Synthesis and biological evaluation of N-arylated-lactam-type iminosugars as potential immunosuppressive agents.

Since the immunosuppressive agents currently used in clinics have significant side effects, it is very important to search for new effective and safe immunosuppressants. Iminosugars as a new class of immunosuppressants are less explored. In this report, 24 new N-arylated iminosugar derivatives, including d-talo and d-galacto epimers, were designed and synthesized, and their immunosuppressive effects were evaluated by MTT assay. The experimental data demonstrated that compound 20 showed the strongest inhibition effect (IC50 = 6.94 µM). Further studies revealed that the inhibitory effects on splenocyte proliferation may come from the suppression of both IFN-γ and IL-4 cytokines. The preliminary structure-activity relationship (SAR) analysis suggested that N-arylated d-galacto-type iminosugars showed better inhibitory activities than d-talo-type analogues. The SAR analysis also showed that the inhibition effect of iminosugars can be improved by decreasing the polarity or increasing the hydrophobicity. These results may be beneficial to the discovery of new iminosugar derivatives as immunosuppressive agents.

Shell-sheddable, pH-sensitive supramolecular nanoparticles based on ortho ester-modified cyclodextrin and adamantyl PEG.

We report a new type of pH-sensitive supramolecular aggregates which possess a programmable character of sequential dePEGylation and degradation. As a platform of designing and building multifunctional supramolecular nanoparticles, a family of 6-OH ortho ester-modified ß-cyclodextrin (ß-CD) derivatives have been synthesized via the facile reaction between ß-CD and cyclic ketene acetals with different alkyl lengths. These asymmetric acid-labile ß-CD derivatives formed amphiphilic supramolecules with adamantane-modified PEG through host-guest interaction in polar solvents such as ethanol. The supramolecules can self-assemble in water to form acid-labile supramolecular aggregates. The results of TEM and light scattering measurements demonstrate that the size and morphology of the aggregates are influenced by the alkyl or PEG length and the host-guest feed ratio. By carefully balancing the alkyl and PEG lengths and adjusting the host-guest ratio, well-dispersed vesicles (50-100 nm) or sphere-like nanoparticles (200-500 nm) were obtained. Zeta potential measurements reveal that these supramolecular aggregates are capable of being surface-functionalized via dynamic host-guest interaction. The supramolecular aggregates were stable at pH 8.4 for at least 12 h as proven by the (1)H NMR and LLS measurements. However, rapid dePEGylation occurred at pH 7.4 due to the hydrolysis of the ortho ester linkages locating at the interface, which resulted in aggregation of the dePEGylated hydrophobic inner cores. Upon further decreasing the pH to 6.4, the hydrophobic cores were further degraded due to the acid-accelerated hydrolysis of the ortho esters. The incubation stability of the acid-labile supramolecular aggregates in neutral buffer could be improved by incorporating hydrophobic poly(ε-caprolactone) into the core of the aggregates.

Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases.

Regioselective and enantioselective hydroxylation of propargylic C-H bonds are useful reactions but often lack appropriate catalysts. Here a green and efficient asymmetric hydroxylation of primary and secondary C-H bonds at propargylic positions has been established. A series of optically active propargylic alcohols were prepared with high regio- and enantioselectivity (up to 99% ee) under mild reaction conditions by using P450tol, while the C≡C bonds in the molecule remained unreacted. This protocol provides a green and practical method for constructing enantiomerically chiral propargylic alcohols. In addition, we also demonstrated that the biohydroxylation strategy was able to scaled up to 2.25 mmol scale with the production of chiral propargyl alcohol 2a at a yield of 196 mg with 96% ee, which's an important synthetic intermediate of antifungal drug Ravuconazole.

[Bioactivity assay of bupleurum injection for inhibiting PGE2 release in vitro].

OBJECTIVE: To establish the in vitro model of PGE2 released by hypothalamic neurocytes under rrIL-1beta in vitro interference, and investigate the correlation of the PGE2 content and the effect of the drug effect concentration in the model under the effect of Bupleurum injection. METHOD: Hypothalamic neurocytes were cultured in vitro, and added with rrIL-1beta (40 microg x L(-1)) stimulation. Cell sap was collected at different time points. ELISA was adopted to determine the content of PGE2 in cell sap collected at different time points. Hypothalamic neurocytes were cultured in vitro, added with rrIL-1beta (40 microg x L(-1)) stimulation and then different concentrations of Bupleurum injection. The changes in the content of PGE2 in cell supernatant were detected by ELISA. An analysis was made on the linear relationship between the sample concentration and the inhibition rate of PGE2. RESULT: The rrIL-1 cells could stimulate in vitro cultured hypothalamic neurocytes to release PGE2 and reach the peak at 10 h. Bupleurum injection could significantly interfere the release of PGE2 in the in vitro model (P < 0.01, P < 0.05), with a certain linear relationship between the interference effect and the effect concentration of Bupleurum injection (r = 0.911, P < 0.01). CONCLUSION: The rrIL-1 cells could stimulate in vitro cultured hypothalamic neurocytes to release PGE2, with a good correlation between the inhibition and generation effects of PGE2 and the drug concentration.

ACE inhibitory activities of two peptides derived from Volutharpa ampullacea perryi hydrolysate and their protective effects on H2O2 induced HUVECs injury.

The purpose of this study is to explore the effects of IVTNWDDMEK and VGPAGPRG, two angiotensin I-converting enzyme (ACE) inhibitory peptides purified from Volutharpa ampullacea perryi, on ACE's two domains and on nitric oxide (NO), endothelin-1(ET-1) production in human vascular endothelial cells (HUVECs). In addition, we sought to investigate the effects of these two peptides on HUVECs injury induced by H2O2. The results indicated that the inhibition of the ACE C-domain was significantly higher than that of the ACE N-domain by these two peptides. Molecular dynamics (MD) analysis revealed that the hydrogen bonds interactions between ACE and two peptides, the chelation between peptides and Zn2+ both play important role, which might contribute significantly to the ACE inhibitory activity. Two peptides significantly increase NO and ET-1 production in a dose-dependent manner and protects against hydrogen peroxide-induced HUVEC cell injury. The reported results also show that two peptides up-regulated the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1), and reduce the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA). Our study indicated that IVTNWDDMEK and VGPAGPRG could be potent ACE inhibitors and Volutharpa ampullacea perryi is a good source of bioactive peptides, which provided a theoretical basis for the broad application of two selected peptides as functional food with anti-hypertensive activity.

Study on the domain selective inhibition of angiotensin-converting enzyme (ACE) by food-derived tyrosine-containing dipeptides.

In this article, the selective inhibition of several tyrosine-containing dipeptides on N and C domain of ACE (angiotensin-converting enzyme) was studied, and the interaction mode of ACE and inhibitors was simulated by molecular docking. MTT assay was used to detect the effect of dipeptide on human umbilical vein endothelial cells (HUVEC). The results showed that the food-derived dipeptides AY (Ala-Tyr), LY (Leu-Tyr), and IY (Ile-Tyr) containing tyrosine at the C-terminal were favorable structures for selective inhibition of ACE C-domain. These dipeptides showed competitive and mixed inhibition patterns, while the dipeptides EY (Glu-Tyr), RY (Arg-Tyr), FY (Phe-Tyr), and SY (Ser-Tyr) showed noncompetitive inhibition. Food-derived dipeptides containing tyrosine have no cytotoxicity on HUVEC cells, which provides a basis for the application of food-derived tyrosine dipeptides as antihypertensive peptides. This study provides a theoretical basis for exploring the selective inhibition mechanism of ACE inhibitory peptides containing tyrosine residue. PRACTICAL APPLICATIONS: Angiotensin-converting enzyme (ACE) is a two-domain dipeptidyl carboxypeptidase, which is a key enzyme to regulate blood pressure. ACE has two active sites, C- and N-domain, which have high catalytic activity. Although the amino acid sequences of the two active sites have 60% similarity, there are some differences in structure and function. The action mechanism of ACE domain should be clarified, and the structure-activity relationship between inhibitors and ACE domain has not been systematically studied. The aim of this study was to identify the selective inhibitory effect of food-derived tyrosine dipeptides on the domain of ACE. This provides a new idea for finding new antihypertensive drugs with less side effects.

ROS-Activated Ratiometric Fluorescent Polymeric Nanoparticles for Self-Reporting Drug Delivery.

Reactive oxygen species (ROS)-responsive theranostic nanomedicines have attracted wide interest in recent years because ROS stress is implicated in some pathological disorders such as inflammatory diseases and cancers. In this article, we report a kind of innovative ROS-responsive theranostic polymeric nanoparticles that are able to load hydrophobic drugs and to fluorescently self-report the in vitro or intracellular drug release under ROS triggering. The fluorescent nanoparticles were formed by amphiphilic block copolymers consisting of a poly(ethylene glycol) (PEG) segment and an oxidation-responsive hydrophobic block. The copolymers with different hydrophobic block lengths were synthesized by the atom transfer radical polymerization of a phenylboronic ester-containing acrylic monomer with a small fraction of a ROS-activatable 1,8-naphthalimide-based fluorescent monomer, using PEG-Br as the macroinitiator. The copolymer nanoparticles were stable in neutral phosphate buffer but degraded upon H2O2 triggering, with the degradation rate depending on the hydrophobic block length and the concentration of H2O2. The degradation of nanoparticles was accompanied by a colorimetric change of the fluorophore from blue to green, which affords the nanoparticles the ability to detecting H2O2 by a ratiometric fluorescent approach. Moreover, the nanoparticles could encapsulate doxorubicin (DOX) and the H2O2-triggered DOX release was well associated with the change in ratiometric fluorescence. Confocal laser scanning microscope results reveal that the fluorescent nanoparticles were internalized into A549 cells through the endocytosis pathway. The ROS-stimulated degradation of the nanoparticles and intracellular DOX release and the fate of the degraded polymers could be monitored by ratiometric fluorescent imaging. Finally, the naked nanoparticles and the degradation products are cytocompatible, whereas the DOX-loaded ones exhibit concentration-dependent cytotoxicity. Of importance, the stimulation with exogenous H2O2 or lipopolysaccharide enhanced obviously the cell-killing capability of the DOX-loaded nanoparticles because of the ROS-enhanced intracellular DOX release.

New Mutation of Coenzyme Q10 Monooxygenase 6 Causing Podocyte Injury in a Focal Segmental Glomerulosclerosis Patient.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function, which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments. The pathogenesis of FSGS has not been completely elucidated; however, recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis. Here, we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism. METHODS: Using next-generation sequencing and Sanger sequencing, we screened the causative gene that was linked to FSGS in this study. The patient's total blood RNA was extracted to validate the messenger RNA (mRNA) expression of coenzyme Q10 monooxygenase 6 (COQ6) and validated it by immunohistochemistry. COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then, F-actin was determined using immunofluorescence staining. Cell apoptosis was evaluated by flow cytometry, the expression of active caspase-3 was determined by Western blot, and mitochondrial function was detected by MitoSOX. RESULTS: Using whole-exome sequencing and Sanger sequencing, we screened a new causative gene, COQ6, NM_182480: exon1: c.G41A: p.W14X. The mRNA expression of COQ6 in the proband showed decreased. Moreover, the expression of COQ6, which was validated by immunohistochemistry, also had the same change in the proband. Finally, we focused on the COQ6 gene to clarify the mechanism of podocyte injury. Flow cytometry showed significantly increased in apoptotic podocytes, and Western blotting showed increases in active caspase-3 in si-COQ6 podocytes. Meanwhile, reactive oxygen species (ROS) levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group. CONCLUSIONS: This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6, which could cause respiratory chain defect, increase the generation of ROS, destroy the podocyte cytoskeleton, and induce apoptosis. It provides basic theoretical basis for the screening of FSGS in the future.

Supersensitive Oxidation-Responsive Biodegradable PEG Hydrogels for Glucose-Triggered Insulin Delivery.

Reactive oxygen species (ROS)-responsive polymers and hydrogels represent an emerging family of intelligent materials owing to the key functions of ROS in physiological processes or pathological diseases. Nonetheless, the weaknesses such as low sensitivity, slow response, instability, and low mechanical strength are associated with the limited ROS-responsive polymeric or supramolecular hydrogels. In this study, a novel type of oxidation-responsive degradable hydrogels was fabricated by the redox-initiated radical polymerization of a 4-arm-poly(ethylene glycol) (PEG) acrylic macromonomer that possesses a H2O2-cleavable phenylboronic acid linker in each of the arms. The macroscopic hydrogels have the features of good cytocompatibility, moderate mechanical strength, and fast response toward H2O2 of low concentration, owing to the covalently cross-linked hydrophilic PEG network and high sensitivity of the linker. They could encapsulate biomacromolecules, such as insulin and glucose oxidase (GOx), with high efficacy, affording a new glucose-responsive insulin-delivery platform on the basis of enzymatic transformation of a biochemical signal (glucose) into an oxidative stimulus (H2O2). Interestingly, in vitro results demonstrate that the same GOx-loaded hydrogel exhibited disparate degradation modes under different triggering molecules, that is, bulk degradation by H2O2 and surface erosion by glucose. Moreover, compared to the macroscopic hydrogel, the nanogel with a diameter of ∼160 nm prepared by inverse emulsion polymerization showed a much higher degradation rate even under triggering of 20 µM H2O2, a pathologically available concentration in vivo.

Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies.

Based on the complete genome of Cyanothece ATCC 51142, the oriCs of both the circular and linear chromosomes in Cyanothece ATCC 51142 have been predicted by utilizing a web-based system Ori-Finder. Here, we provide experimental support for the results of Ori-Finder to identify the replication origins of Cyanothece ATCC 51142 and their interactions with the initiator protein, DnaA. The two replication origins are composed of three characteristically arranged DnaA boxes and an AT-rich stretch, and the oriC in the circular chromosome is followed by the dnaN gene. The dnaA gene is located downstream of the origin of the circular chromosome and it expresses a typical DnaA protein that is divided into four domains (I, II, III, IV), as with other members of the DnaA protein family. We purify DnaA (IV) and characterize the interaction of the purified protein with the replication origins, so as to offer experimental support for the prediction. The results of the electrophoretic mobility shift assay and DNase I footprint assay demonstrate that the C-terminal domain of the DnaA protein from Cyanothece ATCC 51142 specifically binds the oriCs of both the circular and linear chromosomes, and the DNase I footprint assay demonstrates that DnaA (IV) exhibits hypersensitive affinity with DnaA boxes in both oriCs.

Acid-Sensitive Polypseudorotaxanes Based on Ortho Ester-Modified Cyclodextrin and Pluronic F-127.

We demonstrate a new type of acid-sensitive amphiphilic polypseudorotaxanes (PPRs) formed via inclusion complexation between Pluronic F127 and the hydrophobic ß-cyclodextrin (CD) derivative in alcoholic solvents. The 6-OH ortho ester-substituted hydrophobic ß-CD derivative (EMD-CD) was prepared by "click" reaction of ß-CD with 2-ethylidene-4-methyl-1,3-dioxalane under mild conditions. The water-insoluble EMD-CD (host) is capable of forming PPRs with F127 (guest) in ethanol or methanol but not in water, which is confirmed by 1H NMR, wide-angle X-ray diffraction, small-angle X-ray scattering, and the time-dependent threading kinetics. Depending on the host/guest ratio, the PPRs self-assembled into sheet-like structure or vesicular nanoparticles with different sizes in water. These PPR assemblies were stable at pH 8.4 but quickly dissociated into biocompatible products in neutral or in acidic buffers due to the hydrolysis of the ortho ester groups. Good biocompatibility, ease of fabrication, and extremely pH-sensitive character make the PPRs promising carriers for anticancer drug delivery. Moreover, the present work provides an alternative method for the preparation of PPRs composed of water-insoluble CD derivatives.

Oxidation-Promoted Degradation of Aliphatic Poly(carbonate)s via Sequential 1,6-Elimination and Intramolecular Cyclization.

We report a new type of oxidation-promoted fast-degradable aliphatic poly(carbonate)s (PCs) prepared by the ring-opening polymerization (ROP) of a six-membered cyclic carbonate containing a phenylboronic pinacol ester. The ROP of this monomer catalyzed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) proceeded rapidly at ambient temperature with a good control over molecular weight and polydispersity at high monomer conversion. The H2O2-induced decomposition of this cyclic monomer and its noncyclic carbonate analogue was first studied by 1H NMR in order to clearly demonstrate the degradation mechanism of the PCs. The results of 1H NMR, GPC, and Nile Red fluorescence measurements revealed that the PC nanoparticles formulated by the o/w emulsion method were stable in neutral buffer, but upon triggering with H2O2, they underwent rapid surface degradation via the consecutive processes of oxidation, 1,6-elimination, release of CO2, and intramolecular cyclization. The degradation rates of the nanoparticles were dependent on the concentration of H2O2, and the nanoparticles were even sensitive to 0.5 mM of H2O2.

Oxidation-responsive polymers for biomedical applications.

Reactive oxygen species (ROS) play key roles in many physiological processes, such as cell signaling and host innate immunity. However, when they are overproduced, ROS may damage biomolecules in vivo and cause diseases such as cardiovascular or neurodegenerative diseases, cancer, and so forth. Oxidative stress is usually implicated in various inflammatory tissues, representing an important target for the development of various therapeutic strategies. Therefore, various probes for the in vitro detection of ROS or the in vivo diagnosis of the oxidative stress-relevant diseases have been developed. Oxidation-responsive polymers have also attracted great interest due to their potential applications in biomedical fields. In this feature article, we summarize six types of oxidation-responsive polymers based on different oxidation-responsive motifs. Poly(propylene sulfide)s, selenium-based polymers, aryl oxalate- and phenylboronic ester-containing polymers are discussed in detail, while poly(thioketal)s and proline-containing polymeric scaffolds are briefly introduced.

Oxidation-Accelerated Hydrolysis of the Ortho Ester-Containing Acid-Labile Polymers.

We report a versatile method to tune the hydrolysis of the ortho ester-containing block copolymers by covalently incorporating oxidation-sensitive phenylboronic ester units. A series of block copolymers which contain a polyethylene glycol (PEG) block and a hydrophobic segment composed of different amounts of pendent ortho ester and phenylboronic ester groups were synthesized. These copolymers can self-assemble into narrowly dispersed micelle-like nanoparticles in phosphate buffer. The kinetics of phenylboronic ester oxidation and ortho ester hydrolysis in the nanoparticles were studied at different pH and H2O2 concentration. The results indicated that the phenylboronic ester oxidation rate was faster than the ortho ester hydrolysis rate at neutral pH, and both processes were accelerated with increasing H2O2 concentration. Nanoparticles which are extremely sensitive to the biorelevant concentration of H2O2 (50 µM) at pH 7.4 were obtained, suggesting great promise for inflammation-specific drug delivery.