Lysine-targeting inhibition of amyloid ß oligomerization by a green perilla-derived metastable chalcone in vitro and in vivo.

Oligomers of amyloid ß (Aß) represent an early aggregative form that causes neurotoxicity in the pathogenesis of Alzheimer's disease (AD). Thus, preventing Aß 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 Aß aggregation. In this study, we observed the site-specific inhibition of 42-mer Aß (Aß42) oligomerization by the green perilla-derived chalcone 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC), which was converted to its decomposed flavonoids (dDDC, 1-3) via nucleophilic aromatic substitution with water molecules. DDC suppressed Aß42 fibrillization and slowed the transformation of the ß-sheet structure, which is rich in Aß42 aggregates. To validate the contribution of dDDC to the inhibitory effects of DDC on Aß42 aggregation, we synthesized 1-3 and identified 3, a catechol-type flavonoid, as one of the active forms of DDC. 1H-15N SOFAST-HMQC NMR revealed that 1-3 as well as DDC could interact with residues between His13 and Leu17, which were near the intermolecular ß-sheet (Gln15-Ala21). The nucleation in Aß42 aggregates involves the rate-limiting formation of low-molecular-weight oligomers. The formation of a Schiff base with dDDC at Lys16 and Lys28 in the dimer through autoxidation of dDDC was associated with the suppression of Aß42 nucleation. Of note, in two AD mouse models using immunoaffinity purification-mass spectrometry, adduct formation between dDDC and brain Aß was observed in a similar manner as reported in vitro. The present findings unraveled the lysine-targeting inhibitory mechanism of metastable dietary ingredients regarding Aß oligomerization.

Optimization of the Linker Length in the Dimer Model of E22P-Aß40 Tethered at Position 38.

Since amyloid ß (Aß) 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-Aß40 dimer) with an l,l-2,6-diaminopimelic acid linker (n = 3) at position 38, which exhibited moderate cytotoxicity. We synthesized four additional linkers (n = 2, 4, 5, 7) to determine the most appropriate distance between the two Aß40 monomers for a toxic dimer model. Each di-Fmoc-protected two-valent amino acid was synthesized from a corresponding dialdehyde or cycloalkene followed by ozonolysis, using a Horner-Wadsworth-Emmons reaction and asymmetric hydrogenation. Then, the corresponding Aß40 dimer models with these linkers at position 38 were synthesized using the solid-phase Fmoc strategy. Their cytotoxicity toward SH-SY5Y cells suggested that the shorter the linker length, the stronger the cytotoxicity. Particularly, the E22P,G38DAA-Aß40 dimer (n = 2) formed protofibrillar aggregates and exhibited the highest cytotoxicity, equivalent to E22P-Aß42, the most cytotoxic analogue of Aß42. Ion mobility-mass spectrometry (IM-MS) measurement indicated that all dimer models except the E22P,G38DAA-Aß40 dimer existed as stable oligomers (12-24-mer). NativePAGE analysis supported the IM-MS data, but larger oligomers (30-150-mer) were also detected after a 24 h incubation. Moreover, E22P,G38DAA-Aß40, E22P,G38DAP-Aß40, and E22P,G38DAZ-Aß40 (n = 5) dimers suppressed long-term potentiation (LTP). Overall, the ability to form fibrils with cross ß-sheet structures was key to achieving cytotoxicity, and forming stable oligomers less than 150-mer did not correlate with cytotoxicity and LTP suppression.

Synthesis and Characterization of Propeller- and Parallel-Type Full-Length Amyloid ß40 Trimer Models.

Oligomers of the amyloid ß (Aß) protein play a critical role in the pathogenesis of Alzheimer's disease. However, their heterogeneity and lability deter the identification of their tertiary structures and mechanisms of action. Aß trimers and Aß dimers may represent the smallest aggregation unit with cytotoxicity. Although propeller-type trimer models of E22P-Aß40 tethered by an aromatic linker have recently been synthesized, they unexpectedly exhibited little cytotoxicity. To increase the flexibility of trimeric propeller-type models, we designed and synthesized trimer models with an alkyl linker, tert-butyltris-l-alanine (tButA), at position 36 or 38. In addition, we synthesized two parallel-type trimer models tethered at position 38 using alkyl linkers of different lengths, α,α-di-l-norvalyl-l-glycine (di-nV-Gly) and α,α-di-l-homonorleucyl-l-glycine (di-hnL-Gly), based on the previously reported toxic dimer model. The propeller-type E22P,V36tButA-Aß40 trimer (4), which was designed to mimic the C-terminal anti-parallel ß-sheet structures proposed by the structural analysis of 150 kDa oligomers of Aß42, and the parallel-type E22P,G38di-nV-Gly-Aß40 trimer (6) showed significant cytotoxicity against SH-SY5Y cells and aggregative ability to form protofibrillar species. In contrast, the E22P,G38tButA-Aß40 trimer (5) and E22P,G38di-hnL-Gly-Aß40 trimer (7) exhibited weak cytotoxicity, though they formed quasi-stable oligomers observed by ion mobility-mass spectrometry and native polyacrylamide gel electrophoresis. These results suggest that 4 and 6 could have some phase of the structure of toxic Aß oligomers with a C-terminal hydrophobic core and that the conformation and/or aggregation process rather than the formation of stable oligomers contribute to the induction of cytotoxicity.

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 N-glycan structures by ultraperformance liquid chromatography-connected ion mobility mass spectrometry (UPLC/IM-MS). To substantiate this methodology, we here examine 71 pyridylaminated (PA-) N-linked oligosaccharides including isomeric pairs. A data set on collision drift times, retention times, and molecular mass was collected for these PA-oligosaccharides. For standardization of the observables, LC retention times were normalized into glucose units (GU) using pyridylaminated α-1,6-linked glucose oligomers as reference, and drift times in IM-MS were converted into collision cross sections (CCS). To evaluate the CCS value of each PA-oligosaccharide, we introduced a CCS index which is defined as a CCS ratio of a target PA-glycan to the putative standard PA-glucose oligomer of the same m/z. We propose a strategy for practical structural analysis of N-linked glycans based on the database of m/z, CCS index, and normalized retention time (GU).

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). METHODS: The physicochemical characteristics of the modified oligonucleotides were determined using IM-MS. Each oligonucleotide was evaluated by confirming the multivalent charge state drift times, collision cross-section (CCS) values, and CCS widths. RESULTS: By plotting the m/z for oligonucleotides of different lengths and the CCS values at each charge state, a bottoming-out shape plot at one charge per 4.0-3.5 bases was confirmed. Moreover, significant differences were observed in the CCS values between the PS-modified and unmodified oligonucleotides. The PS-modified oligonucleotide showed a wider CCS range that was proportional to the PS modification ratio of the oligonucleotide sequence. CONCLUSIONS: The TWIMS results showed a correlation between the length and modification of oligonucleotides and the CCS values. In addition, it suggested that each charge state of the oligonucleotide ion has different physicochemical properties.

Detection of Amyloid ß Oligomers with RNA Aptamers in AppNL-G-F/NL-G-F 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 ß-protein (Aß42), 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 Aß42 oligomerization impede aptamer research. We previously developed three RNA aptamers (E22P-AbD4, -AbD31, and -AbD43) with high affinity for protofibrils (PFs) derived from a toxic Aß42 dimer. Notably, these aptamers recognized diffuse staining, which likely originated from PFs or higher-order oligomers with curvilinear structures in a knock-in AppNL-G-F/NL-G-F mouse, carrying the Arctic mutation that preferentially induced the formation of PFs, in addition to a PS2Tg2576 mouse. To determine which oligomeric sizes were mainly altered by the aptamer, ion mobility-mass spectrometry (IM-MS) was carried out. One aptamer, E22P-AbD43, formed adducts with the Aß42 monomer and dimer, leading to suppression of further oligomerization. These findings support the utility of these aptamers as diagnostics for AD.

Synthetic and biochemical studies on the effect of persulfidation on disulfide dimer models of amyloid ß42 at position 35 in Alzheimer's etiology.

Protein persulfidation plays a role in redox signaling as an anti-oxidant. Dimers of amyloid ß42 (Aß42), 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. In order to verify whether persulfidation has an effect in AD, herein we report a chemical approach by synthesizing disulfide dimers of Aß42 and their evaluation of biochemical properties. A homoCys-disulfide dimer model at position 35 of Aß42 formed a partial ß-sheet structure, but its neurotoxicity was much weaker than that of the corresponding monomer. In contrast, the congener with an alkyl linker generated ß-sheet-rich 8-16-mer oligomers with potent neurotoxicity. The length of protofibrils generated from the homoCys-disulfide dimer model was shorter than that of its congener with an alkyl linker. Therefore, the current data do not support the involvement of Aß42 persulfidation in Alzheimer's disease.

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-ß- 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. The reactions were monitored by an ultraperformance liquid chromatography system using an amide-based wide-pore column. The products were characterized by liquid chromatography coupled with tandem mass spectrometry. The side reactions were suppressed by adding glycan oxazoline in a stepwise manner under slightly acidic conditions. Through a combination of an azide-carrying glycan transfer reaction under optimized conditions and a bio-orthogonal reaction, a potent cytotoxic agent monomethyl auristatin E was site-specifically conjugated at N-glycosylated Asn297 with a drug-to-antibody ratio of 4. The prepared antibody-drug conjugate exhibited cytotoxicity against HER2-expressing cells.

Synthesis and biochemical characterization of quasi-stable trimer models of full-length amyloid ß40 with a toxic conformation.

Here, we report the first synthesis of quasi-stable trimer models of full-length Aß40 with a toxic conformation using a 1,3,5-phenyltris-l-alanyl linker at position 34, 36, or 38. The only trimer to exhibit weak neurotoxicity against SH-SY5Y cells was the one which was linked at position 38. This suggests that such a propeller-type trimer model is not prone to forming oligomers with potent neurotoxicity, which is in contrast with its corresponding dimer model.

Role of the carboxy groups of triterpenoids in their inhibition of the nucleation of amyloid ß42 required for forming toxic oligomers.

Herein we report that a preferable inhibition of the nucleation phase of Aß42, 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 Aß42. 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 ß40 Oligomers with Significant Neurotoxicity.

The formation of soluble oligomers of amyloid ß42 and 40 (Aß42, Aß40) 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 Aß42, 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 Aß42, Aß40 dimers can also contribute to AD progression because of the more abundance of Aß40 monomer in biological fluids. Here, we describe the synthesis and characterization of three dimer models of the toxic-conformation constrained E22P-Aß40 using l,l-2,6-diaminopimeric acid (DAP) or l,l-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel ß-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aß40 dimer (1) and E22P-A30DAZ-Aß40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity toward SH-SY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aß40 dimer (3) formed ß-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility-mass spectrometry suggested that high molecular-weight oligomers (12-24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel ß-sheet, triggers the formation of toxic Aß oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.

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. Furthermore, it is important to ascertain whether an NCE inhibits multiple CYP isoforms. Hence, using a cocktail-substrate approach that we previously established (in which the activity of 8 CYP isoforms is simultaneously evaluated in a single run), we evaluated the IC50 values of direct inhibitors and TDI parameters (kobs, shifted IC50, KI and kinact) of time-dependent inhibitors that affect multiple CYP isoforms. The IC50 values for 8 CYP isoforms obtained using the cocktail-substrate approach were nearly identical to values previously reported. The TDI parameters for CYP1A2, 2C9, 2C19, 2D6, and CYP3A4/5 obtained using the cocktail-substrate approach were also nearly identical to those obtained using a single-substrate approach. Thus, the cocktail-substrate approach is useful for evaluating DI and TDI in the early stages of drug discovery and development processes.

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. A cocktail of 9 probe substrates for the 8 major CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5) was incubated with human liver microsomes. Each substrate-derived metabolite was simultaneously analyzed by multiple reactions monitoring with a single ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run using stable isotope-labeled internal standards. The ultra-fast UPLC gradient allowed each metabolite to be separated within 1 min, providing quantitative linearity of over 2 orders of magnitude. CYP inhibition by 8 well-known inhibitors was confirmed by comparing single substrates with the substrate cocktail. The inhibition curve profiles and IC50 values for all CYPs in the cocktail substrate were similar to those of single substrates. UPLC-MS/MS using a CYP substrate cocktail is a reliable and robust high-throughput method to accurately assess CYP inhibition potencies of newly developed drugs.

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. In contrast, the spectra calculated by taking into account the statistical distribution of the torsion angles of the individual central residues in the sequences Ala-Gly-Pro, Gly-Pro-Gly, Pro-Gly-Gly, Gly-Gly-Ala, and Gly-Ala-Gly from PDB data could reproduce the observed spectra well. This indicates that the statistical distribution of the torsion angles should be considered for the structural model of (GPGGA)6G similar to the case of the model peptide of elastin.