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Paus, L.V. de; Yu, A.; Janssen, A.P.A.; Berg, R.J.B.H.N. van den; Heitman, L.H.; Stelt, M. van der 2024

Discovery of a photoaffinity probe that captures the active conformation of the cannabinoid CB2 receptor

Article / Letter to editor

open access

The cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor... Show moreThe cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI-121 that stabilized the inactive conformation of the CB2R. Here, we report the structure-based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click-chemistry with fluorophore-azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function. Show less

Doelman, W.; Ligthart, N.A.M.; Plassche, M.A.T. van de; Geus, M.A.R. de; Reinalda, L.; Isendoorn, M.M.E.; ... ; Kasteren, S.I. van 2024

Synthesis of peptides containing a combination of free and 2-trans-cyclooctene carbamate protected lysine residues

Article / Letter to editor

open access

Uddin, M.J.; Overkleeft, H.S.; Lentz, C.S. 2023

Activity-based protein profiling in methicillin-resistant staphylococcus aureus reveals the broad reactivity of a carmofur-derived probe

Article / Letter to editor

open access

Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity... Show moreActivity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity-based probes to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also has antimicrobial and anti-biofilm activity. Carmofur probes were originally designed to target human acid ceramidase, a member of the NTN hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatography/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 20 enzymes from diverse families awaiting further characterization, including the NTN hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labeled serine hydrolases, thus displaying a reactivity profile similar to that of carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes, which could lead to side effects and/or contribute to therapeutic efficacy. Show less

Schada von Borzyskowski, L. 2023

Taking synthetic biology to the seas: from blue chassis organisms to marine aquaforming

Article / Letter to editor

open access

Oceans cover 71 % of Earth's surface and are home to hundreds of thousands of species, many of which are microbial. Knowledge about marine microbes has strongly increased in the past decades due to... Show moreOceans cover 71 % of Earth's surface and are home to hundreds of thousands of species, many of which are microbial. Knowledge about marine microbes has strongly increased in the past decades due to global sampling expeditions, and hundreds of detailed studies on marine microbial ecology, physiology, and biogeochemistry. However, the translation of this knowledge into biotechnological applications or synthetic biology approaches using marine microbes has been limited so far. This review highlights key examples of marine bacteria in synthetic biology and metabolic engineering, and outlines possible future work based on the emerging marine chassis organisms Vibrio natriegens and Halomonas bluephagenesis. Furthermore, the valorization of algal polysaccharides by genetically enhanced microbes is presented as an example of the opportunities and challenges associated with blue biotechnology. Finally, new roles for marine synthetic biology in tackling pressing global challenges, including climate change and marine pollution, are discussed. Show less

Leeuwen, T. van; Doelman, W.; Kieboom, R.W.R. van den; Florea, B.I.; Kasteren, S.I. van 2023

Bioorthogonal peptide enrichment from complex samples using a rink-amide-based catch-and-release strategy

Article / Letter to editor

open access

Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of... Show moreUptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics - the ideal analysis tool in this case - requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures. Show less

Su, Q.; Schroder, S.P.; Lelieveld, L.T.; Guimaraes Da Lomba Ferraz, M.J.; Verhoek, M.; Boot, R.G.; ... ; Kuo, C.L. 2023

Corrigendum: xylose-configured cyclophellitols as selective inhibitors for glucocerebrosidase

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This article corrects the following:

Xylose-Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase
- Volume 22Issue 21ChemBioChem
- pages: 3090-3098
- First Published online: September 13,...

This article corrects the following:

Xylose-Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase
- Volume 22Issue 21ChemBioChem
- pages: 3090-3098
- First Published online: September 13, 2021

Show less

Su, Q.; Schroder, S.P.; Lelieveld, L.T.; Guimaraes Da Lomba Ferraz, M.J.; Verhoek, M.; Boot, R.G.; ... ; Kuo, C.-L. 2023

Corrigendum: Xylose-configured cyclophellitols as selective inhibitors for glucocerebrosidase

Article / Letter to editor

open access

Auberson, Y.P.; Arimondo, P.B.; Duca, M.; Essig, S.; Grether, U.; Rufer, A.C; ... ; Zhang, A.X. 2023

EFMC - trends that link medicinal chemistry and chemical biology to translational drug discovery

Article / Letter to editor

open access

Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed... Show moreGround-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research. Show less

Su, Q.; Schröder, S.P.; Lelieveld, L.T.; Guimaraes Da Lomba Ferraz M.J.; Verhoek, M.; Boot, R.G.; ... ; Kuo, C.L. 2022

Corrigendum: Xylose-configured cyclophellitols as selective inhibitors for glucocerebrosidase

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Huppelschoten, Y.; Mukhopadhyay, R.; Buchardt, J.; Nielsen, T.E.; Vertegaal, A.C.O.; Ovaa, H.; Noort, G.J.V. van 2022

In-plate chemical synthesis of isopeptide-linked SUMOylated peptide fluorescence polarization reagents for high-throughput screening of SENP preferences

Article / Letter to editor

open access

Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by... Show moreSmall ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83 isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a gamma-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1-3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide-linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel-based read-out. Show less

Buijs, N.; Vlaming, H.C.; Haren, M.J. van; Martin, N.I. 2022

Synthetic studies with bacitracin A and preparation of analogues containing alternative zinc binding groups

Article / Letter to editor

open access

Duszenko, N.; Willigen, D.M. van; Bunschoten, A.; Velders, A.H.; Roestenberg, M.; Leeuwen, F.W.B. van 2022

Chemically enhanced immunogenicity of bacteria by supramolecular functionalization with an adjuvant

Article / Letter to editor

open access

Many pathogens blunt immune responses because they lack immunogenic structural features, which typically results in disease. Here, we show evidence suggesting that pathogen immunogenicity can be... Show moreMany pathogens blunt immune responses because they lack immunogenic structural features, which typically results in disease. Here, we show evidence suggesting that pathogen immunogenicity can be chemically enhanced. Using supramolecular host-guest chemistry, we complexed onto the surface of a poorly immunogenic bacterium (Staphylococcus aureus) a TLR7 agonist-based adjuvant. "Adjuvanted" bacteria were readily recognized by macrophages and induced a more pro-inflammatory immunophenotype. Future applications of this concept could yield treatment modalities that bolster the immune system's response to pathogenic microbes. Show less

Huppelschoten, Y.; Elhebieshy, A.F.; Hameed, D.S.; Sapmaz, A.; Buchardt, J.; Nielsen, T.E.; ... ; Noort, G.J.V. van 2022

Total Chemical Synthesis of a Functionalized GFP Nanobody

Article / Letter to editor

metadata only

Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs... Show moreChemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional in vitro validation in pull-down and confocal microscopy settings. Show less

Zhang, Y.; Marechal, N.; Haren, M.J. van; Troffer-Charlier, N.; Cura, V.; Cavarelli, J.; Martin, N.I. 2021

Structural studies provide new insights into the role of lysine acetylation on substrate recognition by CARM1 and inform the design of potent peptidomimetic inhibitors

Article / Letter to editor

open access

Janssen, G.V.; Zhang, S.S.; Merkx, R.; Schiesswohl, C.; Chatterjee, C.; Darwin, K.H.; ... ; Ovaa, H. 2021

Development of Tyrphostin analogues to study inhibition of the Mycobacterium tuberculosis Pup proteasome system**

Article / Letter to editor

open access

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on... Show moreTuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds. Show less

Duca, M.; Gillingham, D.; Olsen, C.A.; Sbardella, G.; Skaanderup, P.R.; Stelt, M. van der; ... ; Auberson, Y.P. 2021

The chemical biology-medicinal chemistry continuum: EFMC's vision

Article / Letter to editor

open access

The European Federation for Medicinal chemistry and Chemical biology (EFMC) is a federation of learned societies. It groups organizations of European scientists working in a dynamic field spanning... Show moreThe European Federation for Medicinal chemistry and Chemical biology (EFMC) is a federation of learned societies. It groups organizations of European scientists working in a dynamic field spanning chemical biology and medicinal chemistry. New ideas, tools, and technologies emerging from a wide array of scientific disciplines continuously energize this rapidly evolving area. Medicinal chemistry is the design, synthesis, and optimization of biologically active molecules aimed at discovering new drug candidates - a mission that in many ways overlaps with the scope of chemical biology. Chemical biology is by now a mature field of science for which a more precise definition of what it encompasses, in the frame of EFMC, is timely. This article discusses chemical biology as currently understood by EFMC, including all activities dealing with the design and synthesis of biologically active chemical tools and their use to probe, characterize, or influence biological systems. Show less

Madern, J.M.; Kim, R.Q.; Misra, M.; Dikic, I.; Zhang, Y.; Ovaa, H.; ... ; Noort, G.J.V. van 2021

Synthesis of stable NAD+ mimics as inhibitors for the legionella pneumophila phosphoribosyl biquitylating enzyme SdeC

Article / Letter to editor

open access

D'Amico, F.; Mukhopadhyay, R.; Ovaa, H.; Mulder, M.P.C. 2021

Targeting TRIM proteins: a quest towards drugging an emerging protein class

Article / Letter to editor

open access

The ubiquitylation machinery regulates several fundamental biological processes from protein homeostasis to a wide variety of cellular signaling pathways. As a consequence, its dysregulation is... Show moreThe ubiquitylation machinery regulates several fundamental biological processes from protein homeostasis to a wide variety of cellular signaling pathways. As a consequence, its dysregulation is linked to diseases including cancer, neurodegeneration, and autoimmunity. With this review, we aim to highlight the therapeutic potential of targeting E3 ligases, with a special focus on an emerging class of RING ligases, named tri-partite motif (TRIM) proteins, whose role as targets for drug development is currently gaining pharmaceutical attention. TRIM proteins exert their catalytic activity as scaffolds involved in many protein-protein interactions, whose multidomains and adapter-like nature make their druggability very challenging. Herein, we give an overview of the current understanding of this class of single polypeptide RING E3 ligases and discuss potential targeting options. Show less

Dijk, J.H.M. van; Hooij, A. van; Groot, L.M.; Geboers, J.; Moretti, R.; Verhard-Seymonsbergen, E.; ... ; Geluk, A. 2021

Synthetic phenolic glycolipids for application in diagnostic tests for leprosy

Article / Letter to editor

open access

Point-of-care (POC) diagnostic tests for the rapid detection of individuals infected with Mycobacterium leprae, the causative pathogen of leprosy, represent efficient tools to guide therapeutic and... Show morePoint-of-care (POC) diagnostic tests for the rapid detection of individuals infected with Mycobacterium leprae, the causative pathogen of leprosy, represent efficient tools to guide therapeutic and prophylactic treatment strategies in leprosy control programs, thus positively contributing to clinical outcome and reducing transmission of this infectious disease. Levels of antibodies directed against the M. leprae-specific phenolic glycolipid I (PGL-I) closely correlate with an individual's bacterial load and a higher risk of developing leprosy. We describe herein the assembly of a set of PGL glycans carrying the characteristic phenol aglycon and featuring different methylation patterns. The PGL trisaccharides were applied to construct neoglycoproteins that were used to detect anti-PGL IgM antibodies in leprosy patients. ELISAs and quantitative lateral-flow assays based on up-converting nanoparticles (UCP-LFAs) showed that the generated PGL-I and PGL-II trisaccharide neoglycoconjugates can be applied for the detection of anti M. leprae IgM antibodies in POC tests. Show less

Marneth, K.; Elst, H. van den; Cramer-Blok, A.J.; Codee, J.D.C.; Overkleeft, H.S.; Aerts, J.M.F.G.; ... ; Ben Bdira F. 2021

Tuning the transglycosylation reaction of a GH11 xylanase by a delicate enhancement of its thumb flexibility

Article / Letter to editor

open access

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific... Show moreGlycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX "thumb" were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts. Show less

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