Mechanism-Based Allylic Carbasugar Chlorides That Form Covalent Intermediates with α- and ß-Galactosidases.

Glycoside hydrolases have been implicated in a wide range of human conditions including lysosomal storage diseases. Consequently, many researchers have directed their efforts towards identifying new classes of glycoside hydrolase inhibitors, both synthetic and from natural sources. A large percentage of such inhibitors are reversible competitive inhibitors that bind in the active site often due to them possessing structural features, often a protonatable basic nitrogen atom, that mimic the enzymatic transition state. We report that mechanism-based small molecule galacto-like configured cyclohexenyl carbasugars form reversible covalent complexes with both α-galactosidase and ß-galactosidase. In addition, we show that the ß-galactosidase from Aspergillus oryzae reacts with three different carbasugar inhibitors, with three different second-order rate constants (kinact/Ki), to give the same enzyme-carbasugar covalent intermediate. The surprising observation that the α-galacto-configured inhibitor covalently labels the A. oryzae ß-galactosidase highlights the catalytic versatility of glycoside hydrolases. We expect that cyclohexenyl covalent inhibitors will become an important class of compounds in the chemical biologist's tool box.

Synthesis of C-glycoside analogues of isopropyl ß-D-1-thiogalactopyranoside (IPTG) and 1-ß-D-galactopyranosyl-2-methylpropane. Conformational analysis and evaluation as inhibitors of the lac repressor in E. coli and as galactosidase inhibitors.

Isopropyl 1-thio-ß-D-galactopyranoside (IPTG, 1) is used widely as an inducer of protein expression in E. coli and 1-ß-D-galactopyranosyl-2-methylpropane (2), a C-glycoside analogue of 1, has also been identified as an inducer. Here, synthesis and study of mimetics of 1 and 2, 1-ß-D-galactopyranosyl-2-methylpropan-1-ols and two cyclic acetals derivatives, that constrain the presentation of the iPr group in various geometries is described. Conformational analysis of C-glycosides in protic solvent is performed using (i) Desmond metadynamics simulations (OPLS4) and (ii) use of 3JHH values obtained by 1H-NMR spectroscopy. 1-ß-D-Galactopyranosyl-2-methylpropane (2) is an effective protein expression inducer when compared to the new mimetics, which were less effective or did not induce expression. 1-ß-D-Galactopyranosyl-2-methylpropane (2) led to significantly reduced proteolysis during protein expression, compared to IPTG suggesting that recombinant protein purification will be easier to achieve with 2, yielding proteins with higher quality and activity. IPTG reduced bacterial growth to a greater degree than 2 compared to the control. IPTG's isopropyl group was observed by molecular dynamics (MD) simulations to be flexible in the binding pocket, deviating from its crystal structure binding mode, without impacting other interactions. The MD simulations predicted that 1-ß-D-galactopyranosyl-2-methylpropane (2) was more likely than IPTG to bind the repressor with a conformation favoured in protic solvent, while maintaining interactions observed for IPTG. MD simulations predicted that isobutanol derivatives may disrupt interactions associated with IPTG's binding mode. The compounds were also evaluated as inhibitors of galactosidases, with 2 being the more potent inhibitor of the E. coli ß-galactosidase. The constrained cyclic acetals showed similar inhibition constants to IPTG indicating E. coli ß-galactosidase can recognize galactopyranoses with varying presentation of the iPr group.

A concise synthetic approach for isoiminosugars.

Isoiminosugars are highly biological active substances. Herein, we report a concise synthetic approach for this class of compounds. The key step relies on a stereospecific 1,2-hydride shift in O-2 tosylated glycopyranosides leading to C-2 branched glycofuranosides. This approach enables a 4-step synthesis of powerful ß-galactosidase inhibitor 4-epi-isofagomine starting from a simple d-glucopyranoside.

Design and synthesis of 6-C-alkyl-DMDP type nanomolar inhibitors of ß-galactosidase and ß-glucosidase based on broussonetine S and related derivatives.

Broussonetine S (9), its C-1' and C-10' stereoisomers, and their corresponding enantiomers have been synthesized from enantiomeric arabinose-derived cyclic nitrones, with cross metathesis (CM), epoxidation and Keck asymmetric allylation as key steps. Glycosidase inhibition assays showed that broussonetine S (9) and its C-10' epimer (10'-epi-9) were nanomolar inhibitors of bovine liver ß-galactosidase and ß-glucosidase; while their C-1' stereoisomers were 10-fold less potent towards these enzymes. The glycosidase inhibition results and molecular docking calculations revealed the importance of the configurations of pyrrolidine core and C-1' hydroxyl for inhibition potency and spectra. Together with the docking calculations we previously reported for α-1-C-alkyl-DAB derivatives, we designed and synthesized a series of 6-C-alkyl-DMDP derivatives with very simple alkyl chains. The inhibition potency of these derivatives was enhanced by increasing the length of the side chain, and maintained at nanomolar scale inhibitions of bovine liver ß-glucosidase and ß-galactosidase after the alkyl groups are longer than eight or ten carbons for the (6R)-C-alkyl-DMDP derivatives and their 6S epimers, respectively. Molecular docking calculations indicated that each series of 6-C-alkyl-DMDP derivatives resides in the same active site of ß-glucosidase or ß-galactosidase with basically similar binding conformations, and their C-6 long alkyl chains extend outwards along the hydrophobic groove with similar orientations. The increasing inhibitions of ß-glucosidase and ß-galactosidase with the number of carbon atoms in the side chains may be explained by improved adaptability of longer alkyl chains in the hydrophobic grooves. In addition, the lower ß-glucosidase and ß-galactosidase inhibitions of (6S)-C-alkyl-DMDP derivatives than their C-6 R stereoisomers can be attributed to the misfolding of their alkyl chains and resulted decreased adaptability in the hydrophobic groove. The work reported herein is valuable for design and development of more potent and selective inhibitors of ß-galactosidase and ß-glucosidase, which have potential in treatment of lysosomal storage diseases. Furthermore, part of the 6-C-alkyl-DMDP derivatives and their enantiomers were also tested as potential anti-cancer agents; all the compounds tested were found with moderate cytotoxic effects on MKN45 cells, which would indicate potential applications of these iminosugars in development of novel anticancer agents.

Pharmacological Chaperones for ß-Galactosidase Related to GM1 -Gangliosidosis and Morquio B: Recent Advances.

A short survey on selected ß-galactosidase inhibitors as potential pharmacological chaperones for GM1 -gangliosidosis and Morquio B associated mutants of human lysosomal ß-galactosidase is provided highlighting recent developments in this particular area of lysosomal storage disorders and orphan diseases.

Mineral Analysis, In Vitro Evaluation of Alpha-Amylase, Alpha-Glucosidase, and Beta-Galactosidase Inhibition, and Antibacterial Activities of Juglans regia L. Bark Extracts.

In traditional medicine, various parts of the plant Juglans regia L. are used to treat several pathological conditions including diabetes and infectious and periodontal diseases. This includes the bark of Juglans regia. The present study is aimed at evaluating for the first time the mineral composition, investigating the antidiabetic and antibacterial properties of Moroccan J. regia bark, and finally determining the correlations between the chemical composition of the tested extracts and their biological activities. The mineral composition was determined using inductively coupled plasma atomic emission spectroscopy. Then, nine extracts were prepared by different methods and modalities of extractions and investigated for their antidiabetic activities, via tests of inhibition of alpha-amylase, alpha-glucosidase, and beta-galactosidase enzymes, and for their antibacterial activities against six strains involved in infectious diseases and periodontology. Finally, the correlation between the chemical compositions of the different extracts prepared and their antidiabetic and antibacterial potencies was determined by Principal Component Analysis (PCA). J. regia is an important source of mineral elements, mainly Fe (19849.8), K (3487.8), Mg (2631.03), and P (691.02) mg/kg plant material. All the extracts of J. regia possess antidiabetic activity, and in particular, the macerated acetone extract gave the highest inhibitory activity against alpha-amylase (IC50 value of 5445.33 ± 82.58 µg/mL), alpha-glucosidase (IC50 value of 323.7 ± 1.71 µg/mL), and beta-galactosidase (IC50 value of 811.2 ± 8.32 µg/mL). For the results of antibacterial activity, the macerated acetone extract at the concentration of 80 mg/mL was found to be the most active by inducing inhibition diameters of 12, 17, 18, 11, 14.5, and 16 mm against Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Listeria innocua, respectively. PCA allowed us to deduce that the extracts richer in polyphenols, in particular, the two acetone and ethanol macerates, have a better antidiabetic activity against alpha-glucosidase as well as a better antibacterial activity. The results of the present study revealed that the aqueous and organic macerate extracts showed a better antidiabetic activity and justified the use of J. regia bark as an antibacterial and antiseptic agent in traditional Moroccan medicine in the treatment of dental affections.

A versatile toolbox for investigating biological processes based on quinone methide chemistry: From self-immolative linkers to self-immobilizing agents.

Quinone methide (QM) species have been included in the design of various functional molecules. In this review, we present a comprehensive overview of bioanalytical tools based on QM chemistry. In the first part, we focus on self-immolative linkers that have been incorporated into functional molecules such as prodrugs and fluorescent probes. In the latter half, we outline how the highly electrophilic property of QMs, enabling them to react rapidly with neighboring nucleophiles, has been applied to develop inhibitors or labeling probes for enzymes, as well as self-immobilizing fluorogenic probes with high spatial resolution. This review systematically summarizes the versatile QM toolbox available for investigating biological processes.

Supramolecular Interaction of Molecular Cage and ß-Galactosidase: Application in Enzymatic Inhibition, Drug Delivery and Antimicrobial Activity.

Enzyme inhibitors play a crucial role in diagnosis of a wide spectrum of diseases related to bacterial infections. We report here the effect of a water-soluble self-assembled PdII8 molecular cage towards ß-galactosidase enzyme activity. The molecular cage is composed of a tetrapyridyl donor (L) and cis-[(en)Pd(NO3 )2 ] (en=ethane-1,2-diamine) acceptor and it has a hydrophobic internal cavity. We have observed that the acceptor moiety mainly possesses the ability to inactivate the ß-galactosidase enzyme activity. Kinetic investigation revealed the mixed mode of inhibition. This inhibition strategy was extended to control the growth of methicillin-resistant Staphylococcus aureus. The internalization of the Pd(II) cage inside the bacteria was confirmed when bacterial solutions were incubated with curcumin loaded cage. The intrinsic green fluorescence of curcumin made the bacteria glow when put under an optical microscope. Furthermore, this curcumin loaded molecular cage shows an enhanced antibacterial activity. Thus, PdII8 molecular cage is quite attractive due to its dual role as enzyme inhibitor and drug carrier.

Synthesis of "All-Cis" Trihydroxypiperidines from a Carbohydrate-Derived Ketone: Hints for the Design of New ß-Gal and GCase Inhibitors.

Pharmacological chaperones (PCs) are small compounds able to rescue the activity of mutated lysosomal enzymes when used at subinhibitory concentrations. Nitrogen-containing glycomimetics such as aza- or iminosugars are known to behave as PCs for lysosomal storage disorders (LSDs). As part of our research into lysosomal sphingolipidoses inhibitors and looking in particular for new ß-galactosidase inhibitors, we report the synthesis of a series of alkylated azasugars with a relative "all-cis" configuration at the hydroxy/amine-substituted stereocenters. The novel compounds were synthesized from a common carbohydrate-derived piperidinone intermediate 8, through reductive amination or alkylation of the derived alcohol. In addition, the reaction of ketone 8 with several lithium acetylides allowed the stereoselective synthesis of new azasugars alkylated at C-3. The activity of the new compounds towards lysosomal ß-galactosidase was negligible, showing that the presence of an alkyl chain in this position is detrimental to inhibitory activity. Interestingly, 9, 10, and 12 behave as good inhibitors of lysosomal ß-glucosidase (GCase) (IC50 = 12, 6.4, and 60 µM, respectively). When tested on cell lines bearing the Gaucher mutation, they did not impart any enzyme rescue. However, altogether, the data included in this work give interesting hints for the design of novel inhibitors.

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi ß-Galactosidase.

The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit ß-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its ß-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated ß-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel ß-galactosidase activity in the Golgi apparatus of human cells; however, the protein responsible for this activity could not be identified. Inhibitor-derived chemical probes can serve as powerful tools to identify the responsible protein. In this study, we first constructed a cell-based high-throughput screening (HTS) system for Golgi ß-galactosidase inhibitors, and then screened inhibitors from two compound libraries using the HTS system, in vitro assay, and cytotoxicity assay. An isoflavone derivative was identified among the final Golgi ß-galactosidase inhibitor compound hits. Molecular docking simulations were performed to redesign the isoflavone derivative into a more potent inhibitor, and six designed derivatives were then synthesized. One of the derivatives, ARM07, exhibited potent inhibitory activity against ß-galactosidase, with an IC50 value of 14.8 µM and competitive inhibition with Ki value of 13.3 µM. Furthermore, the in vitro and cellular inhibitory activities of ARM07 exceeded those of deoxygalactonojirimycin. ARM07 may contribute to the development of affinity-based chemical probes to identify the protein responsible for the newly discovered Golgi ß-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.

Reduction of senescence-associated beta-galactosidase activity by vitamin E in human fibroblasts depends on subjects' age and cell passage number.

Cell senescence is due to the permanent cell cycle arrest that occurs as a result of the inherent limited replicative capacity toward the Hayflick limit (replicative senescence), or in response to various stressors (stress-induced premature senescence, SIPS). With the acquisition of the senescence-associated secretory phenotype (SASP), cells release several molecules (cytokines, proteases, lipids), and express the senescence-associated beta-galactosidase (SA-ß-Gal). Here we tested whether vitamin E affects SA-ß-Gal in an in vitro model of cell ageing. Skin fibroblasts from human subjects of different age (1, 13, 29, 59, and 88 years old) were cultured until they reached replicative senescence. At different passages (Passages 2, 9, 13, and 16), these cells were treated with vitamin E for 24 hr. Vitamin E reduced SA-ß-Gal in all cells at passage 16, but at earlier passage numbers it reduced SA-ß-Gal only in cells isolated from the oldest subjects. Therefore, short time treatment with vitamin E decreases SA-ß-Gal in cells both from young and old subjects when reaching replicative senescence; but in cells isolated from older subjects, a decrease in SA-ß-Gal by vitamin E occurs also at earlier passage numbers. The possible role of downregulation of CD36 by vitamin E, a scavenger receptor essential for initiation of senescence and SASP, is discussed.

Assessment of skin sensitizing potential of metals with ß-galactosidase-expressing E. coli culture system.

It has been a challenge to develop in vitro alternative test methods for accurate prediction of metallic products which may exert skin sensitization, as several test methods adopted by OECD were relatively ineffective in assessing the capacity for metallic compounds to exert sensitizing reactions, compared with organic test substances. Based upon these findings, a system that incorporates ß-galactosidase producing E. coli cultures was tested for its predictive capacity to well-known metallic sensitizers. In this system, E. coli cells were incubated with metal salts at various concentrations and ß-galactosidase suppression by each test metal was determined. Fourteen local lymph node assay (LLNA) categorized metal salts were examined. Although color interference from metal salts was minimal, a fluorometric detection system was also employed using 4-methylumbelliferyl galactopyranoside as a substrate for ß-galactosidase to avoid the color interference, concomitantly with the original UV-spectrometric method. Data demonstrated that two detection methods were comparable and complementary. In addition, most of the metallic sensitizers were correctly identified at 0.6 and 0.8 mM concentrations. Despite the lower specificity obtained in the current study and small number of substances tested, the developed method appears to be a relatively simple and effective in vitro method for detecting metallic sensitizers. When 61 chemicals tested in the ß-galactosidase producing E. coli cultures including the present study were collectively analyzed, the prediction capacity was as high as other OECD-adopted tests: 95.6% of sensitivity, 66.7% of specificity, and 88.5% of accuracy. It is important to emphasize that animals or mammalian cell cultures were not required in the current method, which are in accordance with the EU guidelines on restricted or banned animal testing.

Antiadhesive Nanosomes Facilitate Targeting of the Lysosomal GlcNAc Salvage Pathway through Derailed Cancer Endocytosis.

Activated endocytosis of extracellular macromolecules and their intracellular trafficking to lysosomes is an essential metabolic mechanism in cancer cells during their rapid proliferation. Cancer cells reuse a vast amount of N-acetylglucosamine (GlcNAc) supplied from the GlcNAc salvage pathway for the accelerated synthesis of a pivotal uridine diphosphate (UDP)-GlcNAc. A method to inactivate key glycosidases in lysosomes could critically contribute to the development of potent anticancer therapy. Here we demonstrate that "nanosomes" made of core metals covered by an antiadhesive mixed self-assembled monolayer allow for avoiding nonspecific surface protein corona and targeted molecular delivery through activated endocytosis. Nanosomes carrying suicide substrates showed that lysosomal glycosidases such as ß-hexosaminidase and ß-galactosidase in cancer cells are promising targets for novel anticancer therapeutic nanomedicine that induce apoptotic cell death through lysosomal membrane permeabilization. The advantage of this method is evident because multivalent surface loading by antiadhesive nanosomes makes it possible to highlight "weak interactions" such as carbohydrate-lectin interactions independent of surface protein corona.

Mechanism of antimicrobial peptide NP-6 from Sichuan pepper seeds against E. coli and effects of different environmental factors on its activity.

A novel antimicrobial peptide named NP-6 was identified in our previous work. Here, the mechanisms of the peptide against Escherichia coli (E. coli) were further investigated, as well as the peptide's resistance to temperature, pH, salinity, and enzymes. The transmission electron microscopy (TEM), confocal laser scanning microcopy (CLSM), and flow cytometric (FCM) analysis, combined with measurement of released K+, were performed to evaluate the effect of NP-6 E. coli cell membrane. The influence of NP-6 on bacterial DNA/RNA and enzyme was also investigated. The leakage of K+ demonstrated that NP-6 could increase the permeability of E. coli cell membrane. The ATP leakage, FCM, and CLSM assays suggested that NP-6 caused the disintegration of bacterial cell membrane. The TEM observation indicated that NP-6 could cause the formation of empty cells and debris. Besides, the DNA-binding assay indicated that NP-6 could bind with bacterial genomic DNA in a way that ethidium bromide (EB) did, and suppress the migration of DNA/RNA in gel retardation. Additionally, NP-6 could also affect the activity of ß-galactosidase. Finally, the effect of different surroundings such as heating, pH, ions, and protease on the antimicrobial activity of NP-6 against E. coli was also investigated. Results showed that the peptide was heat stable in the range of 60~100 °C and performed well at pH 6.0~8.0. However, the antimicrobial activity of NP-6 decreased significantly in the presence of Mg2+/Ca2+, and after incubation with trypsin/proteinase K. The results will provide a theoretical support in the further application of NP-6.

Fine air pollution particles induce endothelial senescence via redox-sensitive activation of local angiotensin system.

Fine dust (FD) is a form of air pollution and is responsible for a wide range of diseases. Specially, FD is associated with several cardiovascular diseases (CVDs); long-term exposure to FD was shown to decrease endothelial function, but the underlying mechanism remains unclear. We investigated whether exposure to FD causes premature senescence-associated endothelial dysfunction in endothelial cells (ECs) isolated from porcine coronary arteries. The cells were treated with different concentrations of FD and senescence associated-beta galactosidase (SA-ß-gal) activity, cell cycle progression, expression of endothelial nitric oxide synthase (eNOS), oxidative stress level, and vascular function were evaluated. We found that FD increased SA-ß-gal activity, caused cell cycle arrest, and increased oxidative stress, suggesting the premature induction of senescence; on the other hand, eNOS expression was downregulated and platelet aggregation was enhanced. FD exposure impaired vasorelaxation in response to bradykinin and activated the local angiotensin system (LAS), which was inhibited by treatment with the antioxidant N-acetyl cysteine (NAC) and angiotensin II receptor type 1 (AT1) antagonist losartan (LOS). NAC and LOS also suppressed FD-induced SA-ß-gal activity, increased EC proliferation and eNOS expression, and improved endothelial function. These results demonstrate that FD induces premature senescence of ECs and is associated with increased oxidative stress and activation of LAS. This study can serve as a pharmacological target for prevention and/or treatment of air pollution-associated CVD.

A multi-pumping flow analysis system for ß-galactosidase activity assays.

A fully mechanized Arduino-controlled multi-pumping flow analysis system and procedure for the determination of ß-galactosidase activity are proposed. The applied bioanalytical method is based on the determination of p-nitrophenol formed in the course of enzyme-catalyzed hydrolysis of p-nitrophenyl-galactopyranosides. The photometric detection is performed using dedicated flow-through optoelectronic detector made of paired light emitting diodes. The developed bioanalytical system was applied for evaluation of optimal enzyme detection conditions (pH, temperature and reaction time), selection of appropriate substrate for the assays, comparison of enzymes of different origins (isoenzymes), detection of ß-galactosidase inhibitor and finally to the determination of enzyme activity in some dietary supplements dedicated for people suffering from lactose intolerance. Depending on measurements conditions the developed bioanalytical system allows determination of ß-galactosidase in the wide range of activity (up to 15 U/mL at detection limit ca 0.01 U/mL) with high sample flowthroughput (up to 30 detections per hour). Additionally, the potential utility of the developed analytical system for amyloglucosidase activity assays has been demonstrated.

Isolation from Stevia rebaudiana of DMDP acetic acid, a novel iminosugar amino acid: synthesis and glycosidase inhibition profile of glycine and ß-alanine pyrrolidine amino acids.

DMDP acetic acid [N-carboxymethyl-2,5-dideoxy-2,5-imino-D-mannitol] 5 from Stevia rebaudiana is the first isolated natural amino acid derived from iminosugars bearing an N-alkyl acid side chain; it is clear from GCMS studies that such derivatives with acetic and propionic acids are common in a broad range of plants including mulberry, Baphia, and English bluebells, but that they are very difficult to purify. Reaction of unprotected pyrrolidine iminosugars with aqueous glyoxal gives the corresponding N-acetic acids in very high yield; Michael addition of both pyrrolidine and piperidine iminosugars and that of polyhydroxylated prolines to tert-butyl acrylate give the corresponding N-propionic acids in which the amino group of ß-alanine is incorporated into the heterocyclic ring. These easy syntheses allow the identification of this new class of amino acid in plant extracts and provide pure samples for biological evaluation. DMDP N-acetic and propionic acids are potent α-galactosidase inhibitors in contrast to potent ß-galactosidase inhibition by DMDP.

Safeguarding the catalytic activity and stability of polyaniline chitosan silver nanocomposite bound beta-galactosidase against product inhibitors and structurally related compound.

In this study, an attempt has been made to evaluate the effect of products of ß-galactosidase (ßGS) catalyzed reaction i.e. glucose and galactose and their structurally related compound vitamin C (VC) on the catalytic activity of native and PANI-CS-NC and PANI-CS-Ag-NC adsorbed ßGS. Results indicated a decline in catalytic activity of soluble enzyme in the presence of all investigated compounds. The order of inhibition was found to be VC < glucose < galactose. However, the immobilized preparations were found more resistant to inactivation caused by the added compounds. About 48% activity was retained by PANI-CS-Ag-NC-ßGS in the presence of galactose (5%, w/v), while the native enzyme exhibited only 18% of its original activity. A significant decrease in absorbance and fluorescence intensity was evaluated in soluble enzyme incubated in the presence of all investigated compounds. Three-dimensional fluorescence graphs, CD and FT-IR spectroscopic studies illustrated noteworthy conformational changes in the secondary structure and microenvironment of the soluble enzyme in the presence of VC and tested sugars. These results suggest that both PANI-CS-NC and PANI-CS-Ag-NC bound ßGS are more resistant to the exposure caused by the higher concentration of added glucose, galactose, and VC and, therefore, can be effectively utilized for the production of a hassle-free lactose nano-biosensor.

Impact of Elastin-Derived Peptide VGVAPG on Matrix Metalloprotease-2 and -9 and the Tissue Inhibitor of Metalloproteinase-1, -2, -3 and -4 mRNA Expression in Mouse Cortical Glial Cells In Vitro.

Degradation products of elastin, i.e. elastin-derived peptides (EDPs), are involved in various physiological and pathological processes. EDPs are detectable in cerebrospinal fluid in healthy people and in patients after ischemic stroke. However, to date, no studies concerning the role of EDP in the nervous system were conducted. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play important roles during the repair phases of cerebral ischemia, particularly during angiogenesis and reestablishment of cerebral blood flow. Therefore, the aim of this study was to investigate the impact of the specific elastin-derived peptide VGVAPG on Mmp-2, -9 and Timp-1, -2, -3 and -4 mRNA expression in mouse cortical glial cells in vitro. Primary glial cells were maintained in DMEM/F12 without phenol red supplemented with 10% fetal bovine serum and the cells were exposed to 50 nM, 1 and 50 µM of the VGVAPG peptide. After 3 and 6 h of exposition to the peptide, expression of Mmp-2, -9 and Timp-1, -2, -3 and -4 mRNA was measured. Moreover, siRNA gene knockdown, cytotoxicity and apoptosis measurement were included in our experiments, which showed that VGVAPG in a wide range of concentrations exhibited neither proapoptotic nor cytotoxic properties in mouse glial cells in vitro. The peptides enhanced mRNA expression of Timp-2 and Timp-3 genes in an elastin-binding protein (EBP)-dependent manner. However, changes in mRNA expression of Mmp-2, Mmp-9 and Timp-4 were partially EBP-dependent. The decrease in mRNA expression of Timp-1 was EBP-independent. However, further studies underlying the VGVAPG peptide's mechanism of action in the nervous system are necessary.

4-epi-Isofagomine derivatives as pharmacological chaperones for the treatment of lysosomal diseases linked to ß-galactosidase mutations: Improved synthesis and biological investigations.

(5aR)-5a-C-pentyl-4-epi-isofagomine 1 is a powerful inhibitor of lysosomal ß-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B. We report herein an improved synthesis of this compound and analogs (5a-C-methyl, pentyl, nonyl and phenylethyl derivatives), and a crystal structure of a synthetic intermediate that confirms its configuration resulting from the addition of a Grignard reagent. These compounds were evaluated as glycosidase inhibitors and their potential as chaperones for mutant lysosomal galactosidases determined. Based on these results and on docking studies, the 5-C-pentyl derivative 1 was selected as the optimal structure for further investigations: this compound induces the maturation of mutated ß-galactosidase in fibroblasts of a GM1-gangliosidosis patient and promote the decrease of keratan sulfate and oligosaccharide load in patient cells. Compound 1 is clearly capable of restoring ß-galactosidase activity and of promoting maturation of the protein, which should result in significant clinical benefit. These properties strongly support the development of compound 1 for the treatment of GM1-gangliosidosis and Morquio disease type B patients harboring ß-galactosidase mutations sensitive to pharmacological chaperoning.