25 results on '"Ovaa, H"'
Search Results
2. Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.
- Author
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Gan J, de Vries J, Akkermans JJLL, Mohammed Y, Tjokrodirijo RTN, de Ru AH, Kim RQ, Vargas DA, Pol V, Fasan R, van Veelen PA, Neefjes J, van Dam H, Ovaa H, Sapmaz A, and Geurink PP
- Subjects
- Ubiquitination, Ubiquitin, Cysteine, Protein Processing, Post-Translational, Cysteine Proteases
- Abstract
Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45 , comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.
- Published
- 2023
- Full Text
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3. Chemical Toolkit for PARK7: Potent, Selective, and High-Throughput.
- Author
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Jia Y, Kim RQ, Kooij R, Ovaa H, Sapmaz A, and Geurink PP
- Subjects
- Coloring Agents, Humans, Protein Deglycase DJ-1, Up-Regulation, Parkinson Disease
- Abstract
The multifunctional human Parkinson's disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson's disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors.
- Published
- 2022
- Full Text
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4. 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
5. Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-Terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos.
- Author
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Kooij R, Liu S, Sapmaz A, Xin BT, Janssen GMC, van Veelen PA, Ovaa H, Dijke PT, and Geurink PP
- Subjects
- Animals, Cell Survival, Fluorescent Dyes chemical synthesis, Fluorescent Dyes pharmacology, HEK293 Cells, Humans, Molecular Structure, Small Molecule Libraries chemical synthesis, Small Molecule Libraries pharmacology, Ubiquitin Thiolesterase antagonists & inhibitors, Zebrafish Proteins antagonists & inhibitors, Fluorescent Dyes chemistry, Small Molecule Libraries chemistry, Ubiquitin Thiolesterase analysis, Ubiquitin Thiolesterase metabolism, Zebrafish embryology, Zebrafish Proteins analysis, Zebrafish Proteins metabolism
- Abstract
Many reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an in vivo animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson's and Alzheimer's diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both in vitro and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity.
- Published
- 2020
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6. Manno- epi -cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.
- Author
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Armstrong Z, Kuo CL, Lahav D, Liu B, Johnson R, Beenakker TJM, de Boer C, Wong CS, van Rijssel ER, Debets MF, Florea BI, Hissink C, Boot RG, Geurink PP, Ovaa H, van der Stelt M, van der Marel GM, Codée JDC, Aerts JMFG, Wu L, Overkleeft HS, and Davies GJ
- Subjects
- Cyclohexanols chemical synthesis, Cyclohexanols chemistry, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors chemistry, Humans, Mannosidases metabolism, Molecular Structure, Cyclohexanols pharmacology, Drug Discovery, Enzyme Inhibitors pharmacology, Mannosidases antagonists & inhibitors
- Abstract
Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan
5 GlcNAc2 to produce GlcNAcMan3 GlcNAc2 , the precursor for all complex N -glycans, including the branched N -glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno- epi -cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno- epi -cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.- Published
- 2020
- Full Text
- View/download PDF
7. Chemical Tools and Biochemical Assays for SUMO Specific Proteases (SENPs).
- Author
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Jia Y, Claessens LA, Vertegaal ACO, and Ovaa H
- Subjects
- Amino Acid Sequence, Animals, Biological Assay methods, Drug Evaluation, Preclinical methods, Humans, Molecular Structure, Neoplasms metabolism, Neoplasms therapy, Protease Inhibitors pharmacology, Protein Processing, Post-Translational, Signal Transduction, Structure-Activity Relationship, Sumoylation, Peptide Hydrolases chemistry, Peptide Hydrolases metabolism, Protease Inhibitors chemistry, Protease Inhibitors metabolism
- Abstract
SUMOylation is a reversible and highly dynamic post-translational modification of target proteins by small ubiquitin-like modifiers (SUMO). It is orchestrated by SUMO-activating, -conjugating, and -ligating enzymes in a sequential manner and is important in regulating a myriad of predominantly nuclear processes. DeSUMOylation is achieved by SUMO-specific proteases (SENPs). Deregulation of SUMOylation and deSUMOylation results in cellular dysfunction and is linked to various diseases, including cancer. In recent years, SENPs have emerged as potential therapeutic targets. In this review, we will describe the inhibitors and activity-based probes of SENPs. Furthermore, we will summarize the biochemical assays available for evaluating the activity of SENPs to identify inhibitors.
- Published
- 2019
- Full Text
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8. Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.
- Author
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Resnick E, Bradley A, Gan J, Douangamath A, Krojer T, Sethi R, Geurink PP, Aimon A, Amitai G, Bellini D, Bennett J, Fairhead M, Fedorov O, Gabizon R, Gan J, Guo J, Plotnikov A, Reznik N, Ruda GF, Díaz-Sáez L, Straub VM, Szommer T, Velupillai S, Zaidman D, Zhang Y, Coker AR, Dowson CG, Barr HM, Wang C, Huber KVM, Brennan PE, Ovaa H, von Delft F, and London N
- Subjects
- HEK293 Cells, Humans, Ligands, Models, Molecular, Molecular Weight, Protein Conformation, Time Factors, Drug Evaluation, Preclinical methods, Electrons
- Abstract
Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
- Published
- 2019
- Full Text
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9. 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
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10. Selective PKCδ Inhibitor B106 Elicits Uveal Melanoma Growth Inhibitory Effects Independent of Activated PKC Isoforms.
- Author
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Heijkants R, Teunisse A, de Vries J, Ovaa H, and Jochemsen A
- Subjects
- Enzyme Activation, Humans, Protein Kinase C-delta metabolism, Uveal Neoplasms enzymology, Carbazoles pharmacology, Chromans pharmacology, Protein Kinase C-delta antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Uveal Neoplasms pathology
- Abstract
In uveal melanoma (UM) cells, the protein kinase C (pathway) is almost generally constitutively activated as a result of an activating mutation in either the GNAQ or the GNA11 G-protein. A pan-PKC inhibitor, sotrastaurin (also named AEB071), is in clinical trials for treatment of UM patients with limited success and eliciting adverse effects. Interestingly, genetic interference with expression of just one PKC isoform, e.g., PKCδ, is sufficient to reduce UM cell proliferation. Therefore, we tested the effect of a recently described specific PKCδ inhibitor, B106, on growth and survival of UM cell lines. Surprisingly, we found that B106 efficiently induced apoptosis in several cell lines, but apparently independent of activated PKCδ.
- Published
- 2019
- Full Text
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11. Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer.
- Author
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van der Heden van Noort GJ, Kooij R, Elliott PR, Komander D, and Ovaa H
- Abstract
An optimized large scale and highly reproducible route to orthogonally protected γ-thiolysine is reported. Its utility in the synthesis of bifunctional ubiquitin monomers is demonstrated. These ubiquitin synthons are employed in polymerization reactions giving access to synthetic poly-ubiquitin chains of defined linkage.
- Published
- 2017
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12. A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase.
- Author
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Lahav D, Liu B, van den Berg RJBHN, van den Nieuwendijk AMCH, Wennekes T, Ghisaidoobe AT, Breen I, Ferraz MJ, Kuo CL, Wu L, Geurink PP, Ovaa H, van der Marel GA, van der Stelt M, Boot RG, Davies GJ, Aerts JMFG, and Overkleeft HS
- Subjects
- Drug Evaluation, Preclinical methods, Enzyme Inhibitors chemistry, Glucosylceramidase, Humans, Imino Sugars chemistry, beta-Glucosidase metabolism, Enzyme Assays methods, Enzyme Inhibitors pharmacology, Fluorescence Polarization methods, Imino Sugars pharmacology, beta-Glucosidase antagonists & inhibitors
- Abstract
Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.
- Published
- 2017
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13. How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.
- Author
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Hameed DS, Sapmaz A, and Ovaa H
- Subjects
- Animals, Chemistry Techniques, Synthetic methods, Fluorescence Resonance Energy Transfer, Humans, Molecular Probes chemical synthesis, Molecular Probes chemistry, Molecular Probes metabolism, Peptides chemistry, Peptides metabolism, Ubiquitin chemical synthesis, Ubiquitinated Proteins chemistry, Ubiquitinated Proteins metabolism, Signal Transduction, Ubiquitin chemistry, Ubiquitin metabolism
- Abstract
Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.
- Published
- 2017
- Full Text
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14. Integrating chemical and genetic silencing strategies to identify host kinase-phosphatase inhibitor networks that control bacterial infection.
- Author
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Albers HM, Kuijl C, Bakker J, Hendrickx L, Wekker S, Farhou N, Liu N, Blasco-Moreno B, Scanu T, den Hertog J, Celie P, Ovaa H, and Neefjes J
- Subjects
- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Cell Line, Dual-Specificity Phosphatases genetics, Dual-Specificity Phosphatases metabolism, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Gene Silencing, Humans, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-akt metabolism, Salmonella Infections genetics, Salmonella typhimurium drug effects, Small Molecule Libraries chemistry, Small Molecule Libraries pharmacology, Dual-Specificity Phosphatases antagonists & inhibitors, Host-Pathogen Interactions, Proto-Oncogene Proteins c-akt antagonists & inhibitors, Salmonella Infections drug therapy, Salmonella Infections enzymology, Salmonella typhimurium physiology
- Abstract
Every year three million people die as a result of bacterial infections, and this number may further increase due to resistance to current antibiotics. These antibiotics target almost all essential bacterial processes, leaving only a few new targets for manipulation. The host proteome has many more potential targets for manipulation in order to control bacterial infection, as exemplified by the observation that inhibiting the host kinase Akt supports the elimination of different intracellular bacteria including Salmonella and M. tuberculosis. If host kinases are involved in the control of bacterial infections, phosphatases could be as well. Here we present an integrated small interference RNA and small molecule screen to identify host phosphatase-inhibitor combinations that control bacterial infection. We define host phosphatases inhibiting intracellular growth of Salmonella and identify corresponding inhibitors for the dual specificity phosphatases DUSP11 and 27. Pathway analysis places many kinases and phosphatases controlling bacterial infection in an integrated pathway centered around Akt. This network controls host cell metabolism, survival, and growth and bacterial survival and reflect a natural host cell response to bacterial infection. Inhibiting two enzyme classes with opposite activities-kinases and phosphatases-may be a new strategy to overcome infections by antibiotic-resistant bacteria.
- Published
- 2014
- Full Text
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15. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases.
- Author
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Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, and Ovaa H
- Subjects
- Catalytic Domain, Alkynes chemistry, Cysteine chemistry, Peptide Hydrolases metabolism
- Abstract
Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.
- Published
- 2013
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16. Chemical evolution of autotaxin inhibitors.
- Author
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Albers HM and Ovaa H
- Subjects
- Alternative Splicing, Animals, Biological Assay, Cell Proliferation drug effects, Humans, Isoenzymes antagonists & inhibitors, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Lysophosphatidylcholines metabolism, Lysophospholipids pharmacology, Models, Molecular, Phosphodiesterase Inhibitors chemistry, Phosphoric Diester Hydrolases chemistry, Phosphoric Diester Hydrolases genetics, Signal Transduction drug effects, Lysophospholipids metabolism, Phosphodiesterase Inhibitors pharmacology, Phosphoric Diester Hydrolases metabolism
- Published
- 2012
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17. Probing the specificity and activity profiles of the proteasome inhibitors bortezomib and delanzomib.
- Author
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Berkers CR, Leestemaker Y, Schuurman KG, Ruggeri B, Jones-Bolin S, Williams M, and Ovaa H
- Subjects
- Animals, Boronic Acids chemistry, Bortezomib, Cell Line, Tumor, Cell Survival drug effects, Humans, Mice, Mice, SCID, Proteasome Endopeptidase Complex drug effects, Proteasome Inhibitors chemistry, Pyrazines chemistry, Boronic Acids pharmacology, Proteasome Inhibitors pharmacology, Pyrazines pharmacology
- Abstract
The ubiquitin proteasome system is an attractive pharmacological target for the treatment of cancer. The proteasome inhibitor bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma but is associated with substantial adverse effects and the occurrence of resistance, underscoring the continued need for novel proteasome inhibitors. In this study, bortezomib and the novel proteasome inhibitor delanzomib were compared for their ability to inhibit proteasome activity using both fluorogenic substrates and a recently developed fluorescent proteasome activity probe. Bortezomib and delanzomib were equipotent in inhibiting distinct subunits of the proteasome in a panel of cell lines in vitro. In a preclinical multiple myeloma model, both inhibitors inhibited the proteasome in normal tissues to a similar extent. Tumor proteasome activity was inhibited to a significantly higher extent by delanzomib (60%) compared to bortezomib (32%). In addition, delanzomib was able to overcome bortezomib resistance in vitro. The present findings demonstrate that proteasome activity probes can accurately monitor the effects of proteasome inhibitors on both normal and tumor tissues in preclinical models and can be used as a diagnostic approach to predict resistance against treatment with proteasome inhibitors. Furthermore, the data presented here provide rationale for further clinical development of delanzomib.
- Published
- 2012
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18. A multifunctional protease inhibitor to regulate endolysosomal function.
- Author
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van Kasteren SI, Berlin I, Colbert JD, Keane D, Ovaa H, and Watts C
- Subjects
- Cystatins chemistry, Cysteine Proteases metabolism, Endopeptidases metabolism, Lysosomes enzymology, Lysosomes physiology, Pepstatins chemical synthesis, Pepstatins chemistry, Protease Inhibitors chemical synthesis, Protease Inhibitors chemistry, Structure-Activity Relationship, Lysosomes drug effects, Pepstatins pharmacology, Protease Inhibitors pharmacology
- Abstract
Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin-pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses.
- Published
- 2011
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19. Structure-based design of novel boronic acid-based inhibitors of autotaxin.
- Author
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Albers HM, Hendrickx LJ, van Tol RJ, Hausmann J, Perrakis A, and Ovaa H
- Subjects
- Animals, Binding Sites, Boronic Acids chemistry, Boronic Acids pharmacology, Drug Design, Models, Molecular, Phosphoric Diester Hydrolases chemistry, Pyrophosphatases chemistry, Rats, Stereoisomerism, Structure-Activity Relationship, Boronic Acids chemical synthesis, Pyrophosphatases antagonists & inhibitors
- Abstract
Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in inflammation, fibrosis, and tumor progression, rendering ATX an attractive drug target. We recently described a boronic acid-based inhibitor of ATX, named HA155 (1). Here, we report the design of new inhibitors based on the crystal structure of ATX in complex with inhibitor 1. Furthermore, we describe the syntheses and activities of these new inhibitors, whose potencies can be explained by structural data. To understand the difference in activity between two different isomers with nanomolar potencies, we performed molecular docking experiments. Intriguingly, molecular docking suggested a remarkable binding pose for one of the isomers, which differs from the original binding pose of inhibitor 1 for ATX, opening further options for inhibitor design.
- Published
- 2011
- Full Text
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20. Discovery and optimization of boronic acid based inhibitors of autotaxin.
- Author
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Albers HM, van Meeteren LA, Egan DA, van Tilburg EW, Moolenaar WH, and Ovaa H
- Subjects
- Boronic Acids chemistry, Boronic Acids metabolism, Cell Line, Enzyme Inhibitors chemistry, Enzyme Inhibitors metabolism, Humans, Inhibitory Concentration 50, Lysophosphatidylcholines metabolism, Magnetic Resonance Spectroscopy, Mass Spectrometry, Multienzyme Complexes metabolism, Phosphodiesterase I metabolism, Phosphoric Diester Hydrolases, Pyrophosphatases metabolism, Signal Transduction, Structure-Activity Relationship, Thiazolidinediones chemical synthesis, Thiazolidinediones chemistry, Thiazolidinediones metabolism, Thiazolidinediones pharmacology, Boronic Acids chemical synthesis, Boronic Acids pharmacology, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, Multienzyme Complexes antagonists & inhibitors, Phosphodiesterase I antagonists & inhibitors, Pyrophosphatases antagonists & inhibitors
- Abstract
Autotaxin (ATX) is an extracellular enzyme that hydrolyzes lysophosphatidylcholine (LPC) to produce the lipid mediator lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in diverse physiological and pathological processes, including vascular development, inflammation, fibrotic disease, and tumor progression. Therefore, targeting ATX with small molecule inhibitors is an attractive therapeutic strategy. We recently reported that 2,4-thiazolidinediones inhibit ATX activity in the micromolar range. Interestingly, inhibitory potency was dramatically increased by introduction of a boronic acid moiety, designed to target the active site threonine in ATX. Here we report on the discovery and further optimization of boronic acid based ATX inhibitors. The most potent of these compounds inhibits ATX-mediated LPC hydrolysis in the nanomolar range (IC(50) = 6 nM). The finding that ATX can be targeted by boronic acids may aid the development of ATX inhibitors for therapeutic use.
- Published
- 2010
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21. Nonhydrolyzable ubiquitin-isopeptide isosteres as deubiquitinating enzyme probes.
- Author
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Shanmugham A, Fish A, Luna-Vargas MP, Faesen AC, El Oualid F, Sixma TK, and Ovaa H
- Subjects
- Amino Acid Sequence, Models, Molecular, Molecular Sequence Data, Peptides chemistry, Protein Conformation, Substrate Specificity, Ubiquitin chemistry, Biosensing Techniques methods, Endopeptidases metabolism, Peptides metabolism, Ubiquitin metabolism
- Abstract
We demonstrate that oxime ligation is an efficient, straightforward, and generally applicable strategy for generating nonhydrolyzable ubiquitin (Ub)-isopeptide isosteres. We synthesized nonhydrolyzable K48- and K63-linked Ub-isopeptide isosteres to investigate the selectivity of deubiquitinating enzymes for specific linkages employing surface plasmon resonance spectroscopy. The results indicate that deubiquitinating enzymes specifically recognize the local peptide sequence flanking Ub-branched lysine residues in target proteins. The described strategy allows the systematic investigation of sequence requirements for substrate selectivity of deubiquitinating enzymes.
- Published
- 2010
- Full Text
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22. Class I major histocompatibility complexes loaded by a periodate trigger.
- Author
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Rodenko B, Toebes M, Celie PH, Perrakis A, Schumacher TN, and Ovaa H
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Drug Evaluation, Preclinical, Epitopes immunology, Humans, Kinetics, Ligands, Models, Molecular, Peptides metabolism, Photochemical Processes, Protein Binding drug effects, Protein Multimerization, Protein Structure, Quaternary, T-Lymphocytes immunology, T-Lymphocytes metabolism, Tissue Culture Techniques, Ultraviolet Rays, Histocompatibility Antigens Class I chemistry, Histocompatibility Antigens Class I metabolism, Periodic Acid chemistry, Periodic Acid pharmacology
- Abstract
Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide-MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide-MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide-MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands.
- Published
- 2009
- Full Text
- View/download PDF
23. UV-induced ligand exchange in MHC class I protein crystals.
- Author
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Celie PH, Toebes M, Rodenko B, Ovaa H, Perrakis A, and Schumacher TN
- Subjects
- Amino Acid Sequence, Crystallization, Crystallography, X-Ray, Humans, Ligands, Models, Molecular, Protein Binding radiation effects, Protein Conformation radiation effects, Selenium chemistry, HLA-A2 Antigen chemistry, HLA-A2 Antigen metabolism, Oligopeptides chemistry, Oligopeptides metabolism, Ultraviolet Rays
- Abstract
High-throughput structure determination of protein-ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, "conditional", peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This "in crystallo" exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC beta2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families.
- Published
- 2009
- Full Text
- View/download PDF
24. Profiling proteasome activity in tissue with fluorescent probes.
- Author
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Berkers CR, van Leeuwen FW, Groothuis TA, Peperzak V, van Tilburg EW, Borst J, Neefjes JJ, and Ovaa H
- Subjects
- Animals, Cell Line, Tumor, Cell Survival, Humans, Mice, Molecular Structure, Protein Subunits metabolism, Fluorescent Dyes chemistry, Proteasome Endopeptidase Complex metabolism
- Abstract
With proteasome inhibitors in use in the clinic for the treatment of multiple myeloma and with clinical trials in progress investigating the treatment of a variety of hematologic and solid malignancies, accurate methods that allow profiling of proteasome inhibitor specificity and efficacy in patients are in demand. Here, we describe the development, full biochemical validation, and comparison of fluorescent proteasome activity reporters that can be used to profile proteasome activities in living cells with high sensitivity. Seven of the synthesized probes tested label proteasomes in lysates, although the fluorescent dye used affects their specificity. Two differentially labeled probes tested are suitable for studying proteasome activity in living cells by gel-based assays, by confocal laser scanning microscopy, and by flow cytometry. We established methods using these fluorescent reporters to profile proteasome activity in different mouse tissues, carefully avoiding postlysis artifacts, and we show that proteasome subunit activity is regulated in an organ-specific manner. The techniques described here could be used to study in vivo pharmacological properties of proteasome inhibitors.
- Published
- 2007
- Full Text
- View/download PDF
25. Chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases.
- Author
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Hemelaar J, Galardy PJ, Borodovsky A, Kessler BM, Ploegh HL, and Ovaa H
- Subjects
- B-Lymphocytes metabolism, B-Lymphocytes virology, Binding Sites, Female, Humans, Models, Chemical, Ovarian Neoplasms metabolism, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins chemistry, Substrate Specificity, Endopeptidases chemistry, Proteome, Proteomics methods, Ubiquitin chemistry
- Abstract
Determining the biological function of newly discovered gene products requires the development of novel functional approaches. To facilitate this task, recent developments in proteomics include small molecular probes that target proteolytic enzyme families including serine, threonine, and cysteine proteases. For the families of ubiquitin (Ub) and ubiquitin-like (UBL)-specific proteases, such tools were lacking until recently. Here, we review the advances made in the development of protein-based active site-directed probes that target proteases specific for ubiquitin and ubiquitin-like proteins. Such probes were applied successfully to discover and characterize novel Ub/UBL-specific proteases. Ub/UBL processing and deconjugation are performed by a diverse set of proteases belonging to several different enzyme families, including members of the ovarian tumor domain (OTU) protease family. A further definition of this family of enzymes will benefit from a directed chemical proteomics approach. Some of the Ub/UBL-specific proteases react with multiple Ub/UBLs and members of the same protease family can recognize multiple Ub/UBLs, underscoring the need for tools that appropriately address enzyme specificity.
- Published
- 2004
- Full Text
- View/download PDF
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