Immune response and protective efficacy of the SARS-CoV-2 recombinant spike protein vaccine S-268019-b in mice.

Vaccines that efficiently target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease (COVID-19), are the best means for controlling viral spread. This study evaluated the efficacy of the COVID-19 vaccine S-268019-b, which comprises the recombinant full-length SARS-CoV-2 spike protein S-910823 (antigen) and A-910823 (adjuvant). In addition to eliciting both Th1-type and Th2-type cellular immune responses, two doses of S-910823 plus A-910823 induced anti-spike protein IgG antibodies and neutralizing antibodies against SARS-CoV-2. In a SARS-CoV-2 challenge test, S-910823 plus A-910823 mitigated SARS-CoV-2 infection-induced weight loss and death and inhibited viral replication in mouse lungs. S-910823 plus A-910823 promoted cytokine and chemokine at the injection site and immune cell accumulation in the draining lymph nodes. This led to the formation of germinal centers and the induction of memory B cells, antibody-secreting cells, and memory T cells. These findings provide fundamental property of S-268019-b, especially importance of A-910823 to elicit humoral and cellular immune responses.

Macrocyclic Peptides as a Novel Class of NNMT Inhibitors: A SAR Study Aimed at Inhibitory Activity in the Cell.

Nicotinamide N-methyltransferase (NNMT), which catalyzes the methylation of nicotinamide, is a cytosolic enzyme that has attracted much attention as a therapeutic target for a variety of diseases. However, despite the considerable interest in this target, reports of NNMT inhibitors have still been limited to date. In this work, utilizing in vitro translated macrocyclic peptide libraries, we identified peptide 1 as a novel class of NNMT inhibitors. Further exploration based on the X-ray cocrystal structures of the peptides with NNMT provided a dramatic improvement in inhibitory activity (peptide 23: IC50 = 0.15 nM). Furthermore, by balance of the peptides' lipophilicity and biological activity, inhibitory activity against NNMT in cell-based assay was successfully achieved (peptide 26: cell-based IC50 = 770 nM). These findings illuminate the potential of cyclic peptides as a relatively new drug discovery modality even for intracellular targets.

Artificial Golgi apparatus: globular protein-like dendrimer facilitates fully automated enzymatic glycan synthesis.

Despite the growing importance of synthetic glycans as tools for biological studies and drug discovery, a lack of common methods for the routine synthesis remains a major obstacle. We have developed a new method for automated glycan synthesis that employs the enzymatic approach and a dendrimer as an ideal support within the chemical process. Recovery tests using a hollow fiber ultrafiltration module have revealed that monodisperse G6 (MW = 58 kDa) and G7 (MW = 116 kDa) poly(amidoamine) dendrimers exhibit a similar profile to BSA (MW = 66 kDa). Characteristics of the globular protein-like G7 dendrimer with high solubility and low viscosity in water greatly enhanced throughput and efficiency in automated synthesis while random polyacrylamide-based supports entail significant loss during the repetitive reaction/separation step. The present protocol allowed for the fully automated enzymatic synthesis of sialyl Lewis X tetrasaccharide derivatives over a period of 4 days in 16% overall yield from a simple N-acetyl-d-glucosamine linked to an aminooxy-functionalized G7 dendrimer.

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.

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.

Structural analysis of O-glycopeptides employing negative- and positive-ion multi-stage mass spectra obtained by collision-induced and electron-capture dissociations in linear ion trap time-of-flight mass spectrometry.

Structural analyses of various glycans attached to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on mass spectrometry (MS) combining both collision-induced dissociation (CID) and electron-capture dissociation (ECD) in the positive- and negative-ion modes has been proposed as a simple and direct method of assigning an O-glycan without releasing it from the peptide and of determining the amino acid sequence of the peptide and glycosylation site. The instrument used is an electrospray ionization (ESI) linear ion trap (LIT) time-of-flight (TOF) mass spectrometer with tandem LITs for CID by He gas and ECD. The proposed approach was tested with two synthetic O-glycopeptides binding a sialyl Lewis x (sLe(x)) oligosaccharide and a 3'-sialyl N-acetyllactosamine (3'-SLN) on a serine (S) residue. In the negative-ion mode, the CID MS(2) spectra of O-glycopeptides showed a relatively abundant glycoside-bond cleavage between the core N-acetylglucosamine (GlcNAc) and serine (S) that yields deprotonated C(3)-type fragment ions of O-glycan and deprotonated Z(0)-type peptide ions. The structure of the sLe(x) (3'-SLN) oligosaccharide was simply assigned by comparing the CID MS(3) spectrum derived from the C(3)-type fragment ion with the CID MS(2) spectra of the sLe(x) and sLe(a) (3'- and 6'-SLN) standards (i.e., negative-ion MS(n) spectral matching). The amino acid sequence of the peptide including the glycosylation site was determined from the ECD MS(2) spectrum in the positive-ion mode.

Construction of highly glycosylated mucin-type glycopeptides based on microwave-assisted solid-phase syntheses and enzymatic modifications.

A MUC1-related glycopeptide having five core-2 hexasaccharide branches (C330H527N46O207, MW = 8450.9) was synthesized by a new strategy using a combination of microwave-assisted solid-phase synthesis (MA-SPGS) and enzymatic sugar elongation. Synthesis of a key glycopeptide intermediate was best achieved in a combination of PEGA [poly(ethylene glycol)-poly-(N,N-dimethylacrylamide) copolymer] resin and MA-SPGS using glycosylated amino acid building blocks with high speed and high purity. Deprotection of the glycopeptide intermediate and subsequent glycosyltransferase-catalyzed sugar elongations were performed for generation of the additional diversities with the sugar moieties of glycopeptides using beta1,4-galactosyltransferase (beta1,4-GalT) and two kinds of alpha2,3-sialyltransferases [ST3Gal III; alpha2,3-(N)-SiaT and ST3Gal II; alpha2,3-(O)-SiaT]. These reactions proceeded successfully in the presence of 0.2% Triton X-100 to convert the chemically synthesized trisaccharide glycans to disialylated hexasaccharide.

Combinatorial synthesis of MUC1 glycopeptides: polymer blotting facilitates chemical and enzymatic synthesis of highly complicated mucin glycopeptides.

The chemoselective polymer blotting method allows for rapid and efficient synthesis of glycopeptides based on a "catch and release" strategy between solid-phase and water-soluble polymer supports. We have developed a heterobifunctional linker sensitive to glutamic acid specific protease (BLase). The general procedure consists of five steps, namely (i) the solid-phase synthesis of glycopeptide containing BLase sensitive linker, (ii) subsequent deprotections and the release of the glycopeptide from the resin, (iii) chemoselective blotting of the glycopeptide intermediates in the presence of water-soluble polymers with oxylamino functional groups, (iv) sugar elongations using glycosyltransferases, and (v) the release of target glycopeptides from the polymer platform by selective BLase promoted hydrolysis. The combined use of the solid-phase chemical syntheses of peptides and the enzymatic syntheses of carbohydrates on water-soluble polymers would greatly contribute to the production of complicated glycopeptide libraries, thereby enhancing applicative research. We report here a high-throughput synthetic system for the various types of MUC1 glycopeptides exhibiting a variety of sugar moieties. It is our belief that this concept will become part of the entrenched repertoire for the synthesis of biologically important glycopeptides on the basis of glycosyltransferase reactions in automated and combinatorial syntheses.

Synthesis of optically active trifluoromethylated indolizidine derivatives via stereoselective radical cyclization.

[reaction: see text]. Asymmetric Michael reaction of lithiated trifluoroacetone SAMP-hydrazone with alkylidenemalonates gave addition products stereoselectively. Hydrolyzed enantiomerically pure ketoacids were cyclized to dihydropyridinones. N-Iodopropylation followed by radical cyclization gave optically active trifluoromethylated indolizidinones stereoselectively.