Glycoprotein Semisynthesis by Chemical Insertion of Glycosyl Asparagine Using a Bifunctional Thioacid-Mediated Strategy.

Glycosylation is a major modification of secreted and cell surface proteins, and the resultant glycans show considerable heterogeneity in their structures. To understand the biological processes arising from each glycoform, the preparation of homogeneous glycoproteins is essential for extensive biological experiments. To establish a more robust and rapid synthetic route for the synthesis of homogeneous glycoproteins, we studied several key reactions based on amino thioacids. We found that diacyl disulfide coupling (DDC) formed with glycosyl asparagine thioacid and peptide thioacid yielded glycopeptides. This efficient coupling reaction enabled us to develop a new glycoprotein synthesis method, such as the bifunctional thioacid-mediated strategy, which can couple two peptides with the N- and C-termini of glycosyl asparagine thioacid. Previous glycoprotein synthesis methods required valuable glycosyl asparagine in the early stage and subsequent multiple glycoprotein synthesis routes, whereas the developed concept can generate glycoproteins within a few steps from peptide and glycosyl asparagine thioacid. Herein, we report the characterization of the DDC of amino thioacids and the efficient ability of glycosyl asparagine thioacid to be used for robust glycoprotein semisynthesis.

Cystobactamid 507: Concise Synthesis, Mode of Action, and Optimization toward More Potent Antibiotics.

Lack of new antibiotics and increasing antimicrobial resistance are among the main concerns of healthcare communities nowadays, and these concerns necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids-a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe 1) a concise total synthesis of cystobactamid 507, 2) the identification of the bioactive conformation using noncovalently bonded rigid analogues, and 3) the first structure-activity relationship (SAR) study for cystobactamid 507 leading to new analogues with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor-groove binding as part of the drug-target interaction without showing significant intercalation. By designing a new analogue of cystobactamid 919-2, we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.

Synthetic and Biological Studies on New Urea and Triazole Containing Cystobactamid Derivatives.

Cystobactamids belong to the group of arene-based oligoamides that effectively inhibit bacterial type IIa topoisomerases. Cystobactamid 861-2 is the most active member of these antibiotics. Most amide bonds present in the cystobactamids link benzoic acids with anilines and it was found that some of these amide bonds undergo chemical and enzymatic hydrolysis, especially the one linking ring C with ring D. This work reports on the chemical synthesis and biological evaluation of thirteen new cystobactamids that still contain the methoxyaspartate hinge. However, we exchanged selected amide bonds either by the urea or the triazole groups and modified ring A in the latter case. While hydrolytic stability could be improved with these structural substitutes, the high antibacterial potency of cystobactamid 861-2 could only be preserved in selected cases. This includes derivatives, in which the urea group is positioned between rings A and B and where the triazole is found between rings C and D.

Intravenous injection of l-aspartic acid ß-hydroxamate attenuates choroidal neovascularization via anti-VEGF and anti-inflammation.

Choroidal neovascularization (CNV) is a hallmark of exudative age-related macular degeneration (exAMD) and a major cause of visual loss in AMD. Despite the widespread use of anti-VEGF therapy, serious adverse effects arise from repeated intravitreal injection of anti-VEGF antibodies, which warrant alternative strategy. We report herein that in a CNV murine model created by krypton red laser, intravenous injection of a serine racemase inhibitor, l-Aspartic acid ß-hydroxamate (L-ABH), significantly reduced CNV at the dose 6 mg/kg on the first day before and followed by 3 mg/kg on the third day after laser injury. The CNV volumes were analyzed with isolectin GS-IB4 staining on choroidal/RPE flat mounts on the seventh day after laser injury. Injection of L-ABH did not produce negative effects on retinal function and visual behavior. To dissect the mechanism in vitro, pretreatment with L-ABH in primary RPE cultures significantly reduced production of vascular endothelial growth factor (VEGF) and macrophage chemotactic protein 1 (MCP-1) by TNFα-primed RPEs. Consistent with these observations, L-ABH pretreatment significantly attenuated macrophage migration mediated by TNFα-primed RPE. Collectively, intravenous injection of L-ABH significantly reduced CNV volumes via reducing production of VEGF and MCP-1 by inflammation-primed RPEs.

Stapling of two PEGylated side chains increases the conformational stability of the WW domain via an entropic effect.

Hydrocarbon stapling and PEGylation are distinct strategies for enhancing the conformational stability and/or pharmacokinetic properties of peptide and protein drugs. Here we combine these approaches by incorporating asparagine-linked O-allyl PEG oligomers at two positions within the ß-sheet protein WW, followed by stapling of the PEGs via olefin metathesis. The impact of stapling two sites that are close in primary sequence is small relative to the impact of PEGylation alone and depends strongly on PEG length. In contrast, stapling of two PEGs that are far apart in primary sequence but close in tertiary structure provides substantially more stabilization, derived mostly from an entropic effect. Comparison of PEGylation + stapling vs. alkylation + stapling at the same positions in WW reveals that both approaches provide similar overall levels of conformational stability.

Adaptation of a Bacterial Multidrug Resistance System Revealed by the Structure and Function of AlbA.

To combat the rise of antimicrobial resistance, the discovery of new antibiotics is paramount. Albicidin and cystobactamid are related natural product antibiotics with potent activity against Gram-positive and, crucially, Gram-negative pathogens. AlbA has been reported to neutralize albicidin by binding it with nanomolar affinity. To understand this potential resistance mechanism, we determined structures of AlbA and its complex with albicidin. The structures revealed AlbA to be comprised of two domains, each unexpectedly resembling the multiantibiotic neutralizing protein TipA. Binding of the long albicidin molecule was shared pseudosymmetrically between the two domains. The structure also revealed an unexpected chemical modification of albicidin, which we demonstrate to be promoted by AlbA, and to reduce albicidin potency; we propose a mechanism for this reaction. Overall, our findings suggest that AlbA arose through internal duplication in an ancient TipA-like gene, leading to a new binding scaffold adapted to the sequestration of long-chain antibiotics.

Mn Inhibits GSH Synthesis via Downregulation of Neuronal EAAC1 and Astrocytic xCT to Cause Oxidative Damage in the Striatum of Mice.

Excessive manganese (Mn) can accumulate in the striatum of the brain following overexposure. Oxidative stress is a well-recognized mechanism in Mn-induced neurotoxicity. It has been proven that glutathione (GSH) depletion is a key factor in oxidative damage during Mn exposure. However, no study has focused on the dysfunction of GSH synthesis-induced oxidative stress in the brain during Mn exposure. The objective of the present study was to explore the mechanism of Mn disruption of GSH synthesis via EAAC1 and xCT in vitro and in vivo. Primary neurons and astrocytes were cultured and treated with different doses of Mn to observe the state of cells and levels of GSH and reactive oxygen species (ROS) and measure mRNA and protein expression of EAAC1 and xCT. Mice were randomly divided into seven groups, which received saline, 12.5, 25, and 50 mg/kg MnCl2, 500 mg/kg AAH (EAAC1 inhibitor) + 50 mg/kg MnCl2, 75 mg/kg SSZ (xCT inhibitor) + 50 mg/kg MnCl2, and 100 mg/kg NAC (GSH rescuer) + 50 mg/kg MnCl2 once daily for two weeks. Then, levels of EAAC1, xCT, ROS, GSH, malondialdehyde (MDA), protein sulfhydryl, carbonyl, 8-hydroxy-2-deoxyguanosine (8-OHdG), and morphological and ultrastructural features in the striatum of mice were measured. Mn reduced protein levels, mRNA expression, and immunofluorescence intensity of EAAC1 and xCT. Mn also decreased the level of GSH, sulfhydryl, and increased ROS, MDA, 8-OHdG, and carbonyl in a dose-dependent manner. Injury-related pathological and ultrastructure changes in the striatum of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GSH synthesis through inhibition of EAAC1 and xCT to trigger oxidative damage in the striatum.

A critical analysis of L-asparaginase activity quantification methods-colorimetric methods versus high-performance liquid chromatography.

L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid ß-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations. Graphical abstract ᅟ.

Nicrophorusamides A and B, Antibacterial Chlorinated Cyclic Peptides from a Gut Bacterium of the Carrion Beetle Nicrophorus concolor.

Nicrophorusamides A and B (1 and 2) were discovered from a rare actinomycete, Microbacterium sp., which was isolated from the gut of the carrion beetle Nicrophorus concolor. The structures of the nicrophorusamides were established as new chlorinated cyclic hexapeptides bearing uncommon amino acid units mainly based on 1D and 2D NMR spectroscopic analysis. The absolute configurations of the amino acid residues 5-chloro-l-tryptophan, d-threo-ß-hydroxyasparagine/d-asparagine, l-ornithine, l-allo-isoleucine, d-leucine, and d-valine were determined using Marfey's method and chemical derivatization with 2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate followed by LC/MS analysis. Nicrophorusamide A (1) showed antibacterial activity against several Gram-positive bacteria.

Structure of human immunoproteasome with a reversible and noncompetitive inhibitor that selectively inhibits activated lymphocytes.

Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome ß5i subunit while sparing the constitutive ß5c subunit. Here we report ß5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively, an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with ß5i and ß6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells, and to a lesser extent in activated human T cells. Reversible, ß5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells.

Total Syntheses of Cystobactamids and Structural Confirmation of Cystobactamid 919-2.

The cystobactamids are a family of antibacterial natural products with unprecedented chemical scaffolds that are active against both Gram-positive and Gram-negative pathogens. Herein, we describe the first total synthesis of cystobactamid 919-2 from three fragments. Our convergent synthesis enabled both the confirmation of the correct structure and the determination of the absolute configuration of cystobactamid 919-2.

Eficacia y seguridad de L-asparaginasa erwinia y L-asparaginasa E. Coli pegilada en el tratamiento de pacientes con leucemia linfoblástica aguda que presentam hipersensibilidad a L-asparaginasa E. coli nativa / Efficacy and safety of L-asparaginase erwinia and L-asparaginase E. coli pegylated in the treatment of patients with acute lymphoblastic leukemia who are hypersensitive to L-asparaginase E. coli native

INTRODUÇÃO: Antecedentes: El presente dictamen expone la evaluación de tecnología de la eficacia y seguridad del uso de L-asparaginasa Erwinia y L-asparaginasa E. coli pegilada para el tratamiento de pacientes con leucemia linfoblástica aguda que presentan hipersensibilidad a L-asparaginasa E. coli nativa. Aspectos Generales: La leucemia es el tipo de cáncer más común en niños, representando aproximadamente el 30% de todos los tipos de cáncer diagnosticados en niños; siendo la leucemia linfoblástica aguda (LLA) uno de los dos tipos de leucemias más comunes. Adicionalmente, alrededor del 60% de todos los casos de LLA ocurre en pacientes menores de 20 años. Así, LLA es un tipo de leucemia de alta importancia dentro de población joven. Tecnología Sanitaria de Interés: Las células neoplásicas en la leucemia linfoblástica aguda (LLA) no sintetizan las cantidades necesarias del aminoácido L-asparagina; por lo que requieren de funtes externas (i.e., L-asparagina extracelular). La L-asparaginasa, es una enzima que cataliza la conversión de L-asparagina más agua, en ácido aspártico y amoniaco, ocasionando que los niveles de L-asparagina extracelular disminuyan; y que por los tanto las células d ela LLA no cuenten con L-asparagina extracelular. Así, estas células neoplásicas se quedan sin fuentes de L-asparagina, y no pueden sintetizar proteínas de gran imporancia para su supervivencia, ocasionando su muerte. METODOLOGÍA: Estrategia de Búsqueda: Se realizó una búsqueda de la literatura a la eficacia y seguridad del uso de L-asparaginasa Erwinia y L-asparaginasa E. coli pegilada para el tratamiento de pacientes niños y adultos con leucemia linfoblástica aguda que presentan hipersensibilidad a L-asparaginasa E. coli nativa. Esta búsqueda se realizó utilizando los meta-buscadores: Translating Research into Practice (TRIPDATABASE) Y National Library of Medicine (Pubmed-Medline). RESULTADOS: Sinopsis de la Evidencia: Se realizó la búsqueda bibliográfica y de evidencia científica hasta marzo del 2017 para el sustento del uso de L-asparaginasa Erwinia en el tratamiento de leucemia linfoblástica aguda en pacientes niños y adultos que presentan hipersensibilidad a L-asparaginasa E. coli nativa. Se presente la evidencia disponible según el tipo de publicación priorizada en los criterios de inclusión (i.e., GP, ETS, RS y ECA fase III), siendo los ensayos de fase III o en su defecto ensayos controlados y aleatorizados la principal considerada. CONCLUSIONES: El presente dictamen evaluó la mejor evidencia disponible hasta marco 2017 en relación al uso de L-asparaginasa Erwinia y L-asparaginasa E. coli pegilada para el tratamiento de pacientes niños, adolescentes, y adultos con leucemia linfoblástica aguda, que presentan hipersensibilidad a L-asparaginasa E. coli nativa. El Instituto de Tecnologías Sanitarias-IETSI, aprueba el uso de L-asparaginasa Erwinia como parte del esquema quimioterápico utilizada para el tratamiento de pacientes niños, adolescentes, y adultos con leucemia linfoblástica aguda, que presentan hipersensibilidad grado 2 o más a L-asparaginasa E. coli nativa. La vigencia de este dictamen preliminar es de dos años a partir de la fecha de publicación. Asimismo, el Instituto de Tecnologías Sanitarias-IETSI, no aprueba el uso de L-asparaginasa E. coli pegilada en el tratamiento de pacientes niños, adolescentes, y adultos con leucemia linfoblástica aguda, que presentan hipersensiblidad grado 2 o más a L-asparaginasa E. coli nativa.

Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme.

The biosynthesis of branched alkoxy groups, such as the unique t-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metabolites contain an isopropyl group proposed to be formed using CysS, a cobalamin-dependent radical S-adenosylmethionine (SAM) methyltransferase. Here, we reconstitute the CysS-catalyzed reaction, on p-aminobenzoate thioester substrates, and demonstrate that it not only catalyzes sequential methylations of a methyl group to form ethyl and isopropyl groups but remarkably also sec-butyl and t-butyl groups. To our knowledge, this is the first in vitro reconstitution of a cobalamin-dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t-butyl group.

Isolation of Coralmycins A and B, Potent Anti-Gram Negative Compounds from the Myxobacteria Corallococcus coralloides M23.

Two new potent anti-Gram negative compounds, coralmycins A (1) and B (2), were isolated from cultures of the myxobacteria Corallococcus coralloides M23, together with another derivative (3) that was identified as the very recently reported cystobactamid 919-2. Their structures including the relative stereochemistry were elucidated by interpretation of spectroscopic, optical rotation, and CD data. The relative stereochemistry of 3 was revised to "S*R*" by NMR analysis. The antibacterial activity of 1 was most potent against Gram-negative pathogens, including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae, with MICs of 0.1-4 µg/mL; these MICs were 4-10 and 40-100 times stronger than the antibacterial activities of 3 and 2, respectively. Thus, these data indicated that the ß-methoxyasparagine unit and the hydroxy group of the benzoic acid unit were critical for antibacterial activity.

Endogeous sulfur dioxide protects against oleic acid-induced acute lung injury in association with inhibition of oxidative stress in rats.

The role of endogenous sulfur dioxide (SO2), an efficient gasotransmitter maintaining homeostasis, in the development of acute lung injury (ALI) remains unidentified. We aimed to investigate the role of endogenous SO2 in the pathogenesis of ALI. An oleic acid (OA)-induced ALI rat model was established. Endogenous SO2 levels, lung injury, oxidative stress markers and apoptosis were examined. OA-induced ALI rats showed a markedly downregulated endogenous SO2/aspartate aminotransferase 1 (AAT1)/AAT2 pathway and severe lung injury. Chemical colorimetry assays demonstrated upregulated reactive oxygen species generation and downregulated antioxidant capacity in OA-induced ALI rats. However, SO2 increased endogenous SO2 levels, protected against oxidative stress and alleviated ALI. Moreover, compared with OA-treated cells, in human alveolar epithelial cells SO2 downregulated O2(-) and OH(-) generation. In contrast, L-aspartic acid-ß-hydroxamate (HDX, Sigma-Aldrich Corporation), an inhibitor of endogenous SO2 generating enzyme, promoted free radical generation, upregulated poly (ADP-ribose) polymerase expression, activated caspase-3, as well as promoted cell apoptosis. Importantly, apoptosis could be inhibited by the free radical scavengers glutathione (GSH) and N-acetyl-L-cysteine (NAC). The results suggest that SO2/AAT1/AAT2 pathway might protect against the development of OA-induced ALI by inhibiting oxidative stress.

Cystobactamids: myxobacterial topoisomerase inhibitors exhibiting potent antibacterial activity.

The development of new antibiotics faces a severe crisis inter alia owing to a lack of innovative chemical scaffolds with activities against Gram-negative and multiresistant pathogens. Herein, we report highly potent novel antibacterial compounds, the myxobacteria-derived cystobactamids 1-3, which were isolated from Cystobacter sp. and show minimum inhibitory concentrations in the low µg mL(-1) range. We describe the isolation and structure elucidation of three congeners as well as the identification and annotation of their biosynthetic gene cluster. By studying the self-resistance mechanism in the natural producer organism, the molecular targets were identified as bacterial type IIa topoisomerases. As quinolones are largely exhausted as a template for new type II topoisomerase inhibitors, the cystobactamids offer exciting alternatives to generate novel antibiotics using medicinal chemistry and biosynthetic engineering.

In silico investigation of potential pyruvate kinase M2 regulators from traditional Chinese medicine against cancers.

A recent research in cancer research demonstrates that tumor-specific pyruvate kinase M2 (PKM2) plays an important role in chromosome segregation and mitosis progression of tumor cells. To improve the drug development of TCM compounds, we aim to identify potent TCM compounds as lead compounds of PKM2 regulators. PONDR-Fit protocol was utilized to predict the disordered disposition in the binding domain of PKM2 protein before virtual screening as the disordered structure in the protein may cause the side effect and downregulation of the possibility of ligand to bind with target protein. MD simulation was performed to validate the stability of interactions between PKM2 proteins and each ligand after virtual screening. The top TCM compounds, saussureamine C and precatorine, extracted from Lycium chinense Mill. and Abrus precatorius L., respectively, have higher binding affinities with target protein in docking simulation than control. They have stable H-bonds with residues A:Lys311 and some other residues in both chains of PKM2 protein. Hence, we propose the TCM compounds, saussureamine C and precatorine, as potential candidates as lead compounds for further study in drug development process with the PKM2 protein against cancer.

In silico investigation of potential TRAF6 inhibitor from traditional Chinese medicine against cancers.

It has been indicated that tumor necrosis factor receptor-associated factor-6 (TRAF6) will upregulate the expression of hypoxia-inducible factor-1α (HIF-1α) and promote tumor angiogenesis. TRAF6 proteins can be treated as drug target proteins for a differentiation therapy against cancers. As structural disordered disposition in the protein may induce the side-effect and reduce the occupancy for ligand to bind with target protein, PONDR-Fit protocol was performed to predict the disordered disposition in TRAF6 protein before virtual screening. TCM compounds from the TCM Database@Taiwan were employed for virtual screening to identify potent compounds as lead compounds of TRAF6 inhibitor. After virtual screening, the MD simulation was performed to validate the stability of interactions between TRAF6 proteins and each ligand. The top TCM compounds, tryptophan, diiodotyrosine, and saussureamine C, extracted from Saussurea lappa Clarke, Bos taurus domesticus Gmelin, and Lycium chinense Mill., have higher binding affinities with target protein in docking simulation. However, the docking pose of TRAF6 protein with tryptophan is not stable under dynamic condition. For the other two TCM candidates, diiodotyrosine and saussureamine C maintain the similar docking poses under dynamic conditions. Hence, we propose the TCM compounds, diiodotyrosine and saussureamine C, as potential candidates as lead compounds for further study in drug development process with the TRAF6 protein against cancer.

Insight into HIV of IFN-induced myxovirus resistance 2 (MX2) expressed by traditional Chinese medicine.

Recently, an important topic of the acquired immunodeficiency syndrome (AIDS) had been published in 2013. In this report, the expression of the IFN-induced myxovirus resistance 2 (MX2) had been defined the function to kill the human immunodeficiency virus (HIV). The screening from the Traditional Chinese Medicine (TCM) database by simulating molecular docking and molecular dynamics could select candidate compounds, which may express MX2 against HIV. Saussureamine C, Crotalaburnine, and Precatorine are selected based on the highest docking score and other TCM compounds. The data from molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions, and hydrogen bond with structure variations, this research could assess the interaction between protein and ligand interaction. In addition to the detection of TCM compound efficacy, we suggest that Saussureamine C is better than the others in protein-ligand interaction and the structural variation to express MX2.

A facile synthesis of α-N-ribosyl-asparagine and α-N-ribosyl-glutamine building blocks.

Adenosine diphosphate ribosylation (ADP-ribosylation) is a widely occurring post-translational modification of proteins at nucleophilic side chain of amino acid residues. Elucidation of ADP-ribosylation events would benefit greatly from the availability of well-defined ADP-ribosylated peptides and analogues thereof. In this paper we present a novel approach to the chemical synthesis of ribosylated amino acid building blocks using traceless Staudinger ligation. We describe an efficient and stereoselective synthesis of α-N-ribosyl-asparagine (α-N-ribosyl-Asn) and α-N-ribosyl-glutamine (α-N-ribosyl-Gln) building blocks starting from 5-tert-butyldiphenylsilyl-ß-D-ribofuranosyl azide. The N-glycosyl aminoacids are produced in good yields as pure α-anomers, suitably protected for peptide synthesis.