The chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial... Show moreThe chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial focus was the identification of formaldehyde-induced modifications in diphtheria toxin, which is described in Chapter 2. Subsequently, the methods described in Chapter 2 were applied to study what effects formaldehyde-induced modifications on model proteins have on their susceptibility to enzymatic proteolysis (Chapter 3). During the analysis of these model proteins, unknown formaldehyde-induced modifications were observed. The structural elucidation of these modifications, the discovery of a new type of crosslinks and various other subsequent reaction products are described in Chapter 4. The degradomics analysis described in Chapter 3 was applied to tetanus toxoids to distinguish heat-denaturated toxoids from their original state (Chapter 5). In order to reduce the analysis time and further improve the degradomics approach, an optimized strategy using Tandem Mass Tag multiplexing for the relative quantification of peptides was developed for the analysis of diphtheria toxoids (Chapter 6). Finally, Chapter 7 provides a brief discussion on the results presented in this thesis and offers some perspectives on implementation of the findings for toxoid vaccine development, quality control and further research. Show less
Michiels, T.J.M.; Veen, M.A. van; Meiring, H.D.; Jiskoot, W.; Kersten, G.F.A.; Metz, B. 2021
Currently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared... Show moreCurrently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared to known good products) can be proven in a panel of in vitro assays. By mimicking the in vivo antigen processing in a simplified in vitro approach, it may be possible to distinguish aberrant products from good products. To demonstrate this, heat-exposed diphtheria toxoid was subjected to partial digestion by cathepsin S (an endoprotease involved in antigen processing), and the peptide formation/degradation kinetics were mapped for various heated toxoids. To overcome the limitations associated with the very large number of samples, we used common reference-based tandem mass tag (TMT) labeling. Instead of using one label per condition with direct comparison between the set of labels, we compared multiple labeled samples to a common reference (a pooled sample containing an aliquot of each condition). In this method, the number of samples is not limited by the number of unique TMT labels. This TMT multiplexing strategy allows for a 15-fold reduction of analysis time while retaining the reliability advantage of TMT labeling over label-free quantification. The formation of the most important peptides could be followed over time and compared among several conditions. The changes in enzymatic degradation kinetics of diphtheria toxoid revealed several suitable candidate peptides for use in a quality control assay that can distinguish structurally aberrant diphtheria toxoid from compliant toxoids. Show less
) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher... Show more) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines. Show less
Yang, X.; Michiels, T.J.M.; Jong, C. de; Soethoudt, M.; Dekker, N.; Gordon, E.; ... ; IJzerman, A.P. 2018
Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have... Show moreUsing activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A 2A receptor (hA(2A)R) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA(2A)R ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA(2A)R Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA(2A)R by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA(2A)R, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs. Show less
The structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served... Show moreThe structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA(2A) receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A(2A)-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A(2A) receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine. Show less
A covalent antagonist for the human adenosine A2A receptor Xue Yang, Guo Dong, Thomas J.M. Michiels, Eelke B. Lenselink, Laura Heitman, Julien Louvel, Ad P. IJzerman Abstract The structure of the... Show moreA covalent antagonist for the human adenosine A2A receptor Xue Yang, Guo Dong, Thomas J.M. Michiels, Eelke B. Lenselink, Laura Heitman, Julien Louvel, Ad P. IJzerman Abstract The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine. Show less
The structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served... Show moreThe structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA(2A) receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A(2A)-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A(2A) receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine. Show less
The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as... Show moreThe structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.KEYWORDS: A2A adenosine receptor; Adenosine; Covalent antagonist; G protein-coupled receptors; Radioligand binding Show less
