Mechanistic Insights into the Chaperoning of Human Lysosomal-Galactosidase Activity: Highly Functionalized Aminocyclopentanes and C-5a-Substituted Derivatives of 4-epi-Isofagomine.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid ß-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent ß-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of ß-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

Potent GH20 N-Acetyl-ß-d-hexosaminidase Inhibitors: N-Substituted 3-acetamido-4-amino-5-hydroxymethyl-cyclopentanediols.

From 1,2;3,4-di-O-isopropylidene-d-galactopyranose, a preliminary series of highly functionalized amino(hydroxymethyl)cyclopentanes was easily available. These amine-containing basic carbasugars featuring the d-galacto configuration are potent inhibitors of the GH20 ß-d-hexosaminidases probed and may bear potential as regulators of N-acetyl-d-hexosaminidase activities in vivo.

A new type of pharmacological chaperone for GM1-gangliosidosis related human lysosomal ß-galactosidase: N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols.

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of ß-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

Synthesis of C-5a-chain extended derivatives of 4-epi-isofagomine: Powerful ß-galactosidase inhibitors and low concentration activators of GM1-gangliosidosis-related human lysosomal ß-galactosidase.

From an easily available partially protected formal derivative of 1-deoxymannojirimycin, by hydroxymethyl chain-branching and further elaboration, lipophilic analogs of the powerful ß-d-galactosidase inhibitor 4-epi-isofagomine have become available. New compounds exhibit improved inhibitory activities comparable to benchmark compound NOEV (N-octyl-epi-valienamine) and may serve as leads towards improved and more selective pharmacological chaperones for GM1-gangliosidosis.

Correction: The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0015730.].

Comparison of hybridization methods and real-time PCR: their value in animal cell line characterization.

For biotechnological protein production, the International Conference of Harmonization (ICH) recommends appropriate testing of expression constructs and characterization of producer cell lines in order to assure uniform specifications, improve safety, and confirm stable productivity. Commonly, hybridization analyses are used for the evaluation of genetic stability, gene and transcript copy numbers as well as the integrity of the coding sequence. However, improvements in polymerase chain reaction (PCR) techniques have accelerated the analysis of genetic parameters and have nowadays become the method of choice for the evaluation of gene copy numbers and transcript levels. Nevertheless, Southern and Northern blot analyses are still valuable tools that deliver additional information to PCR results during cell clone characterization studies. In this study, we discuss advantages and drawbacks of hybridization and PCR methods in regard to their applicability and efficiency during the development of recombinant mammalian cell lines. A comparative review of the literature as well as an overview of findings from our own group will be given.

A Morita-Baylis-Hillman based route to C-5a-chain-extended 4-epi-isofagomine type glycosidase inhibitors.

By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,ß-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of ß-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal ß-galactosidase mutant R201C.

N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new family of activity promotors for a GM1-gangliosidosis related human lysosomal ß-galactosidase mutant.

From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of ß-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable ß-galactosidase inhibitors and activity promotors of GM1-gangliosidosis related human lysosomal ß-galactosidase mutant R201C.

From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching at C-4 and suitable modification, lipophilic analogs of the powerful ß-D-galactosidase inhibitor 4-epi-isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities when compared with the unsubstituted parent compound and may serve as leads toward new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

Proteomic analysis of human cataract aqueous humour: Comparison of one-dimensional gel LCMS with two-dimensional LCMS of unlabelled and iTRAQ®-labelled specimens.

In this study, we report a comparative and quantitative analysis by mass spectrometry of the protein content of aqueous humour from cataract (control) patients. In addition to protein profiling, the approach is layered with quantitative proteomics using the iTRAQ® methodology. Aqueous humour from ten clinically-matched patients was collected and depleted of albumin and immunoglobulin G. Pairs of patient material were pooled and divided into three aliquots for subsequent analysis by alternative proteomic approaches. Excluding keratin, trypsin, residual albumin and immunoglobulins, a total of 198 protein groups were identified across the entire study. Relative protein quantitation with iTRAQ® revealed that 88% of the proteins had a maximal ±2-fold differential regulation between 3 of the 4 labelled samples, indicating minimal variation. The identified proteins were categorised by gene ontology and one third of the proteins were annotated as extracellular. The major molecular functions of the proteins in aqueous humour are binding (protein, metal ion, heparin, and DNA) and inhibition of proteolytic activity. Complementary to molecular function, the predominant biological processes for the proteins in aqueous humour are assigned to inflammatory and immune responses, and transport.

The tight junction associated signalling proteins ZO-1 and ZONAB regulate retinal pigment epithelium homeostasis in mice.

Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration.

AAV-Mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa.

In the Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) cannot phagocytose the outer segment discs that are continually shed from photoreceptors. The resulting accumulation of debris in the subretinal space leads to a progressive loss of photoreceptors. The defect results from a mutation in the Mertk gene, which is normally expressed in the RPE. Mertk is a receptor tyrosine kinase, involved in the binding of photoreceptor debris. Mutations in MERTK have also been described in patients with retinitis pigmentosa (RP). Here we demonstrate that subretinal injection of recombinant adeno-associated virus (AAV) expressing the murine Mertk gene can significantly prolong photoreceptor cell survival in the RCS rat. Electroretinographic analysis of treated eyes showed that functional photoreceptors were still present at 9 weeks, when there is virtually no activity in untreated control eyes. Histological analysis of treated eyes revealed a decrease in the amount of debris in the subretinal space, suggesting that RPE function was restored. Moreover, 9 weeks after treatment the number of photoreceptors was 2.5-fold higher in treated than in control eyes. This study provides strong support for the development of AAV-mediated gene therapy for RP caused by mutations in the MERTK gene.