Stop codon readthrough as a treatment option for epidermolysis bullosa-Where we are and where we are going.

In the context of rare genetic diseases caused by nonsense mutations, the concept of induced stop codon readthrough (SCR) represents an attractive avenue in the ongoing search for improved treatment options. Epidermolysis bullosa (EB)-exemplary for this group of diseases-describes a diverse group of rare, blistering genodermatoses. Characterized by extreme skin fragility upon minor mechanical trauma, the most severe forms often result from nonsense mutations that lead to premature translation termination and loss of function of essential proteins at the dermo-epidermal junction. Since no curative interventions are currently available, medical care is mainly limited to alleviating symptoms and preventing complications. Complementary to attempts of gene, cell and protein therapy in EB, SCR represents a promising medical alternative. While gentamicin has already been examined in several clinical trials involving EB, other potent SCR inducers, such as ataluren, may also show promise in treating the hitherto non-curative disease. In addition to the extensively studied aminoglycosides and their derivatives, several other substance classes-non-aminoglycoside antibiotics and non-aminoglycoside compounds-are currently under investigation. The extensive data gathered in numerous in vitro experiments and the perspectives they reveal in the clinical setting will be discussed in this review.

Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond.

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.

Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa.

INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.

Argatroban pharmacokinetics and pharmacodynamics in critically ill cardiac surgical patients with suspected heparin-induced thrombocytopenia.

Only limited data are available on the pharmacokinetic and pharmacodynamic properties of argatroban in critically ill patients under clinical conditions. We determined plasma concentrations of argatroban, and its main metabolite M1, within a time period of 48 hours in 25 critically ill cardiac surgical patients, who were suspected of heparin-induced thrombocytopenia and had the clinical need for anticoagulation. Argatroban infusion was started at 0.5 µg/kg/minute, and adjusted in 0.1-0.25 µg/kg/minute increments when the activated partial thromboplastin time (aPTT) was not within the target range. Median argatroban plasma half-life was 2.7 hours (interquartile range 1.8 to 7.3). Linear regression analysis revealed that argatroban half-life was significantly related to the total bilirubin concentration (R² = 0.66, p< 0.001), as well as to the metabolism of argatroban, which was assessed by the ratio of the areas under the concentration time curves (AUC) of argatroban and M1 (R² = 0.60, p< 0.001). Continuous veno-venous haemodialysis did not significantly affect argatroban plasma half-life. The predictive property of argatroban plasma levels for aPTT was low (R² = 0.28, p< 0.001). Multiple linear regression analysis revealed significant contributions of age and serum albumin levels to the effect of argatroban on aPTT, expressed as the AUC ratio argatroban/aPTT (R² = 0.67, adjusted R² = 0.65, p< 0.001). In conclusion, argatroban plasma half-life is markedly increased in critically ill cardiac surgical patients, and further prolonged by hepatic dysfunction due to impaired metabolism. Patient age and serum albumin concentration significantly contribute to the variability in the anticoagulant activity of argatroban.

CuPc/Au(1 1 0): Determination of the azimuthal alignment by a combination of angle-resolved photoemission and density functional theory.

Here we report on a combined experimental and theoretical study on the structural and electronic properties of a monolayer of Copper-Phthalocyanine (CuPc) on the Au(1 1 0) surface. Low-energy electron diffraction reveals a commensurate overlayer unit cell containing one adsorbate species. The azimuthal alignment of the CuPc molecule is revealed by comparing experimental constant binding energy (kxky )-maps using angle-resolved photoelectron spectroscopy with theoretical momentum maps of the free molecule's highest occupied molecular orbital (HOMO). This structural information is confirmed by total energy calculations within the framework of van-der-Waals corrected density functional theory. The electronic structure is further analyzed by computing the molecule-projected density of states, using both a semi-local and a hybrid exchange-correlation functional. In agreement with experiment, the HOMO is located about 1.2 eV below the Fermi-level, while there is no significant charge transfer into the molecule and the CuPc LUMO remains unoccupied on the Au(1 1 0) surface.

Mixed-valence interactions in triarylamine-gold-nanoparticle conjugates.

Optical and electrochemical investigations of triarylamine redox centres attached to gold nanoparticles via a pi-conjugated bridge show intervalence charge-transfer bands which prove to be surprisingly strong interchromophore interactions.

Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells.

Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.

Differential modulation of the glutamate transporters GLT1, GLAST and EAAC1 by docosahexaenoic acid.

At present, the ability of polyunsaturated fatty acids (PUFAs) to regulate individual glutamate transporter subtypes is poorly understood and very little information exists on the mechanism(s) by which PUFAs achieve their effects on the transport process. Here we investigate the effect of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the activity of the mammalian glutamate transporter subtypes, GLT1, GLAST and EAAC1 individually expressed in human embryonic kidney (HEK) cells. Exposure of cells to 100 muM DHA increased the rate of d-[(3)H]aspartate uptake by over 72% of control in HEK(GLT1) cells, and by 45% of control in HEK(EAAC1) cells. In contrast, exposure of HEK(GLAST) cells to 200 muM DHA resulted in almost 40% inhibition of d-[(3)H]aspartate transport. Removal of extracellular calcium increased the inhibitory potential of DHA in HEK(GLAST) cells. In contrast, in the absence of extracellular calcium, the stimulatory effect of DHA on d-[(3)H]aspartate uptake in HEK(GLT1) and HEK(EAAC1) cells was abolished, and significant inhibition of the transport process by DHA was observed. Inhibition of CaM kinase II or PKC had no effect on the ability of DHA to inhibit transport into HEK(GLAST) cells but abolished the stimulatory effect of DHA on d-[(3)H]aspartate transport into HEK(GLT1) and HEK(EAAC1) cells. Inhibition of PKA had no effect on the modulation of d-[(3)H]aspartate transport by DHA in any of the cell lines. We conclude that DHA differentially modulates the GLT1, GLAST and EAAC1 glutamate transporter subtypes via different mechanisms. In the case of GLT1 and EAAC1, DHA appears to stimulate d-[(3)H]aspartate uptake via a mechanism requiring extracellular calcium and involving CaM kinase II and PKC, but not PKA. In contrast, the inhibitory effect of DHA on GLAST does not require extracellular calcium and does not involve CaM kinase II, PKC or PKA.

A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain.

We have identified a novel carboxyl-terminal splice-variant of the glutamate transporter GLT1, which we denote as GLT1c. Within the rat brain only low levels of protein and message were detected, protein expression being restricted to end feet of astrocytes apposed to blood vessels or some astrocytes adjacent to the ventricles. Conversely, within the retina, this variant was selectively and heavily expressed in the synaptic terminals of both rod- and cone-photoreceptors in both humans and rats. Double-immunolabelling with antibodies to the carboxyl region of GLT1b/GLT1v, which is strongly expressed in apical dendrites of bipolar cells and in cone photoreceptors revealed that in the rat GLT1c was co-localised with GLT1b/GLT1v in cone photoreceptors but not with GLT1b/GLT1v in bipolar cells. GLT1c expression was developmentally regulated, only appearing at around postnatal day 7 in the rat retina, when photoreceptors first exhibit a dark current. Since the glutamate transporter EAAT5 is also expressed in terminals of rod photoreceptor terminals these data indicate that rod photoreceptors express two glutamate transporters with distinct properties. Similarly, cone photoreceptors express two glutamate transporters. We suggest that differential usage of these transporters by rod and cone photoreceptors may influence the kinetics of glutamate transmission by these neurons.

Cloning, transport properties, and differential localization of two splice variants of GLT-1 in the rat CNS: implications for CNS glutamate homeostasis.

At least two splice variants of GLT-1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT-1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT-1, namely GLT-1alpha. We demonstrate that GLT-1alpha is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT-1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT-1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT-1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.

Localization and possible function of the glutamate transporter, EAAC1, in the rat retina.

We investigated the localization and possible function of EAAC1 in the rat retina. Immunocytochemical localization of EAAC1 at the light-microscopic level revealed a fine dust-like labelling pattern across the two synaptic layers. Horizontal cell and subpopulations of amacrine cell somata were labelled, as were some somata within the ganglion cell layer. Some immunoreactive puncta were observed within the cytoplasm of amacrine cells, in regions well away from synaptic sites. At the ultrastructural level, EAAC1 immunolabelled one postsynaptic element at synapses and also processes well away from the synaptic release site. Since EAAC1 was localized away from synaptic sites, we evaluated the role EAAC1 plays in GABA formation by measuring GABA concentrations via reversed-phase high-performance liquid chromatography following incubation of retinae in enzyme and glutamate uptake inhibitors. Incubation of retinae in D-threo-beta-hydroxyaspartate or D/ L-threo-beta-benzyloxyaspartate, which are known to inhibit the glutamate transporters GLAST1, GLT1, and EAAC1, caused a decrease in GABA synthesis by around 50%. Incubation in 6-diazo-5-oxo- L-norleucine, a phosphate-activated glutaminase inhibitor, decreased GABA formation by 40%. Taken together with the anatomical data, the results of this study suggest that EAAC1 plays very little role in GABA synthesis - indeed GABA formation occurs predominantly from glutamine. By virtue of its location both near and well away from synaptic release sites, EAAC1 may regulate glutamate uptake differentially.