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1.
Metab Eng ; 77: 53-63, 2023 05.
Article in English | MEDLINE | ID: mdl-36906118

ABSTRACT

N-linked glycosylation is a crucial post-translational modification of many biopharmaceuticals, including monoclonal antibodies (mAbs), capable of modifying their biological effect in patients and thus considered as a critical quality attribute (CQA). However, expression of desired and consistent glycosylation patterns remains a constant challenge for the biopharmaceutical industry and constitutes the need for tools to engineer glycosylation. Small non-coding microRNAs (miRNAs) are known regulators of entire gene networks and have therefore the potential of being used as tools for modulation of glycosylation pathways and for glycoengineering. Here, we demonstrate that novel identified natural miRNAs are capable of altering N-linked glycosylation patterns on mAbs expressed in Chinese hamster ovary (CHO) cells. We established a workflow for a functional high-throughput screening of a complete miRNA mimic library and identified 82 miRNA sequences affecting various moieties including galactosylation, sialylation, and α-1,6 linked core-fucosylation, an important glycan feature influencing antibody-dependent cytotoxicity (ADCC). Subsequent validation shed light on the intra-cellular mode of action and the impact on the cellular fucosylation pathway of miRNAs reducing core-fucosylation. While multiplex approaches increased phenotypic effects on the glycan structure, a synthetic biology approach utilizing rational design of artificial miRNAs further enhanced the potential of miRNAs as novel, versatile and tune-able tools for engineering of N-linked glycosylation pathways and expressed glycosylation patterns towards favourable phenotypes.


Subject(s)
MicroRNAs , Cricetinae , Animals , Glycosylation , MicroRNAs/genetics , MicroRNAs/metabolism , CHO Cells , Cricetulus , Antibodies, Monoclonal/genetics , Polysaccharides/genetics
2.
Sci Rep ; 9(1): 16947, 2019 11 18.
Article in English | MEDLINE | ID: mdl-31740740

ABSTRACT

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.


Subject(s)
Small Molecule Libraries/pharmacology , alpha-Synuclein/metabolism , Amyloid/antagonists & inhibitors , Amyloid/metabolism , Cell Line , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays/methods , Humans , Intrinsically Disordered Proteins/metabolism , Phagocytosis/drug effects , Protein Folding , Small Molecule Libraries/chemistry , Small Molecule Libraries/toxicity , Surface Plasmon Resonance , alpha-Synuclein/chemistry , alpha-Synuclein/drug effects
3.
Anal Bioanal Chem ; 409(6): 1569-1580, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27909779

ABSTRACT

A key aspect for the further development of matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (MS) is a better understanding of the working principles of MALDI matrices. To address this issue, a chemical compound library of 59 structurally related cinnamic acid derivatives was synthesized. Potential MALDI matrices were evaluated with sulfatides, a class of anionic lipids which are abundant in complex brain lipid mixtures. For each matrix relative mean S/N ratios of sulfatides were determined against 9-aminoacridine as a reference matrix using negative ion mass spectrometry with 355 and 337 nm laser systems. The comparison of matrix features with their corresponding relative mean S/N ratios for sulfatide detection identified correlations between matrix substitution patterns, their chemical functionality, and their MALDI-MS performance. Crystal structures of six selected matrices provided structural insight in hydrogen bond interactions in the solid state. Principal component analysis allowed the additional identification of correlation trends between structural and physical matrix properties like number of exchangeable protons at the head group, MW, logP, UV-Vis, and sulfatide detection sensitivity. Graphical abstract Design, synthesis and mass spectrometric evaluation of MALDI-MS matrix compound libraries allows the identification of matrix structure - MALDI-MS performance relationships using multivariate statistics as a tool.


Subject(s)
Cinnamates/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Sulfoglycosphingolipids/analysis , Crystallography, X-Ray , Models, Molecular , Principal Component Analysis
4.
Anal Chem ; 85(19): 9156-63, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23984824

ABSTRACT

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has become a method of choice in lipid analysis, as it provides localization information for defined lipids that is not readily accessible with nonmass spectrometric methods. Most current MALDI matrices have been found empirically. Nevertheless, preferential matrix properties for many analyte classes are poorly understood and may differ between lipid classes. We used rational matrix design and semiautomated screening for the discovery of new matrices suitable for MALDI-IMS of lipids. Utilizing Smartbeam- and nitrogen lasers for MALDI, we systematically compared doubly substituted α-cyanocinnamic acid derivatives (R(1)-CCA-R(2)) with respect to their ability to serve as negative ion matrix for various brain lipids. We identified 4-phenyl-α-cyanocinnamic acid amide (Ph-CCA-NH2) as a novel negative ion matrix that enables analysis and imaging of various lipid classes by MALDI-MS. We demonstrate that Ph-CCA-NH2 displays superior sensitivity and reproducibility compared to matrices commonly employed for lipids. A relatively small number of background peaks and good matrix suppression effect could make Ph-CCA-NH2 a widely applicable tool for lipid analysis.


Subject(s)
Cinnamates/chemistry , Lipids/analysis , Animals , Brain , Cinnamates/chemical synthesis , Rats , Rats, Sprague-Dawley , Software , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
5.
Bioorg Med Chem Lett ; 19(18): 5321-4, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19692239

ABSTRACT

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1' pocket.


Subject(s)
Chelating Agents/pharmacology , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/pharmacology , Catalytic Domain , Chelating Agents/chemistry , Matrix Metalloproteinase 13/chemistry , Models, Molecular , Protease Inhibitors/chemistry , Protein Binding , Structure-Activity Relationship
6.
Anal Biochem ; 335(1): 50-7, 2004 Dec 01.
Article in English | MEDLINE | ID: mdl-15519570

ABSTRACT

The goal of this study was to explore the applicability of surface plasmon resonance (SPR)-based fragment screening to identify compounds that bind to factor VIIa (FVIIa). Based on pharmacophore models virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa and immobilized these compounds on microarrays. In affinity fingerprinting experiments, a number of compounds were identified to be specifically interacting with FVIIa and shown to fall into four structural classes. The results demonstrate that the chemical microarray technology platform using SPR detection generates unique chemobiological information that is useful for de novo discovery and lead development and allows the detection of weak interactions with ligands of low molecular weight.


Subject(s)
Drug Design , Factor VIIa/antagonists & inhibitors , Factor VIIa/metabolism , Organic Chemicals/chemistry , Peptides/chemistry , Peptides/chemical synthesis , Protein Array Analysis , Chemistry, Pharmaceutical , Combinatorial Chemistry Techniques , Factor VIIa/chemistry , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Weight , Organic Chemicals/chemical synthesis , Protein Binding , Surface Plasmon Resonance
7.
Nucleic Acids Res ; 30(24): 5497-508, 2002 Dec 15.
Article in English | MEDLINE | ID: mdl-12490718

ABSTRACT

Bleomycins (BLMs) are antitumor antibiotics that in the presence of iron and oxygen mediate DNA damage by 4'-hydrogen atom abstraction of pyrimidines 3' to guanines. The resulting 4'-deoxyribose radicals can be trapped by O2 and ultimately result in the formation of base-propenal and gapped DNA with 3'-phosphoglycolate (3'-PG) and 5'-phosphate (5'-P) ends. The role of this lesion in triggering double-strand cleavage of duplex DNA by a single BLM molecule and the mechanism by which this lesion is repaired in vivo remain unsolved problems. The structure of these lesions is an essential step in addressing both of these problems. Duplex DNAs (13mers containing tethered hexaethylene glycol linkers) with GTAC and GGCC cleavage sites have been synthesized in which gaps containing 3'-PG and 5'-P ends at the sites of BLM cleavage have been inserted. The former sequence represents a hot spot for double-strand cleavage, while the latter is a hot spot for single-strand cleavage. Analytical methods to characterize the lesioned products have been developed. These oligonucleotides have been examined using 2D NMR methods and molecular modeling. The studies reveal that the lesioned DNAs are B-form and the 3'-PG and 5'-P are extrahelical. The base opposite the gap and the base pairs adjacent to the gap remain well stacked in the DNA duplex. Titrations of the lesioned GGCC oligomer with HOO-CoBLM leads to a mixture of complexes, in contrast to results of a similar titration with the lesioned GTAC oligomer.


Subject(s)
Glycolates/chemistry , Oligonucleotides/chemistry , Phosphates/chemistry , Bleomycin/chemistry , Bleomycin/pharmacology , Chromatography, High Pressure Liquid/methods , DNA/chemistry , DNA/drug effects , Magnetic Resonance Spectroscopy/methods , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis
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