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The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein.We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators ofABCG2 activity. Bothwild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cellmembrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of diseasecausing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application. [ABSTRACT FROM AUTHOR]

Pseudoxanthoma elasticum (PXE), a prototype of heritable multisystem disorders, is characterised by pathologic mineralisation of connective tissues, with primary clinical manifestations in the skin, eyes and the cardiovascular system. The causative gene was initially identified as ABCC6 which encodes an ABC transporter protein (ABCC6) expressed primarily in the liver and the kidneys. The critical role of ABCC6 in ectopic mineralisation has been confirmed by the development of Abcc6 −/− knock-out mice which recapitulate the features of connective tissue mineralisation characteristic of PXE. Over 300 distinct loss-of-function mutations representative of over 1000 mutant alleles in ABCC6 have been identified by streamlined mutation detection strategies in this autosomal recessive disease. More recently, missense mutations in the GGCX gene, either in compound heterozygous state or digenic with a recurrent ABCC6 nonsense mutation (p.R1141X), have been identified in patients with PXE-like cutaneous findings and vitamin K-dependent coagulation factor deficiency. GGCX encodes a carboxylase which catalyses γ-glutamyl carboxylation of coagulation factors as well as of matrix gla protein (MGP) which in fully carboxylated form serves as a systemic inhibitor of pathologic mineralisation. Collectively, these observations suggest the hypothesis that a consequence of loss-of-function mutations in the ABCC6 gene is the reduced vitamin K-dependent γ-glutamyl carboxylation of MGP, with subsequent connective tissue mineralisation. Further progress in understanding the detailed pathomechanisms of PXE should provide novel strategies to counteract, and perhaps cure, this complex heritable disorder at the genome–environment interface. [ABSTRACT FROM AUTHOR]

The arginine-specific reagent 1,2-cyclohexanedione reacts selectively with the arginine residue of the C-l-phosphate-binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu-43 to Arg-56, the residue modified by cyclohexanedione being Arg-55. This peptide was absent from digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg-55 with loss of activity. Selective isolation of the peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix-bound boric acid groups with vicinal cis-hxdroxyl groups of cyclohexanedione-modified arginine side chains. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to a report that Hungary's research-grant agency for basic science, OTKA, will lose autonomy beginning on January 1, 2015.

14- O -methyl (14- O- Me) group in morphine-6- O -sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14- O -methylmorphine (14- O -MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo . The potency and efficacy of test compound were measured by [ 35 S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14- O -MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile 5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14- O -MeM showed higher efficacy (E max ) and potency (EC 50 ) than morphine in MVD, RVD or [ 35 S]GTPγS binding. In addition, 14- O -MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo , in rat tail-flick test 14- O -MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14- O -MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14- O -MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value. [ABSTRACT FROM AUTHOR]

Opioid antagonists, naloxone and naltrexone have long been used in clinical practice and research. In addition to their low selectivity, they easily pass through the blood-brain barrier. Quaternization of the amine group in these molecules, (e.g. methylnaltrexone) results in negligible CNS penetration. In addition, zwitterionic compounds have been reported to have limited CNS access. The current study, for the first time gives report on the synthesis and the in vitro [competition binding, G-protein activation, isolated mouse vas deferens (MVD) and mouse colon assay] pharmacology of the zwitterionic compound, naltrexone-14- O -sulfate. Naltrexone, naloxone, and its 14- O -sulfate analogue were used as reference compounds. In competition binding assays, naltrexone-14- O -sulfate showed lower affinity for µ, δ or κ opioid receptor than the parent molecule, naltrexone. However, the μ/κ opioid receptor selectivity ratio significantly improved, indicating better selectivity. Similar tendency was observed for naloxone-14- O -sulfate when compared to naloxone. Naltrexone-14- O -sulfate failed to activate [ 35 S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile 5,6 deltorphin II and U69593), similarly to the reference compounds. Schild plot constructed in MVD revealed that naltrexone-14- O -sulfate acts as a competitive antagonist. In mouse colon, naltrexone-14- O -sulfate antagonized the inhibitory effect of morphine with lower affinity compared to naltrexone and higher affinity when compared to naloxone or naloxone-14- O -sulfate. In vivo (mouse tail-flick test), subcutaneously injected naltrexone-14- O -sulfate antagonized morphine's antinociception in a dose-dependent manner, indicating it's CNS penetration, which was unexpected from such zwitter ionic structure. Future studies are needed to evaluate it's pharmacokinetic profile. [ABSTRACT FROM AUTHOR]

Background: Pseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PPi). It is unknown how ABCC6 genotype affects plasma PPi. Methods: We studied the association of ABCC6 genotype (192 patients with biallelic pathogenic ABCC6 mutations) and PPi levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PPi level and disease severity. Results: Our findings confirm low PPi in PXE compared with healthy controls (0.53 ± 0.15 vs. 1.13 ± 0.29 µM, p < 0.01). The PPi of patients correlated with increasing age (β: 0.05 µM, 95% CI: 0.03–0.06 per 10 years) and was higher in females (0.55 ± 0.17 vs. 0.51 ± 0.13 µM in males, p = 0.03). However, no association between PPi and PXE phenotypes was found. When adjusted for age and sex, no association between PPi and ABCC6 genotype was found. Conclusions: Our data suggest that the relationship between ABCC6 mutations and reduced plasma PPi may not be as direct as previously thought. PPi levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PPi and between PPi and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading. [ABSTRACT FROM AUTHOR]

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4P2O7 is absorbed in humans. Here, we report that gelatin‐encapsulated Na2H2P2O7 has similar absorption properties in healthy volunteers and people affected by PXE. The sodium‐free K2H2P2O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6−/− mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing. [ABSTRACT FROM AUTHOR]

Objective The pathogenesis of calcinosis cutis, a disabling complication of SSc, is poorly understood and effective treatments are lacking. Inorganic pyrophosphate (PPi) is a key regulator of ectopic mineralization, and its deficiency has been implicated in ectopic mineralization disorders. We therefore sought to test the hypothesis that SSc may be associated with reduced circulating PPi, which might play a pathogenic role in calcinosis cutis. Methods Subjects with SSc and age-matched controls without SSc were recruited from the outpatient rheumatology clinics at Rutgers and Northwestern Universities (US cohort), and from the Universities of Szeged and Debrecen (Hungarian cohort). Calcinosis cutis was confirmed by direct palpation, by imaging or both. Plasma PPi levels were determined in platelet-free plasma using ATP sulfurylase to convert PPi into ATP in the presence of excess adenosine 5' phosphosulfate. Results Eighty-one patients with SSc (40 diffuse cutaneous, and 41 limited cutaneous SSc) in the US cohort and 45 patients with SSc (19 diffuse cutaneous and 26 limited cutaneous SSc) in the Hungarian cohort were enrolled. Calcinosis was frequently detected (40% of US and 46% of the Hungarian cohort). Plasma PPi levels were significantly reduced in both SSc cohorts with and without calcinosis (US: P  = 0.003; Hungarian: P  < 0.001). Conclusions Circulating PPi are significantly reduced in SSc patients with or without calcinosis. Reduced PPi may be important in the pathophysiology of calcinosis and contribute to tissue damage with chronic SSc. Administering PPi may be a therapeutic strategy and larger clinical studies are planned to confirm our findings. [ABSTRACT FROM AUTHOR]

Abstract: The human multidrug resistance ABC transporters provide a protective function in our body against a large number of toxic compounds. These proteins, residing in the plasma membrane, perform an active, ATP-dependent extrusion of such xenobiotics. However, the same proteins are also used by the tumor cells to fight various anticancer agents. ABCG2 is an important member of the multidrug resistance proteins, an ‘ABC half transporter’, which functions as a homodimer in the cell membrane. In this review, we provide a basic overview of ABCG2 function in physiology and drug metabolism, but concentrate on the discussion of mutations and polymorphisms discovered in this protein. Interestingly, a single nucleotide mutation, changing amino acid 482 from arginine to threonine or glycine in ABCG2, results in a major increase in the catalytic activity and a wider drug recognition by this protein. Still, this mutation proved to be an in vitro artifact, produced only in heavily drug-selected cell lines. In contrast, at least two, but possibly more polymorphic variants of ABCG2 were found to be present in large human populations with different ethnic background. However, currently available experimental data regarding the cellular expression, localization and function of these ABCG2 variants are strongly contradictory. Since, the proteins produced by these variant alleles may differently modulate cancer treatment, general drug absorption and toxicity, may represent risk factors in fetal toxicity, or alter the differentiation of stem cells, their exact characterization is a major challenge in this field. [Copyright &y& Elsevier]

Connective tissue calcifying diseases (CTCs) are characterized by abnormal calcium deposition in connective tissues. CTCs are caused by multiple factors including chronic diseases (Type II diabetes mellitus, chronic kidney disease), the use of pharmaceuticals (e.g. warfarin, glucocorticoids) and inherited rare genetic diseases such as pseudoxanthoma elasticum (PXE), generalized arterial calcification in infancy (GACI) and Keutel syndrome (KTLS). This review explores our current knowledge of these rare inherited CTCs, and highlights the most promising avenues for pharmaceutical intervention. Advancing our understanding of rare inherited forms of CTC is not only essential for the development of therapeutic strategies for patients suffering from these diseases, but also fundamental to delineating the mechanisms underpinning acquired chronic forms of CTC. [ABSTRACT FROM AUTHOR]

Abstract: Human ABCG2 is a plasma membrane glycoprotein that provides physiological protection against xenobiotics. ABCG2 also significantly influences biodistribution of drugs through pharmacological tissue barriers and confers multidrug resistance to cancer cells. Moreover, ABCG2 is the molecular determinant of the side population that is characteristically enriched in normal and cancer stem cells. Numerous tumors depend on unregulated EGFR signaling, thus inhibition of this receptor by small molecular weight inhibitors such as gefitinib, and the novel second generation agents vandetanib, pelitinib and neratinib, is a promising therapeutic option. In the present study, we provide detailed biochemical characterization regarding the interaction of these EGFR inhibitors with ABCG2. We show that ABCG2 confers resistance to gefitinib and pelitinib, whereas the intracellular action of vandetanib and neratinib is unaltered by the presence of the transporter. At higher concentrations, however, all these EGFR inhibitors inhibit ABCG2 function, thereby promoting accumulation of ABCG2 substrate drugs. We also report enhanced expression of ABCG2 in gefitinib-resistant non-small cell lung cancer cells, suggesting potential clinical relevance of ABCG2 in acquired drug resistance. Since ABCG2 has important impact on both the pharmacological properties and anti-cancer efficiencies of drugs, our results regarding the novel EGFR inhibitors should provide useful information about their therapeutic applicability against ABCG2-expressing cancer cells depending on EGFR signaling. In addition, the finding that these EGFR inhibitors efficiently block ABCG2 function may help to design novel drug-combination therapeutic strategies. [Copyright &y& Elsevier]

Abstract: The ABCG2 multidrug transporter protein has been identified as a key player in cancer drug resistance and xenobiotic elimination, as its actively transported substrates include anticancer drugs, intermediates of heme metabolism, xenobiotics, and also drug conjugates. Several transported substrates at higher concentrations, and some anticancer agents even at low concentrations directly inhibit the ABCG2 transporter, thus it is difficult to provide estimation for pharmacologically important ABCG2-dependent interactions. In addition, as documented here, in mutant variants of the transporter, inhibitors of the wild-type ABCG2 may become actively transported substrates. In this paper we describe a rapid in vitro assay to identify transport modulation by measuring the cell surface interaction of a conformation sensitive monoclonal antibody (5D3) with ABCG2 in intact cells. As documented, in conjunction with membrane ATPase, transport and cytotoxicity measurements, this assay provides a reliable estimate of concentration-dependent modulation of ABCG2 by newly emerging pharmacophores. A high-throughput, 96-well plate assay platform is also provided. [Copyright &y& Elsevier]

Abstract: In this review, we summarize the currently available information on the membrane topology of some key members of the human ABC protein subfamilies, and present the predicted domain arrangements. In the lack of high-resolution structures for eukaryotic ABC transporters this topology is based only on prediction algorithms and biochemical data for the location of various segments of the polypeptide chain, relative to the membrane. We suggest that topology models generated by the available prediction methods should only be used as guidelines to provide a basis of experimental strategies for the elucidation of the membrane topology. [Copyright &y& Elsevier]

The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein. [ABSTRACT FROM AUTHOR]

Multidrug resistance is frequently observed when treating cancer patients with chemotherapeutic agents. A variety of ATP binding cassette (ABC) transporters, localized in the cell membrane, cause this phenomenon by extruding a variety of chemotherapeutic agents from the tumor cells. However, the major physiological role of the multidrug transporters is the protection of our cells and tissues against xenobiotics, and these transporters play a key role in drug availability, metabolism and toxicity. Three major groups of ABC transporters are involved in multidrug resistance: the classical P-glycoprotein MDR1, the multidrug resistance associated proteins (MRP1, MRP2, and probably MRP3, MRP4 and MRP5), and the ABCG2 protein, an ABC half-transporter. All these proteins were shown to catalyze an ATP-dependent active transport of chemically unrelated compounds. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. By examining the interactions of the multidrug transporters with pharmacological and toxic agents, a prediction for the cellular and tissue distribution of these compounds can be achieved. Oral bioavailability, entering the blood–brain and blood-CSF barrier, reaching the fetus through the placenta, liver and kidney secretion, cellular entry for affecting intracellular targets, are all questions, which can be addressed by basic in vitro studies on the multidrug resistance proteins. Investigation of the substrate interactions and modulation of multidrug transporters may pave the way for predictive toxicology and pharmacogenomics. Here we show that by using in vitro assay systems it is possible to measure the interactions of multidrug transporters with various drugs and toxic agents. We focus on the characterisation of the MRP1 and MRP3 proteins, their relevance in chemoresistance of cancer and in drug metabolism and toxicity. [Copyright &y& Elsevier]

Specific tyrosine kinase inhibitors (TKIs) are rapidly developing clinical tools applied for the inhibition of malignant cell growth and metastasis formation. Most of these newly developed TKI molecules are hydrophobic, thus rapidly penetrate the cell membranes to reach intracellular targets. However, a large number of tumor cells overexpress multidrug transporter membrane proteins, which efficiently extrude hydrophobic drugs and thus may prevent the therapeutic action of TKIs. In the present work, we demonstrate that the most abundant and effective cancer multidrug transporters, MDR1 and MRP1, directly interact with several TKIs under drug development or already in clinical trials. This interaction with the transporters does not directly correlate with the hydrophobicity or molecular structure of TKIs, and shows a large variability in transporter selectivity and affinity. We suggest that performing enzyme- and cell-based multidrug transporter interaction tests for TKIs may greatly facilitate drug development, and allow the prediction of clinical TKI resistance based on this mechanism. Moreover, diagnostics for the expression of specific multidrug transporters in the malignant cells, combined with information on the interactions of the drug transporter proteins with TKIs, should allow a highly effective, individualized clinical treatment for cancer patients. [Copyright &y& Elsevier]

Effective treatments for chronic pain without abuse liability are urgently needed. One in 5 adults suffer chronic pain and half of these patients report inefficient treatment. Mu opioid receptor agonists (MOP), including oxycodone, tramadol and morphine, are often prescribed to treat chronic pain, however, use of drugs targeting MOP can lead to drug dependency, tolerance and overdose deaths. Kappa opioid receptor (KOP) agonists have antinociceptive effects without abuse potential; however, they have not been utilised clinically due to dysphoria and sedation. We hypothesise that mixed opioid receptor agonists targeting the KOP and delta opioid receptor (DOP) would have a wider therapeutic index, with the rewarding effects of DOP negating the negative effects of KOP. MP1104, an analogue of 3-Iodobenzoyl naltrexamine, is a novel mixed opioid receptor agonist with potent antinociceptive effects mediated via KOP and DOP in mice without rewarding or aversive effects. In this study, we show MP1104 has potent, long-acting antinociceptive effects in the warm-water tail-withdrawal assay in male and female mice and rats; and is longer acting than morphine. In the paclitaxel-induced neuropathic pain model in mice, MP1104 reduced both mechanical and cold allodynia and unlike morphine, did not produce tolerance when administered daily for 23 days. Moreover, MP1104 did not induce sedative effects in the open-field locomotor activity test, respiratory depression in mice using whole-body plethysmography, or have cross-tolerance with morphine. This data supports the therapeutic development of mixed opioid receptor agonists, particularly mixed KOP/DOP agonists, as non-addictive pain medications with reduced tolerance. Image 1 • MP1104 is a mixed opioid receptor agonist. • MP1104 exerts long-acting antinociceptive effects in the warm-water tail-withdrawal assay. • In a paclitaxel-induced neuropathic pain model, MP1104 reduced responding to non-disease levels. • MP1104 did not induce sedative or respiratory side-effects. [ABSTRACT FROM AUTHOR]

The present work represents the in vitro (potency, affinity, efficacy) and in vivo (antinociception, constipation) opioid pharmacology of the novel compound 14-methoxycodeine-6-O-sulfate (14-OMeC6SU), compared to the reference compounds codeine-6-O-sulfate (C6SU), codeine and morphine. Based on in vitro tests (mouse and rat vas deferens, receptor binding and [35S]GTPγS activation assays), 14-OMeC6SU has µ-opioid receptor-mediated activity, displaying higher affinity, potency and efficacy than the parent compounds. In rats, 14-OMeC6SU showed stronger antinociceptive effect in the tail-flick assay than codeine and was equipotent to morphine, whereas C6SU was less efficacious after subcutaneous (s.c.) administration. Following intracerebroventricular injection, 14-OMeC6SU was more potent than morphine. In the Complete Freund's Adjuvant-induced inflammatory hyperalgesia, 14-OMeC6SU and C6SU in s.c. doses up to 6.1 and 13.2 µmol/kg, respectively, showed peripheral antihyperalgesic effect, because co-administered naloxone methiodide, a peripherally acting opioid receptor antagonist antagonized the measured antihyperalgesia. In addition, s.c. C6SU showed less pronounced inhibitory effect on the gastrointestinal transit than 14-OMeC6SU, codeine and morphine. This study provides first evidence that 14-OMeC6SU is more effective than codeine or C6SU in vitro and in vivo. Furthermore, despite C6SU peripheral antihyperalgesic effects with less gastrointestinal side effects the superiority of 14-OMeC6SU was obvious throughout the present study. [ABSTRACT FROM AUTHOR]

Calcification of various tissues is a significant health issue associated with aging, cancer and autoimmune diseases. There are both environmental and genetic factors behind this phenomenon and understanding them is essential for the development of efficient therapeutic approaches. Pseudoxanthoma elasticum (PXE) is a rare genetic disease, a prototype for calcification disorders, resulting from the dysfunction of ABCC6, a transport protein found in the membranes of cells. It is identified by excess calcification in a variety of tissues (e.g., eyes, skin, arteries) and currently it has no cure, known treatments target the symptoms only. Preclinical studies of PXE have been successful in mice, proving the usefulness of animal models for the study of the disease. Here, we present a new zebrafish ( Danio rerio ) model for PXE. By resolving some ambiguous assemblies in the zebrafish genome, we show that there are two functional and one non-functional paralogs for ABCC6 in zebrafish ( abcc6a, abcc6b.1 , and abcc6b.2 , respectively). We created single and double mutants for the functional paralogs and characterized their calcification defects with a combination of techniques. Zebrafish deficient in abcc6a show defects in their vertebral calcification and also display ectopic calcification foci in their soft tissues. Our results also suggest that the impairment of abcc6b.1 does not affect this biological process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Czimer, Porok, Csete, Gyüre, Lavró, Fülöp, Chen, Gyergyák, Tusnády, Burgess, Mócsai, Váradi and Varga.)

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The COVID-19 epidemic hit everyone, professionals and civilians alike. The possibility of a worldwide pandemic has long been theorized by epidemiologists, infectologists on the one hand, and sociologists and behavioral scientists dealing with communication and social habits on the other. Yet, faced with real-time events, daily infections and mortality statistics, almost everyone feels uninformed or disturbingly inexperienced. This summary aims to provide an overview of the latest scientific evidences. Of course, the incomplete material, compiled in late March 2020, will certainly contain a few elements that likely will be outdated in a few weeks. The authors hope that in the next publication we will all read much better and more hopeful prospects. Orv Hetil. 2020; 161(17): 644–651., (© Szerző(k)/The Author(s))

Purpose: Sigma-1 receptors (S1Rs) are overexpressed in almost all human cancers, especially in breast cancers. 1-(4-Iodophenyl)-3-(2-adamantyl)guanidine (IPAG) is a validated high-affinity S1R antagonist. The objective of the current study is to evaluate the potential of iodine-124-labeled IPAG ([ 124 I]IPAG) to image S1R-overexpressing tumors., Procedures: [ 124 I]IPAG was synthesized from a tributyltin precursor dissolved in ethanol using chloramine-T as oxidant. Purity was analyzed using HPLC. In vitro and in vivo studies were performed using the breast cancer cell line MCF-7. Competitive inhibition studies were performed using haloperidol and cold IPAG. Tumors were established in athymic nude mice by injecting 10 7 cells subcutaneously. Mice were imaged on micro-positron emission tomography (PET) at 4, 24, 48, 72, and 144 h post i.v. injection. Biodistribution studies were performed at same time points. In vivo tracer dilution studies were performed using excess of IPAG and haloperidol. The efficacy of [ 124 I]IPAG to image tumors was evaluated in LNCaP tumor-bearing mice as well., Results: [ 124 I]IPAG was synthesized in quantitative yield and in vitro studies indicated that [ 124 I]IPAG binding was specific to S1R. PET imaging studies in MCF7 tumor-bearing mice reveal that [ 124 I]IPAG accumulates in tumor and is preferentially retained while clearing from non-target organs. The tumor to background increases with time, and tumors could be clearly visualized starting from 24 h post administration. Similar results were obtained in mice bearing LNCaP tumors. In vivo tracer dilution studies showed that the uptake of [ 124 I]IPAG could be competitively inhibited by excess of IPAG and haloperidol., Conclusions: [ 124 I]IPAG was synthesized successfully in high yields, and in vitro and in vivo studies demonstrate specificity of [ 124 I]IPAG. [ 124 I]IPAG shows specific accumulation in tumors with increasing tumor to background ratio at later time points and therefore has high potential for imaging S1R-overexpressing cancers.

Pseudoxanthoma elasticum is a heritable disease caused by ABCC6 deficiency. Patients develop ectopic calcification in skin, eyes, and vascular tissues. ABCC6, primarily found in liver and kidneys, mediates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi), a potent inhibitor of calcification. Pseudoxanthoma elasticum patients and Abcc6 -/- mice display reduced PPi levels in plasma and peripheral tissues. Pseudoxanthoma elasticum is currently incurable, although some palliative treatments exist. In recent years, we have successfully developed therapeutic methodologies to compensate the PPi deficit in animal models and humans. Here, we inadvertently discovered that modulating dietary PPi can also be an effective approach to reducing calcification in Abcc6 -/- mice. Our findings were prompted by a change in institutional rodent diet. The new chow was enriched in PPi, which increased plasma PPi, and significantly reduced mineralization in Abcc6 -/- mice. We also found that dietary PPi is readily absorbed in humans. Our results suggest that the consumption of food naturally or artificially enriched in PPi represents a possible intervention to mitigate calcification progression in pseudoxanthoma elasticum, that dietary preferences of patients may explain pseudoxanthoma elasticum heterogeneous manifestations, and that animal chow has the potential to influence data reproducibility., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A library-friendly approach to generate new scaffolds is decisive for the development of molecular probes, drug like molecules and preclinical entities. Here, we present the design and synthesis of novel heterocycles with spiro-2,6-dioxopiperazine and spiro-2,6-pyrazine scaffolds through a three-component reaction using various amino acids, ketones, and isocyanides. Screening of select compounds over fifty CNS receptors including G-protein coupled receptors (GPCRs), ion channels, transporters, and enzymes through the NIMH psychoactive drug screening program indicated that a novel spiro-2,6-dioxopyrazine scaffold, UVM147, displays high binding affinity at sigma-1 (σ 1 ) receptor in the nanomolar range. In addition, molecular docking of UVM147 at the human σ 1 receptor have shown that it resides in the same binding site that was occupied by the ligand 4-IBP used to obtain a crystal structure of the human sigma-1 (σ 1 ) receptor., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Ever since Garrod deduced the existence of inborn errors in 1901, a vast array of metabolic diseases has been identified and characterized in molecular terms. In 2018 it is difficult to imagine that there is any uncharted backyard left in the metabolic disease landscape. Nevertheless, it took until 2013 to identify the cause of a relatively frequent inborn error, pseudoxanthoma elasticum (PXE), a disorder resulting in aberrant calcification. The mechanism found was not only biochemically interesting but also points to possible new treatments for PXE, a disease that has remained untreatable. In this review we sketch the tortuous road that led to the biochemical understanding of PXE and to new ideas for treatment. We also discuss some of the controversies still haunting the field., (Copyright © 2018 Elsevier Ltd. All rights reserved.)