Thiophene bioisosteres of GluN2B selective NMDA receptor antagonists: Synthesis and pharmacological evaluation of [7]annuleno[b]thiophen-6-amines

Thiophene bioisosteres of potent GluN2B receptor negative allosteric modulators were prepared and evaluated pharmacologically. The five-step synthesis of 4,5,7,8-tetrahydro[7]annuleno[b]thiophen-6-one (10) was considerably improved by carboxylation of thiophene-3-carboxylic acid (8) in the first reaction step. Reductive amination and alkylation led to three homologous series of secondary and tertiary phenylalkylamines 5, 11 and 12. Metalation, reaction with 1-formylpiperidine and subsequent reduction provided hydroxymethyl derivatives 15 and 16, which had been designed as bioisosteres of phenols. 2-Bromo derivatives 18 were obtained by bromination of ketone 10 with NBS and subsequent reductive amination. High GluN2B affinity was achieved with [7]annuleno[b]thiophenes bearing a 3-phenylpropylamino or 4-phenylbutylamino moiety (e.g. 5c: K_i = 5.9 nM; 11d: K_i = 9.0 nM). Tertiary ethylamines 12 showed lower GluN2B affinity than tertiary methylamines 11 or secondary amines 5 (e.g. 5c: K_i = 5.9 nM; 11c: K_i = 6.0; 12c: K_i = 51 nM). A Br-atom or a hydroxymethyl moiety in 2-position were less tolerated by the GluN2B receptor. Very similar relationships between the structure and GluN2B affinity and structure and σ affinity, in particular σ₂ affinity, were detected. A slight preference for the ifenprodil binding site of GluN2B receptors over σ₁ and σ₂ receptors was found for methylamines 11c (≈2-fold) and 11d (≈1.5–2-fold) as well as for bromo derivative 18c (≈3-fold).     

Multivariate data analysis of growth medium trends affecting antibody glycosylation

Use of multivariate data analysis for the manufacturing of biologics has been increasing due to more widespread use of data-generating process analytical technologies (PAT) promoted by the US FDA. To generate a large dataset on which to apply these principles, we used an in-house model CHO DG44 cell line cultured in automated micro bioreactors alongside PAT with four commercial growth media focusing on antibody quality through N-glycosylation profiles. Using univariate analyses, we determined that different media resulted in diverse amounts of terminal galactosylation, high mannose glycoforms, and aglycosylation. Due to the amount of in-process data generated by PAT instrumentation, multivariate data analysis was necessary to ascertain which variables best modeled our glycan profile findings. Our principal component analysis revealed components that represent the development of glycoforms into terminally galacotosylated forms (G1F and G2F), and another that encompasses maturation out of high mannose glycoforms. The partial least squares model additionally incorporated metabolic values to link these processes to glycan outcomes, especially involving the consumption of glutamine. Overall, these approaches indicated a tradeoff between cellular productivity and product quality in terms of the glycosylation. This work illustrates the use of multivariate analytical approaches that can be applied to complex bioprocessing problems for identifying potential solutions.     

Molecular basis for the selective recognition and ubiquitination of centromeric histone H3 by yeast E3 ligase Psh1

Centromeres are chromosomal loci marked by histone variant Cen H3(centromeric histone H3) and essential for genomic stability and cell division. The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeast Cen H3(Cse4) for ubiquitination and controls the cellular level of Cse4 for proteolysis,but the underlying mechanism remains largely unknown. Here, we show that Psh1 uses a Cse4-binding domain(CBD, residues 1-211) to interact with Cse4-H4 instead of H3-H4, yielding a dissociation constant(K_d) of 27 nM. Psh1 recognizes Cse4-specific residues in the L1 loop and a2 helix to ensure Cse4 binding and ubiquitination. We map the Psh1-binding region of Cse4-H4 and identify a wide range of Cse4-specific residues required for the Psh1-mediated Cse4 recognition and ubiquitination. Further analyses reveal that histone chaperone Scm3 can impair Cse4 ubiquitination by abrogating Psh1-Cse4 binding. Together, our study reveals a novel Cse4-binding mode distinct from those of known Cen H3 chaperones and elucidates the mechanism by which Scm3 competes with Psh1 for Cse4 binding.     

Condensation reactions catalyzed by α-N-acetylgalactosaminidase from Aspergillus niger yielding α-N-acetylgalactosaminides

Abstract

Extracellular α-N-acetylgalactosaminidase from Aspergillus niger catalyzed glycosylation yielding a series of 2-acetamido-2-deoxy-α-D-galactobiosides using 2-acetamido-2-deoxy-D-galactopyranose as a glycosyl donor. The isomers α-D-GalpNAc-(1→6)-D-GalpNAc, α-D-GalpNAc-(1→3)-D-GalpNAc and α-D-GalpNAc-(1→6)-D-GalfNAc were isolated and spectrally characterized. The purified enzyme was further used for the glycosylation of free amino acids (serine and threonine) and their N-(tert-butoxycarbonyl)-protected analogs to synthesize the Tn antigen (GalpNAc-α-O-Ser/Thr) and its N-(tert-butoxycarbonyl)-protected derivatives.     

How independent are TSE agents from their hosts?

Central to understanding the nature TSE agents (or prions) is how their genetic information is distinguished from the host. Are TSEs truly infectious diseases with host-independent genomes, or are they aberrations of a host component derived from the host genome? Recent experiments tested whether glycosylation of host PrP affects TSE strain characteristics. Wild-type mice were infected with 3 TSE strains passaged through transgenic mice with PrP devoid of glycans at 1 or both N-glycosylation sites. Strain-specific characteristics of 1 TSE strain changed but did not change for 2 others. Changes resulted from the selection of mutant TSE strains in a novel replicative environment. In general the properties of established TSEs support the genetic independence of TSE agents from the host, and specifically the primary structure of PrP does not directly encode TSE agent properties. However sporadic TSEs, challenge this independency. The prion hypothesis explains emerging TSEs relatively successfully but poorly accounts for the diversity and mutability of established TSE strains, or how many different infectious conformations are sustained thermodynamically. Research on early changes in RNA expression and events at the ribosome may inform the debate on TSE agent properties and their interaction with host cell machinery.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Genetic variants and signatures of selective sweep of Hanwoo population (Korean native cattle)

Although there have been many studies of native Korean cattle, Hanwoo, there have been no selective sweep studies in these animals. This study was performed to characterize genetic variation and identify selective signatures. We sequencedthe genomes of 12 cattle, and identified 15125420 SNPs, 1768114 INDELs, and 3445 CNVs. The SNPs, INDELs, and CNVs were similarly distributed throughout the genome, and highly variable regions were shown to contain the BoLA family and GPR180, which are related to adaptive immunity. We also identified the domestication footprints of the Hanwoo population by searching for selective sweep signatures, which revealed the RCN2 gene related to BPV resistance. The results of this study may contribute to genetic improvement of the Hanwoo population in Korea. [BMB Reports 2013; 46(7):346-351]     

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC–MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.     

Discovery of selective farnesoid X receptor agonists for the treatment of hyperlipidemia from traditional Chinese medicine based on virtual screening and in vitro validation

Abstract

Farnesoid X receptor (FXR), a bile acid receptor, has important roles in maintaining bile acid and cholesterol homeostasis, which is an attractive target for hyperlipidemia. Present study aimed to discover potential selective FXR agonists over G-protein coupled bile acid receptor 1 (GPBAR1, TGR5) from traditional Chinese medicine (TCM) by using virtual screening, in vitro studies and molecular dynamics simulation (MD). Ligand-based pharmacophore model for FXR was firstly built to screen FXR agonists from the Traditional Chinese Medicine Database (TCMD). Then, 21 FXR crystal structures were clustered in two types and two representative structures (PDB ID: 3OMM and 3P89) were, respectively, used to carry out molecular docking to refine the screened result. Moreover, the pharmacophore model for GPBAR1 was built to screen selective FXR agonists with no activity on GPBAR1. A set of 24 candidate selective FXR agonists which fitvalue of FXR pharmacophore model and docking score of 3OMM and 3P89 were in the top 100 and cannot match the pharmacophore model for GPBAR1 were obtained. By the lipid-lowering activity test in HepG2 cell lines, Arctigenin was identified to be potential selective FXR agonist with the activity of 20?μmol·L^?1. After down-regulating FXR, Arctigenin could increase the mRNA of FXR while exerted no effect on the mRNA of GPBAR1. MD was further used to interpret the mechanism of Arctigenin with the representative structures. This research provided a new screening procedure for finding selective candidate compounds and appropriate docking models of a target by considering the structure diversity of PDB structures, which was applied to discovery novel selective FXR agonists to treat hyperlipidemia.

Communicated by Ramaswamy H. Sarma