Separation and Characterization of Therapeutic Oligonucleotide Isomer Impurities by Cyclic Ion Mobility Mass Spectrometry.

Therapeutic oligonucleotides such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA) are among the most remarkable modalities in modern medicine. ASOs and siRNA are composed of single- or double-stranded 15-25 mer synthesized oligonucleotides, which can be used to modulate gene expression. Liquid chromatography-mass spectrometry (LC/MS) is a necessary technique for the quality control of therapeutic oligonucleotides; it is used to evaluate the quantities of target oligonucleotides and their impurities.

Lysine-targeting inhibition of amyloid β oligomerization by a green perilla-derived metastable chalcone and .

Oligomers of amyloid β (Aβ) represent an early aggregative form that causes neurotoxicity in the pathogenesis of Alzheimer's disease (AD). Thus, preventing Aβ aggregation is important for preventing AD. Despite intensive studies on dietary compounds with anti-aggregation properties, some identified compounds are susceptible to autoxidation and/or hydration upon incubation in water, leaving unanswered issues regarding which active structures in metastable compounds are actually responsible for the inhibition of Aβ aggregation.

Optimization of the Linker Length in the Dimer Model of E22P-Aβ40 Tethered at Position 38.

Since amyloid β (Aβ) oligomers are more cytotoxic than fibrils, various dimer models have been synthesized. We focused on the C-terminal region that could form a hydrophobic core in the aggregation process and identified a toxic conformer-restricted dimer model (E22P,G38DAP-Aβ40 dimer) with an l,l-2,6-diaminopimelic acid linker ( = 3) at position 38, which exhibited moderate cytotoxicity. We synthesized four additional linkers ( = 2, 4, 5, 7) to determine the most appropriate distance between the two Aβ40 monomers for a toxic dimer model.

A Data Set of Ion Mobility Collision Cross Sections and Liquid Chromatography Retention Times from 71 Pyridylaminated N-Linked Oligosaccharides.

Determination of the glycan structure is an essential step in understanding structure-function relationships of glycans and glycoconjugates including biopharmaceuticals. Mass spectrometry, because of its high sensitivity and mass resolution, is an excellent means of analyzing glycan structures. We previously proposed a method for rapid and precise identification of -glycan structures by ultraperformance liquid chromatography-connected ion mobility mass spectrometry (UPLC/IM-MS).

Physicochemical property evaluation of modified oligonucleotides by traveling-wave ion mobility mass spectrometry.

Rationale: Therapeutic oligonucleotides have molecular weights of more than 6000 Da. They typically contain chemically modified structures such as phosphorothioate (PS) and a locked nucleic acid (LNA). To determine the effect of the length and chemical modification on the physicochemical properties, various nucleic acids with different lengths and modified structures were analyzed using traveling-wave ion mobility mass spectrometry (TWIMS).

Detection of Amyloid β Oligomers with RNA Aptamers in App Mice: A Model of Arctic Alzheimer's Disease.

RNA aptamers have garnered attention for diagnostic applications due to their ability to recognize diverse targets. Oligomers of 42-mer amyloid β-protein (Aβ42), whose accumulation is relevant to the pathology of Alzheimer's disease (AD), are among the most difficult molecules for aptamer recognition because they are prone to aggregate in heterogeneous forms. In addition to designing haptens for in vitro selection of aptamers, the difficulties involved in determining their effect on Aβ42 oligomerization impede aptamer research.

Synthetic and biochemical studies on the effect of persulfidation on disulfide dimer models of amyloid β42 at position 35 in Alzheimer's etiology.

Protein persulfidation plays a role in redox signaling as an anti-oxidant. Dimers of amyloid β42 (Aβ42), which induces oxidative stress-associated neurotoxicity as a causative agent of Alzheimer's disease (AD), are minimum units of oligomers in AD pathology. Met35 can be susceptible to persulfidation through its substitution to homoCys residue under the condition of oxidative stress.

Characterization of Antibody Products Obtained through Enzymatic and Nonenzymatic Glycosylation Reactions with a Glycan Oxazoline and Preparation of a Homogeneous Antibody-Drug Conjugate via Fc N-Glycan.

Glycan engineering of antibodies has received considerable attention. Although various endo-β- N-acetylglucosaminidase mutants have been developed for glycan remodeling, a side reaction has been reported between glycan oxazoline and amino groups. In this study, we performed a detailed characterization for antibody products obtained through enzymatic and nonenzymatic reactions with the aim of maximizing the efficiency of the glycosylation reaction with fewer side products.

Role of the carboxy groups of triterpenoids in their inhibition of the nucleation of amyloid β42 required for forming toxic oligomers.

Herein we report that a preferable inhibition of the nucleation phase of Aβ42, related to the formation of toxic oligomers, by triterpenoids from medicinal herbs originates from a salt bridge of their carboxy groups with Lys16 and 28 in Aβ42. Such a direct interaction targeting the monomer, dimer, and trimer suppressed further oligomerization. In contrast, the corresponding congeners without carboxy groups failed to do so.

Synthetic Models of Quasi-Stable Amyloid β40 Oligomers with Significant Neurotoxicity.

The formation of soluble oligomers of amyloid β42 and 40 (Aβ42, Aβ40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aβ42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the C-terminal region. However, in addition to Aβ42, Aβ40 dimers can also contribute to AD progression because of the more abundance of Aβ40 monomer in biological fluids.

Cocktail-substrate approach-based high-throughput assay for evaluation of direct and time-dependent inhibition of multiple cytochrome P450 isoforms.

Avoiding drug-drug interactions (DDIs) mediated through inhibition of cytochrome P450 (CYP) activity is highly desirable. Direct inhibition (DI) of CYP through new chemical entities (NCEs) or time-dependent inhibition (TDI) through reactive metabolites should be elucidated at an early stage of drug discovery research. In particular, TDI of CYP occurring through reactive metabolites may be irreversible and even sustained, causing far more serious DDIs for TDIs than for DIs.

Reliable high-throughput method for inhibition assay of 8 cytochrome P450 isoforms using cocktail of probe substrates and stable isotope-labeled internal standards.

A significant number of new chemical entities (NCEs) disappear due to cytochrome P450 (CYP)-mediated clinical drug-drug interactions in drug discovery. Therefore, a high throughput assay of CYP activities is necessary in order to evaluate the inhibitory or inducible potencies of CYP isoforms with NCEs in early drug discovery. Here, we developed and validated a high-throughput assay to simultaneously monitor the in vitro activities of 8 CYP isoforms.

Solid-state NMR analysis of a peptide (Gly-Pro-Gly-Gly-Ala)6-Gly derived from a flagelliform silk sequence of Nephila clavipes.

Solid-state NMR is especially useful when the structures of peptides and proteins should be analyzed by taking into account the structural distribution, that is, the distribution of the torsion angle of the individual residue. In this study, two-dimensional spin-diffusion solid-state NMR spectra of 13C-double-labeled model peptides (GPGGA)6G of flagelliform silk were observed for studying the local structure in the solid state. The spin-diffusion NMR spectra calculated by assuming the torsion angles of the beta-spiral structure exclusively could not reproduce the observed spectra.