7 results on '"van Doodewaerd BR"'
Search Results
2. Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes.
- Author
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van Tol BDM, van Doodewaerd BR, Lageveen-Kammeijer GSM, Jansen BC, Talavera Ormeño CMP, Hekking PJM, Sapmaz A, Kim RQ, Moutsiopoulou A, Komander D, Wuhrer M, van der Heden van Noort GJ, Ovaa H, and Geurink PP
- Subjects
- Ubiquitination, Ubiquitin metabolism, Ubiquitins metabolism, Polyubiquitin metabolism, Deubiquitinating Enzymes metabolism
- Abstract
Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
3. Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.
- Author
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Ali M, van Gent ME, de Waal AM, van Doodewaerd BR, Bos E, Koning RI, Cordfunke RA, Drijfhout JW, and Nibbering PH
- Subjects
- Humans, Staphylococcus aureus, Antimicrobial Peptides, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents chemistry, Polylactic Acid-Polyglycolic Acid Copolymer pharmacology, Peptides pharmacology, Bacteria, Biofilms, Anti-Infective Agents pharmacology, Nanoparticles chemistry
- Abstract
Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of the peptide. The aim of this study was to investigate the physical and functional characteristics of SAAP-148 NPs and to compare the selectivity index of the formulated peptide with that of the peptide in solution. SAAP-148 NPs displayed favorable physiochemical properties [size = 94.1 ± 23 nm, polydispersity index (PDI) = 0.08 ± 0.1, surface charge = 1.65 ± 0.1 mV, and encapsulation efficiency (EE) = 86.7 ± 0.3%] and sustained release of peptide for up to 21 days in PBS at 37 °C. The antibacterial and cytotoxicity studies showed that the selectivity index for SAAP-148 NPs was drastically increased, by 10-fold, regarding AMR Staphylococcus aureus and 20-fold regarding AMR Acinetobacter baumannii after 4 h. Interestingly, the antibiofilm activity of SAAP-148 NPs against AMR S. aureus and A. baumannii gradually increased overtime, suggesting a dose-effect relationship based on the peptide's in vitro release profile. Using 3D human skin equivalents (HSEs), dual drug SAAP-148 NPs and the novel antibiotic halicin NPs provided a stronger antibacterial response against planktonic and cell-associated bacteria than SAAP-148 NPs but not halicin NPs after 24 h. Confocal laser scanning microscopy revealed the presence of SAAP-148 NPs on the top layers of the skin models in close proximity to AMR S. aureus at 24 h. Overall, SAAP-148 NPs present a promising yet challenging approach for further development as treatment against bacterial infections.
- Published
- 2023
- Full Text
- View/download PDF
4. Encapsulation of SAAP-148 in Octenyl Succinic Anhydride-Modified Hyaluronic Acid Nanogels for Treatment of Skin Wound Infections.
- Author
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van Gent ME, van Baaren T, Kłodzińska SN, Ali M, Dolezal N, van Doodewaerd BR, Bos E, de Waal AM, Koning RI, Drijfhout JW, Nielsen HM, and Nibbering PH
- Abstract
Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204-253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37-41% in 72 h) upon reconstitution. Lyophilized SAAP-148-loaded nanogels showed similar antimicrobial activity as SAAP-148 against planktonic and biofilm-residing AMR Staphylococcus aureus and Acinetobacter baumannii . Importantly, formulated SAAP-148 showed reduced cytotoxicity against human erythrocytes, primary human skin fibroblasts and human keratinocytes. Additionally, lyophilized SAAP-148-loaded nanogels eradicated AMR S. aureus and A. baumannii colonizing a 3D human epidermal model, without inducing any cytotoxicity in contrast to SAAP-148. These findings indicate that OSA-HA nanogels increase SAAP-148's therapeutic potential for treatment of skin wound infections.
- Published
- 2023
- Full Text
- View/download PDF
5. Exploring the Versatility of the Covalent Thiol-Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease.
- Author
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Mons E, Kim RQ, van Doodewaerd BR, van Veelen PA, Mulder MPC, and Ovaa H
- Subjects
- Deubiquitinating Enzymes chemistry, HEK293 Cells, Humans, Pargyline chemistry, Propylamines chemistry, Ubiquitin Thiolesterase chemistry, Alkynes chemistry, Cysteine Proteases chemistry, Pargyline analogs & derivatives, Sulfhydryl Compounds chemistry
- Abstract
Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CD
WT and catalytically inactive mutant USP16CDC205A . Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.- Published
- 2021
- Full Text
- View/download PDF
6. The Alkyne Moiety as a Latent Electrophile in Irreversible Covalent Small Molecule Inhibitors of Cathepsin K.
- Author
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Mons E, Jansen IDC, Loboda J, van Doodewaerd BR, Hermans J, Verdoes M, van Boeckel CAA, van Veelen PA, Turk B, Turk D, and Ovaa H
- Subjects
- Alkynes chemistry, Cathepsin K metabolism, Cysteine Proteinase Inhibitors chemical synthesis, Cysteine Proteinase Inhibitors chemistry, Humans, Models, Molecular, Molecular Structure, Small Molecule Libraries chemical synthesis, Small Molecule Libraries chemistry, Alkynes pharmacology, Cathepsin K antagonists & inhibitors, Cysteine Proteinase Inhibitors pharmacology, Small Molecule Libraries pharmacology
- Abstract
Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation ( k
inact ) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.- Published
- 2019
- Full Text
- View/download PDF
7. Neurotoxicity fingerprinting of venoms using on-line microfluidic AChBP profiling.
- Author
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Slagboom J, Otvos RA, Cardoso FC, Iyer J, Visser JC, van Doodewaerd BR, McCleary RJR, Niessen WMA, Somsen GW, Lewis RJ, Kini RM, Smit AB, Casewell NR, and Kool J
- Subjects
- Carrier Proteins, Chromatography, Liquid, Humans, Nicotinic Antagonists, Peptides chemistry, Snake Venoms chemistry, Tandem Mass Spectrometry, Microfluidic Analytical Techniques methods, Neurotoxins toxicity, Peptides isolation & purification, Snake Venoms toxicity
- Abstract
Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
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