Expansion of Antibacterial Spectrum of Muraymycins toward Pseudomonas aeruginosa.

It is urgent to develop novel anti-Pseudomonas agents that should also be active against multidrug resistant P. aeruginosa. Expanding the antibacterial spectrum of muraymycins toward P. aeruginosa was investigated by the systematic structure-activity relationship study. It was revealed that two functional groups, a lipophilic side chain and a guanidino group, at the accessory moiety of muraymycins were important for the anti-Pseudomonas activity, and analogue 29 exhibited antibacterial activity against a range of P. aeruginosa strains with the minimum inhibitory concentration values of 4-8 µg/mL.

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics.

BACKGROUND: Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA. New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed. METHODS: To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach. RESULTS: Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells. CONCLUSION: The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation. GENERAL SIGNIFICANCE: These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents.

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring.

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.

Conformational restriction approach to ß-secretase (BACE1) inhibitors: effect of a cyclopropane ring to induce an alternative binding mode.

Improvement of a drug's binding activity using the conformational restriction approach with sp³ hybridized carbon is becoming a key strategy in drug discovery. We applied this approach to BACE1 inhibitors and designed four stereoisomeric cyclopropane compounds in which the ethylene linker of a known amidine-type inhibitor 2 was replaced with chiral cyclopropane rings. The synthesis and biologic evaluation of these compounds revealed that the cis-(1S,2R) isomer 6 exhibited the most potent BACE1 inhibitory activity among them. X-ray structure analysis of the complex of 6 and BACE1 revealed that its unique binding mode is due to the apparent CH-π interaction between the rigid cyclopropane ring and the Tyr71 side chain. A derivatization study using 6 as a lead molecule led to the development of highly potent inhibitors in which the structure-activity relationship as well as the binding mode of the compounds clearly differ from those of known amidine-type inhibitors.

Synthesis of pacidamycin analogues via an Ugi-multicomponent reaction.

The second-generation synthesis of 3'-hydroxypacidamycin D (2) has been accomplished via an Ugi-four component reaction at a late stage of the synthesis. This approach provided ready access to a range of analogues including diastereomers of the diaminobutylic acid residue and hybrid-type analogues of mureidomycins. Biological evaluations of these analogues indicated that the stereochemistry at the diaminobutylic acid residue has a crucial impact on both the MraY biochemical inhibition and whole-cell antibacterial activity.

Total synthesis and biological evaluation of pacidamycin D and its 3'-hydroxy analogue.

Full details of the total synthesis of pacidamycin D (4) and its 3'-hydroxy analogue 32 are described. The chemically labile Z-oxyacyl enamide moiety is the most challenging chemical structure found in uridylpeptide natural products. Key elements of our approach to the synthesis of 4 include the efficient and stereocontrolled construction of the Z-oxyvinyl halides 6 and 7 and their copper-catalyzed cross-coupling with the tetrapeptide carboxamide 5, a thermally unstable compound containing a number of potentially reactive functional groups. This synthetic route also allowed us to easily prepare 3'-hydroxy analogue 32. The assemblage by cross-coupling of the Z-oxyvinyl halide 6 and the carboxamide 5 at a late stage of the synthesis provided ready access to a range of uridylpeptide antibiotics and their analogues, despite their inherent labile nature with potential epimerization, simply by altering the tetrapeptide moiety.

Total synthesis of pacidamycin D by Cu(I)-catalyzed oxy enamide formation.

The first total synthesis of pacidamycin D, which is expected to be a good candidate as an antibacterial agent against P. aeruginosa, is described. The key elements of our approach feature an efficient and stereocontrolled construction of the Z-oxyvinyl iodide and copper-catalyzed cross-coupling with the tetrapeptide carboxamide.

A strategy for neuraminidase inhibitors using mechanism-based labeling information.

A potent inhibitor for Vibrio cholerae neuraminidase (VCNA) was developed by using a novel two-step strategy, a target amino acid validation using mechanism-based labeling information, and a potent inhibitor search using a focused library. The labeling information suggested the hidden dynamics of a loop structure of VCNA, which can be a potential target of the novel inhibitor. A focused library composed of 187 compounds was prepared from a 9-azide derivative of 2,3-dehydro-N-acetylneuraminic acid (DANA) to interrupt the function of the loop of the labeled residues. Inhibitor 3 c showed potent inhibition properties and was the strongest inhibitor with FANA, a N-trifluoroacetyl derivative of DANA. Validation studies of the inhibitor with a detergent and a Lineweaver-Burk plot suggested that the 9-substitution group would interact hydrophobically with the target loop moiety, adding a noncompetitive inhibition property to the DANA skeleton. This information enabled us to design compound 4 having the combined structure of 3 c and FANA. Compound 4 showed the most potent inhibition (K(i) =73 nM, mixed inhibition) of VCNA with high selectivity among the tested viral, bacterial, and mammal neuraminidases.

An efficient approach to the discovery of potent inhibitors against glycosyltransferases.

We describe a standardized approach for searching potent and selective inhibitors of glycosyltransferases by high throughput quantitative MALDI-TOFMS-based screening of focused compound libraries constructed by 1,3-dipolar cycloaddition of the desired azidosugar nucleotides with various alkynes. An aminooxy-functionalized reagent with a stable isotope was conjugated with oligosaccharides to afford glycopeptides as acceptor substrates with improved ion sensitivity. Enhanced ionization potency of new substrates allowed for MALDI-TOFMS-based facile and quantitative analysis of enzymatic glycosylation in the presence of glycosyl donor substrates. A non-natural synthetic sugar nucleotide was identified to be the first highly specific inhibitor for rat recombinant alpha2,3-(N)-sialyltransferase (alpha2,3ST, IC(50) = 8.2 microM), while this compound was proved to become a favorable substrate for rat recombinant alpha2,6-(N)-sialyltransferase (alpha2,6ST, K(m) = 125 microM). Versatility of this strategy was demonstrated by identification of two selective inhibitors for human recombinant alpha1,3-fucosyltransferase V (alpha1,3-FucT, K(i) = 293 nM) and alpha1,6-fucosyltransferase VIII (alpha1,6-FucT, K(i) = 13.8 microM).

Identification of glycosylated exendin-4 analogue with prolonged blood glucose-lowering activity through glycosylation scanning substitution.

Exendin-4, a glucagon-like peptide 1 receptor agonist, is a potent therapeutic xenopeptide hormone for the treatment of type 2 diabetes. In order to further improve in vivo activity, we examined the introduction of sialyl N-acetyllactosamine (sialyl LacNAc) to exendin-4. The glycosylated analogue having sialyl LacNAc at position 28 was found to have improved in vivo activity with prolonged glucose-lowering activity.

Highly oriented recombinant glycosyltransferases: site-specific immobilization of unstable membrane proteins by using Staphylococcus aureus sortase A.

Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta1,4-galactosyltransferase or recombinant Helicobacter pylori alpha1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of our strategy based on "site-specific" transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.

Potent inhibitor scaffold against Trypanosoma cruzi trans-sialidase.

The protozoan Trypanosoma cruzi, the causative agent of Chagas' disease, can infect the heart, causing cardiac arrest frequently followed by death. To treat this disease, a potential molecular drug target is T. cruzi trans-sialidase (TcTS). However, inhibitors found to date are not strong enough to serve as a lead scaffold; most inhibitors reported thus far are derivatives of the substrate sialic acid or a transition state analogue known as 2,3-dehydro-3-deoxy-N-acetylneuraminic acid (DANA) with an IC(50) value of more than hundreds of micromolar. Since natural products are highly stereodiversified and often provide highly specific biological activity, we screened a natural product library for inhibitors of TcTS and identified promising flavonoid and anthraquinone derivatives. A structure-activity relationship (SAR) analysis of the flavonoids revealed that apigenin had the minimal and sufficient structure for inhibition. Intriguingly, the compound has been reported to possess trypanocidal activity. An SAR analysis of anthraquinones showed that 6-chloro-9,10-dihydro-4,5,7-trihydroxy-9,10-dioxo-2-anthracenecarboxylic acid had the strongest inhibitory activity ever found against TcTS. Moreover, its inhibitory activity appeared to be specific to TcTS. These compounds may serve as potent lead chemotherapeutic scaffolds against Chagas' disease.

An essential epitope of anti-MUC1 monoclonal antibody KL-6 revealed by focused glycopeptide library.

Human serum Krebs von den Lungen-6 (KL-6) antigen, a high-molecular-weight glycoprotein classified as a polymorphic epithelial mucin (MUC1), is a biomarker of diseases such as interstitial pneumonia, lung adenocarcinoma, breast cancer, colorectal adenocarcinoma, and hepatocellular carcinoma. Anti-KL-6 monoclonal antibody (anti-KL-6 MAb) is therefore a potential diagnostic and therapeutic reagent. Although glycosylation at Thr/Ser residues of the tandem-repeating MUC1 peptides appears to determine the disease-associated antigenic structures of KL-6, an essential epitope structure recognized by anti-KL-6 MAb remains unclear. In the present study, a novel compound library of synthetic MUC1 glycopeptides allowed the first rapid and precise evaluation of the specific epitope structure of anti-KL-6 MAb by combined use of a tailored glycopeptides library and common ELISA protocol. We demonstrated that the minimal antigenic structure, an essential epitope, recognized by anti-KL-6 MAb is a heptapeptide sequence Pro-Asp-Thr-Arg-Pro-Ala-Pro (PDTRPAP), in which the Thr residue is modified by Neu5Ac alpha2,3Gal beta1,3GalNAc alpha (2,3-sialyl T antigen, core 1-type O-glycan). Anti-KL-6 MAb did not bind with other tumor-relevant antigens, such as GalNAc alpha (Tn), Neu5Ac alpha2,6GalNAc alpha (STn), and Gal beta1,3GalNAc alpha (T), except for Neu5Ac alpha2,3Gal beta1,3(Neu5Ac alpha2,6)GalNAc alpha (2,3/2,6-disialyl T). However, anti-KL-6 MAb could not differentiate the above minimal antigenic glycopeptide from some core 2-based glycopeptides involving this crucial epitope structure and showed a similar binding affinity toward these compounds, indicating that branching at the O-6 position of GalNAc residue does not influence the interaction of anti-KL-6 MAb with some MUC1 glycoproteins involving an essential epitope. Actually, anti-KL-6 MAb reacts with 2,3/2,6-disialyl T having a 2,3-sialyl T component. This is why anti-KL-6 MAb often reacts with various kinds of tumor-derived MUC1 glycoproteins as well as a clinically important MUC1 glycoprotein biomarker of interstitial pneumonia, namely KL-6, originally discovered as a circulating pulmonary adenocarcinoma-associated antigen. In other words, combined use of anti-KL-6 MAb and some probes that can differentiate the sugars substituted at the O-6 position of the GalNAc residue in MUC1 glycopeptides including the PDTRPAP sequence might be a promising diagnostic protocol for individual disease-specific biomarkers. It was also revealed that glycosylation at neighboring Thr/Ser residues outside the immunodominant PDTRPAP motif strongly influences the interaction between anti-KL-6 MAb and MUC1 glycopeptides involving the identified epitope. Our novel strategy will greatly facilitate the processes for the identification of the tumor-specific and strong epitopes of various known anti-MUC1 MAbs and allow for their practical application in the generation of improved antibody immunotherapeutics, diagnostics, and MUC1-based cancer vaccines.

Functional neoglycopeptides: synthesis and characterization of a new class of MUC1 glycoprotein models having core 2-based O-glycan and complex-type N-glycan chains.

An efficient protocol for the construction of MUC1-related glycopeptide analogues having complex O-glycan and N-glycan chains was established by integrating chemical and enzymatic approaches on the functional polymer platforms. We demonstrated the feasibility of sortase A-mediated ligation between two glycopeptide segments by tagging with signal peptides, LPKTGLR and GG, at each C- or N-terminal position. Structural analysis of the macromolecular N,O-glycopeptides was performed by means of ESI-TOFMS (MS/MS) equipped with an electron-captured dissociation device. Immunological assay using MUC1 glycopeptides synthesized in this study revealed that N-glycosylation near the antigenic O-glycosylated PDTR motif did not disturb the interaction between the anti-MUC1 monoclonal antibody and this crucial O-glycopeptide moiety. NMR study indicated that the N-terminal immunodominant region [Ala-Pro-Asp-Thr(O-glycan)-Arg] forms an inverse gamma-turn-like structure, while the C-terminal region composed of N-glycopeptide and linker SrtA-peptide was proved to be an independently random structure. These results indicate that the bulky O- and N-glycan chains can function independently as disease-relevant epitopes and ligands for carbohydrate-binding proteins, when both are combined by an artificial intervening peptide having a possible effect of separating N- and C-terminal regions. The present strategy will greatly facilitate rapid synthesis of multiply functionalized complex neoglycopeptides as new types of convenient tools or models for the investigation of thhe structure-function relationship of various glycoproteins and development of novel class glycopeptide-based biopharmaceuticals, drug delivery systems, and biomedical materials.

Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity.

Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogues with N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and alpha2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepared by chemoenzymatic approaches. We assessed the receptor binding affinity and cAMP production activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addition of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic analysis of the degradation reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addition, the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogues with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogues of other bioactive peptides as desirable pharmaceuticals.

Development of a simple and robust assay to screen for inhibitors of sphingosine kinases.

Sphingosine kinases (SPHKs) catalyze the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), which plays important roles in a wide variety of intra- and extracellular functions. Conventionally, SPHK activity has been determined using radioisotope thin layer chromatography (TLC) and autoradiography to detect the product S1P. Here we describe the development of a simple and robust in vitro SPHK assay in 384-well format with no requirement for any separation steps such as extraction and TLC. The assay is based on (33)P-phosphate transfer from [gamma-(33)P]ATP to sphingosine and subsequent detection of the [(33)P]S1P using AquaBind plates (Asahi Techno Glass, Tokyo, Japan). Enzymatic and inhibition characteristics determined with this assay are in good agreement with previously reported values determined in the conventional TLC assay. K(m) values for D-erythro-sphingosine and ATP were determined to be 17.5 microM and 19.2 microM, respectively. The kinase reaction could be inhibited by ADP and N,N-dimethylsphingosine with a 50% inhibitory concentration of 410 microM and 450 microM, respectively. The established assay format was easily adapted to an automated screening platform and is characterized by a high signal-to-background ratio, small variation, and excellent Z factors.

Development of a simple homogeneous assay to screen for inhibitors of N-acetylglucosamine-6-sulfotransferases.

L-selectin, a leukocyte adhesion molecule, plays a central role in lymphocyte homing to secondary lymphoid tissue and to certain sites of inflammation. Carbohydrate sulfation was implicated in this process, when it was demonstrated that carbohydrate sulfotransferase-mediated sulfation of N-acetylglucosamine (GlcNAc) within sialyl Lewis X of cognate endothelial ligands for L-selectin was an essential modification for L-selectin binding. The recently identified GlcNAc-6-sulfotransferases GlcNAc6ST-1 and -2, which facilitate GlcNAc sulfation by catalyzing the transfer of a sulfonyl group from 3(')-phosphoadenosine 5(')-phosphosulfate (PAPS) to the 6-hydroxy group of the acceptor GlcNAc moiety, contribute to the biosynthesis of the 6-sulfosialyl Lewis X motif. Due to their pivotal role in L-selectin ligand biosynthesis, this enzyme class has recently emerged as an important and relatively unexplored class of potential targets for anti-inflammatory therapy. However, no inhibitors have been reported to date and screening for lead inhibitors has been hampered by the lack of simple assay formats suitable for high-throughput screening. Here, we report the development of a simple homogeneous in vitro sulfotransferase assay using a newly synthesized biotinylated glycoside as a substrate. The assay is based on GlcNAc6ST-2-mediated [35S]sulfate transfer from [35S]PAPS to the biotinylated glycoside and subsequent detection using streptavidin-coated SPA beads. K(m) values with partially purified GlcNAc6ST-2 for PAPS and the biotinylated glycoside were estimated to be 8.4 and 34.5 microM, respectively. The sulfotransferase reaction could be inhibited by 3('),5(')-ADP with an IC(50) of 2.1 microM. The assay can be operated in 384-well format; is characterized by a high signal-to-noise ratio, low variation, and excellent Z factors; and is highly suitable for high-throughput screening.

A versatile high-throughput screen for inhibitors of lipid kinase activity: development of an immobilized phospholipid plate assay for phosphoinositide 3-kinase gamma.

The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K gamma isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening. The authors report the development of a simple and robust 384-well plate assay that is based on(33) P-phosphate transfer from radiolabeled [gamma(33) P]ATP to phosphatidylinositol immobilized on Maxisorp plates. The established assay format for PI3K gamma was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K gamma determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K gamma was observed at pH 7.0. The apparent K(m) value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3 microM (6.0-8.6 microM, 95% confidence interval [CI]). IC(50) values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.