n-Propyl 6-amino-2,6-dideoxy-2,2-difluoro-ß-d-glucopyranoside is a good inhibitor for the ß-galactosidase from E. coli.

A convenient route has been developed for the synthesis of novel 6-amino-2,2-(or 3,3-difluoro)-2-(or 3),6-dideoxy-hexopyranoses. Biological screening showed these compounds as good inhibitors for several glycosidases. Especially n-propyl 6-amino-2,6-dideoxy-2,2-difluoro-ß-d-glucopyranoside (8) was an excellent competitive inhibitor for the ß-galactosidase from E. coli holding a K i of 0.50 µM.

Synthesis and biochemical evaluation of two novel N-hydroxyalkylated cyclosporin A analogs.

The cyclic undecapeptide cyclosporin A (CsA) is a widely used immunosuppressive agent. Its immunosuppressive properties arise from strong binding to cyclophilins (Cyp) followed by inhibition of the protein calcineurin (CaN) by the binary CsA/Cyp complex and subsequent negative regulation of T-cell activation. In the present study we show a novel way to modify the CsA ring by selective N-hydroxyalkylation of the residues Val5 and d-Ala8. Moreover, the influence of these structural CsA modifications on the ability of the CsA analogs to bind Cyp, to inhibit CaN and to penetrate membranes of living cells was investigated. Our results show that the Val5 N-substitution significantly improved compound cell-permeability and markedly diminished CaN inhibition by the binary CsA analog/CypA complex but to a lesser extent Cyp inhibition. In contrast, the N-alkylation of d-Ala8 gave a product with significantly reduced affinity for Cyp but its immunosuppressive effects remained similar to CsA. Possible explanations of the observed experimental data are provided by computational studies.

Screening for Selective Protein Inhibitors by Using the IANUS Peptide Array.

Finding new road blacks: A peptidic inhibitor of calcineurin (CaN)-mediated nuclear factor of activated T cells (NFAT) dephosphorylation, which is developed through a template-assisted IANUS (Induced orgANisation of strUcture by matrix-assisted togethernesS) peptide array, is cell permeable and able to block the translocation of green fluorescent protein-NFAT fusion protein (GFP-NFAT) into the nucleus after stimulation.

Assessing Glomerular Filtration in Small Animals Using [68Ga]DTPA and [68Ga]EDTA with PET Imaging.

PURPOSE: Determining the glomerular filtration rate (GFR) is essential for clinical medicine but also for pre-clinical animal studies. Functional imaging using positron emission tomography (PET) allows repetitive almost non-invasive measurements. The aim of the study was the development and evaluation of easily synthesizable PET tracers for GFR measurements in small animals. PROCEDURES: Diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) were labeled with Ga-68. The binding to blood cells and plasma proteins was tested in vitro. The distribution of the tracers in rats was analyzed by PET imaging and ex vivo measurements. From the time-activity-curve of the blood compartment (heart) and the total tracer mass excreted by the kidney, the GFR was calculated. These values were compared directly with the inulin clearance in the same animals. RESULTS: Both tracers did not bind to blood cells. [68Ga]DPTA but not [68Ga]EDTA showed strong binding to plasma proteins. For this reason, [68Ga]DPTA stayed much longer in the blood and only 30 % of the injected dose was eliminated by the kidney within 60 min whereas the excretion of [68Ga]EDTA was 89 ± 1 %. The calculated GFR using [68Ga]EDTA was comparable to the measured inulin clearance in the same animal. Using [68Ga]-DPTA, the measurements led to values which were 80 % below the normal GFR. The results also revealed that definition of the volume of interest for the blood compartment affects the calculation and may lead to a slight overestimation of the GFR. CONCLUSIONS: [68Ga]EDTA is a suitable tracer for GFR calculation from PET imaging in small animals. It is easy to be labeled, and the results are in good accordance with the inulin clearance. [68Ga]DTPA led to a marked underestimation of GFR due to its strong binding to plasma proteins and is therefore not an appropriate tracer for GFR measurements.

Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes.

The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turnover in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway have been discovered, the ubiquitination mechanism and substrate specificity of N-end rule pathway E3 ubiquitin ligases have remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we used a novel tool to molecularly characterize polyubiquitination live, in real time. We gained mechanistic insights into PRT1 substrate preference and activation by monitoring live ubiquitination using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in-gel fluorescence scanning as well as in real time by fluorescence polarization. The enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc instantaneously and with significantly reduced reagent consumption. We demonstrated that PRT1 is indeed an E3 ligase, which has been hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.

Identification of low abundance cyclophilins in human plasma.

Cylophilins (Cyps) belong to the ubiquitously distributed enzyme class of peptidyl prolyl cis/trans isomerases (EC5.2.1.8), which are foldases capable of accelerating slow steps in the refolding of denatured proteins. At least 20 different Cyp isoenzymes are broadly distributed among all organs and cellular compartments in humans. Extracellularly localized Cyps came into the scientific focus recently because of their involvement in the control of inflammatory diseases, as well as viral and bacterial infections. However, detailed insights into Cyp functions are often hampered by the lack of sensitive detection methods. We present an improved method for affinity purification and detection of Cyp in biotic samples in this manuscript. The procedure takes advantage of two novel cyclosporine A derivatives. Derivative 1 was used to capture Cyps from the sample while derivative 2 was applied for selective release from the affinity matrix. Using this approach, eight different Cyp (CypA, CypB, CypC, Cyp40 (PPID), CypE, CypD (PPIF), CypH, and CypL1) were unambiguously detected in healthy human blood plasma. Moreover, extracellular CypA was found to be partially modified by Nε acetylation on residues Lys44, Lys133, Lys155, as well as Nα  acetylation at the N-terminal Val residue. Nα  acetylation of Ser2 residue was also found for Cyp40.

Elevated exhaled leukotriene B4 in the small airway compartment in children with asthma.

BACKGROUND: Inflammatory processes in the asthmatic lung involve the large and small airway and alveolar sites. Leukotriene B4 (LTB4) is an important disease marker, but its role in inflammation of the small airways in asthma has not been established yet. OBJECTIVE: To distinguish between large and small airway or alveolar LTB4 concentrations in children with asthma using the new technique of fractionated exhaled breath condensate sampling. METHODS: Sixty-eight children (9-17 years old, 33 children with asthma and 35 controls) underwent fractional exhaled nitric oxide (FeNO) measurements, lung function testing, and collection of fractionated exhaled breath condensate using a capnograph-based approach. The LTB4 concentrations in the small airway or alveolar and large airway fractions were correlated to disease status, lung function impairment, and clinical parameters. RESULTS: Children with asthma had significantly higher LTB4 concentrations in the small airway or alveolar fraction than controls (5.58 pg/mL; 95% interquartile range [IQR], 2.0-11.77 pg/mL; vs 2.0 pg/mL; 95% IQR, 2.0-6.2 pg/mL; P = .003). No difference was found between the groups in the large airway fraction. Children with obstructive lung function impairment (forced expiratory volume in 1 second z score <-1.65) had increased small airway or alveolar LTB4 concentrations compared with children without impairment (2.0 pg/mL; 95% IQR, 2.0-9.21 pg/mL; vs 18.32 pg/mL; 95% IQR, 3.7-23.02 pg/mL; P = .04). Children with asthma but without pathologic obstructive lung function still had higher LTB4 concentrations than controls (5.57 pg/mL; 95% IQR, 2.00-10.60 pg/mL; vs 2.00 pg/mL; 95% IQR, 2.00-6.20 pg/mL; P = .01). CONCLUSION: LTB4 is detectable and elevated in the small airway or alveolar fraction of exhaled breath condensate in pediatric asthma. Because of the possibility of detecting elevated levels in patients without lung function impairment in controlled disease, it may be used as a noninvasive marker of small airways disease; however, future long-term studies are needed.

Structure and biomedical applications of amyloid oligomer nanoparticles.

Amyloid oligomers are nonfibrillar polypeptide aggregates linked to diseases, such as Alzheimer's and Parkinson's. Here we show that these aggregates possess a compact, quasi-crystalline architecture that presents significant nanoscale regularity. The amyloid oligomers are dynamic assemblies and are able to release their individual subunits. The small oligomeric size and spheroid shape confer diffusible characteristics, electrophoretic mobility, and the ability to enter hydrated gel matrices or cells. We finally showed that the amyloid oligomers can be labeled with both fluorescence agents and iron oxide nanoparticles and can target macrophage cells. Oligomer amyloids may provide a new biological nanomaterial for improved targeting, drug release, and medical imaging.

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture.

Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H2O2 concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H2O2, therefore acting as a protection against cell death by accidental necrosis.

Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir.

Until recently, there were no effective drugs available blocking coronavirus (CoV) infection in humans and animals. We have shown before that CsA and FK506 inhibit coronavirus replication (Carbajo-Lozoya, J., Müller, M.A., Kallies, S., Thiel, V., Drosten, C., von Brunn, A. Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506. Virus Res. 2012; Pfefferle, S., Schöpf, J., Kögl, M., Friedel, C., Müller, M.A., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Züst, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H.-J., Schwegmann-Weßels, C., Pöhlmann, S., Haas, J., Drosten, C. and von Brunn, A. The SARS-Coronavirus-host interactome: identification of cyclophilins as target for pan-Coronavirus inhibitors. PLoS Pathog., 2011). Here we demonstrate that CsD Alisporivir, NIM811 as well as novel non-immunosuppressive derivatives of CsA and FK506 strongly inhibit the growth of human coronavirus HCoV-NL63 at low micromolar, non-cytotoxic concentrations in cell culture. We show by qPCR analysis that virus replication is diminished up to four orders of magnitude to background levels. Knockdown of the cellular Cyclophilin A (CypA/PPIA) gene in Caco-2 cells prevents replication of HCoV-NL63, suggesting that CypA is required for virus replication. Collectively, our results uncover Cyclophilin A as a host target for CoV infection and provide new strategies for urgently needed therapeutic approaches.

ERp29 deficiency affects sensitivity to apoptosis via impairment of the ATF6-CHOP pathway of stress response.

Endoplasmic reticulum protein 29 (ERp29) belongs to the redox-inactive PDI-Dß-subfamily of PDI-proteins. ERp29 is expressed in all mammalian tissues examined. Especially high levels of expression were observed in secretory tissues and in some tumors. However, the biological role of ERp29 remains unclear. In the present study we show, by using thyrocytes and primary dermal fibroblasts from adult ERp29(-/-) mice, that ERp29 deficiency affects the activation of the ATF6-CHOP-branch of unfolded protein response (UPR) without influencing the function of other UPR branches, like the ATF4-eIF2α-XBP1 signaling pathway. As a result of impaired ATF6 activation, dermal fibroblasts and adult thyrocytes from ERp29(-/-) mice display significantly lower apoptosis sensitivities when treated with tunicamycin and hydrogen peroxide. However, in contrast to previous reports, we could demonstrate that ERp29 deficiency does not alter thyroglobulin expression levels. Therefore, our study suggests that ERp29 acts as an escort factor for ATF6 and promotes its transport from ER to Golgi apparatus under ER stress conditions.

Anti-inflammatory effects of extracellular cyclosporins are exclusively mediated by CD147.

Leukocyte trafficking and recruitment is a critical process in host immune surveillance and in inflammatory diseases. Extracellular cyclophilins (eCyps) have been identified as a novel class of chemotactic mediators. The impact of eCyp/CD147 interactions for the recruitment of leukocytes during inflammation was analyzed using a structurally simplified cell-impermeable eCyp inhibitor. This compound was highly effective at inhibiting leukocyte migration toward CypA in vitro as well as in the recruitment of leukocytes during inflammation in a mouse model of experimentally induced peritonitis and delayed-type hypersensitivity reaction. By using CD147-/- mice in combination with the cell-impermeable eCyp inhibitor, we were able to show that the action of eCyps in inflammation is exclusively mediated by interaction with CD147. Our findings suggest that blocking eCyps may be an effective therapeutic target for reducing inflammatory diseases associated with leukocyte recruitment.

Fine tuning the inhibition profile of cyclosporine A by derivatization of the MeBmt residue.

Unique respect: The biological properties of four CsA derivatives were fine-tuned by tractable modifications of the MeBmt residue. The new CsA derivatives share strong inhibitory activity toward cyclophilins (Cyps), but each is unique with respect to immunosuppressive action and cellular localization. These CsA analogues can be used to study the physiological roles of extracellular Cyps.

Amplification of the inhibitory activity and reversal of the selectivity of miglitol by C(2')-monofluorination.

Selective monofluorination of the α-glycosidase inhibitor and antidiabetic agent miglitol at positions C(2') or C(6) creates competitive inhibitors of glycosidases. Introducing a fluorine substituent at position C(6) results in a reduced binding to the enzyme whereas fluorination at C(2') produces an inhibitor with an activity four times higher than the parent compound. This compound is selective for the α-galactosidase from green coffee beans. Its screening against a panel of human cell lines showed a low cytotoxicity, therefore, making this compound an interesting candidate for further clinical investigations.

Difluorotetrahydropyridothiazinone: a selective ß-galactosidase inhibitor.

Selective difluorination, introducing a lactame moiety (instead of an amine) and a double bond in a trihydroxy-2-thiaquinolizidine derivative reverses the selectivity of the glycosidase inhibitor - a selective inhibitor for an α-glucosidase is altered into an excellent, competitive inhibitor for a ß-galactosidase.

Amplification of the inhibitory activity of miglitol by monofluorination.

Selective monofluorination of the alpha-glycosidase inhibitor and antidiabetic agent Miglitol at position C2 creates an competitive inhibitor of five times higher activity than the parent compound. Its screening against a panel of human cell lines showed a low cytotoxicity therefore making this compound an interesting candidate for further clinical investigations.