrations, K_n=[Tl(en)_n ³⁺]/{[Tl(en)_n−1 ³⁺]·[en]}, at 298 K in 0.5 M NaClO₄ ionic medium in pyridine, were calculated from ²⁰⁵Tl NMR integrals: log K₁=7.6±0.7; log K₂=5.2±0.5 and log K₃=2.64±0.05. Linear correlation between both the ²⁰⁵Tl NMR shifts and spin–spin coupling ²⁰⁵Tl–¹H versus the stability constants has been found and discussed. A single crystal with the composition [Tl(en)₃](ClO₄)₃ was synthesized and its structure determined by X-ray diffraction. The Tl³⁺ ion is coordinated by three ethylenediamine ligands via six N-donor atoms in a distorted octahedral fashion.