43 results on '"Deu E"'
Search Results
2. Farbkodierte Duplexsonographie nach Laserangioplastie von femoro-polplitealen Gefäßverschlüssen
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Steiner, H., Lammer, J., Flückiger, F., Deu, E., Pilger, E., Melzer, G., Gebhardt, J., editor, Hackelöer, B.-J., editor, von Klinggräff, G., editor, and Seitz, K., editor
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- 1990
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3. Transhepatische Behandlung benigner Gallenwegsstenosen
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Lammer, J., Deu, E., Neumayer, K., Steiner, H., Schneider, G. H., editor, and Vogler, E., editor
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- 1988
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4. Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic agents in vivo
- Author
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Deu, E., primary, Chen, I. T., additional, Lauterwasser, E. M. W., additional, Valderramos, J., additional, Li, H., additional, Edgington, L. E., additional, Renslo, A. R., additional, and Bogyo, M., additional
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- 2013
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5. Tuberkulose des Duodenums
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Flückiger, F., primary, Deu, E., additional, and Hausegger, K., additional
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- 1989
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6. Activity-based protein profiling of human and plasmodium serine hydrolases and interrogation of potential antimalarial targets.
- Author
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Davison D, Howell S, Snijders AP, and Deu E
- Abstract
Malaria remains a global health issue requiring the identification of novel therapeutic targets to combat drug resistance. Metabolic serine hydrolases are druggable enzymes playing essential roles in lipid metabolism. However, very few have been investigated in malaria-causing parasites. Here, we used fluorophosphonate broad-spectrum activity-based probes and quantitative chemical proteomics to annotate and profile the activity of more than half of predicted serine hydrolases in P . falciparum across the erythrocytic cycle. Using conditional genetics, we demonstrate that the activities of four serine hydrolases, previously annotated as essential (or important) in genetic screens, are actually dispensable for parasite replication. Of importance, we also identified eight human serine hydrolases that are specifically activated at different developmental stages. Chemical inhibition of two of them blocks parasite replication. This strongly suggests that parasites co-opt the activity of host enzymes and that this opens a new drug development strategy against which the parasites are less likely to develop resistance., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)
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- 2022
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7. Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy.
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van Dalen FJ, Bakkum T, van Leeuwen T, Groenewold M, Deu E, Koster AJ, van Kasteren SI, and Verdoes M
- Abstract
Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 van Dalen, Bakkum, van Leeuwen, Groenewold, Deu, Koster, van Kasteren and Verdoes.)
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- 2021
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8. Correction: Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.
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Tan MSY, Davison D, Sanchez MI, Anderson BM, Howell S, Snijders AP, Edgington-Mitchell LE, and Deu E
- Abstract
[This corrects the article DOI: 10.1371/journal.pone.0227341.].
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- 2020
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9. Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.
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Tan MSY, Davison D, Sanchez MI, Anderson BM, Howell S, Snijders A, Edgington-Mitchell LE, and Deu E
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- Animals, Antimalarials chemistry, Cell Membrane Permeability, Humans, Malaria diagnostic imaging, Malaria drug therapy, Molecular Probes chemistry, Plasmodium falciparum drug effects, Plasmodium falciparum enzymology, Sulfones, Tryptophan, Cysteine Proteases metabolism, Cysteine Proteinase Inhibitors chemistry, Malaria enzymology
- Abstract
Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability., Competing Interests: The authors have declared that no competing interests exist.
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- 2020
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10. Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.
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Sanchez MI, de Vries LE, Lehmann C, Lee JT, Ang KK, Wilson C, Chen S, Arkin MR, Bogyo M, and Deu E
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- Antimalarials toxicity, Cells, Cultured, Drug Evaluation, Preclinical, Humans, Antimalarials pharmacology, Cysteine Proteases, Cysteine Proteinase Inhibitors pharmacology, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases antagonists & inhibitors, Plasmodium falciparum drug effects, Plasmodium falciparum enzymology, Protozoan Proteins antagonists & inhibitors
- Abstract
Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas's disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development., Competing Interests: The authors have declared that no competing interests exist.
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- 2019
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11. Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and covalent inhibitors.
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de Vries LE, Sanchez MI, Groborz K, Kuppens L, Poreba M, Lehmann C, Nevins N, Withers-Martinez C, Hirst DJ, Yuan F, Arastu-Kapur S, Horn M, Mares M, Bogyo M, Drag M, and Deu E
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- Erythrocytes drug effects, Erythrocytes metabolism, Humans, Malaria, Falciparum drug therapy, Malaria, Falciparum metabolism, Models, Molecular, Molecular Structure, Plasmodium falciparum drug effects, Plasmodium falciparum metabolism, Protein Conformation, Substrate Specificity, Amino Acids chemistry, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases metabolism, Erythrocytes parasitology, Malaria, Falciparum parasitology, Peptide Fragments metabolism, Plasmodium falciparum growth & development, Protease Inhibitors pharmacology
- Abstract
Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes, it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of nonoptimal peptide substrates. Finally, our homology modelling and docking studies provide potential structural explanations of the differences in specificity between DPAP1, DPAP3, and CatC, and between substrates and inhibitors in the case of DPAP3. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European BiochemicalSocieties.)
- Published
- 2019
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12. Preliminary Definitions for Sacroiliac Joint Pathologies in the OMERACT Juvenile Idiopathic Arthritis Magnetic Resonance Imaging Score (OMERACT JAMRIS-SIJ).
- Author
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Otobo TM, Conaghan PG, Maksymowych WP, van der Heijde D, Weiss P, Sudol-Szopinska I, Herregods N, Jaremko JL, Meyers AB, Rumsey D, Inarejos EC, Kirkhus E, Stimec J, Panwar J, Thorpe K, Jans L, van Rossum MA, Tolend M, Perez M, Tzaribachev N, Sandhya P, Tse S, Simone A, Swami VG, Touma Z, Lambert R, and Doria AS
- Subjects
- Humans, Inflammation diagnostic imaging, Magnetic Resonance Imaging, Severity of Illness Index, Arthritis, Juvenile diagnostic imaging, Sacroiliac Joint diagnostic imaging, Sacroiliitis diagnostic imaging
- Abstract
Objective: To develop definitions for the assessment of magnetic resonance imaging (MRI) pathologies of the sacroiliac joints (SIJ) in juvenile idiopathic arthritis., Methods: An Outcome Measures in Rheumatology (OMERACT) consensus-driven methodology consisting of iterative surveys and focus group meetings within an international group of rheumatologists and radiologists., Results: Two domains, inflammation and structural, were identified. Definitions for bone marrow edema, joint space inflammation, capsulitis, and enthesitis were derived for joint inflammation; sclerosis, erosion, fatty lesion, and ankylosis were defined for assessing structural joint changes., Conclusion: Preliminary consensus-driven definitions for inflammation and structural elements have been derived, underpinning the ongoing development of the OMERACT Juvenile Arthritis MRI SIJ scoring system (OMERACT JAMRIS-SIJ).
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- 2019
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13. Plasmodium falciparum dipeptidyl aminopeptidase 3 activity is important for efficient erythrocyte invasion by the malaria parasite.
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Lehmann C, Tan MSY, de Vries LE, Russo I, Sanchez MI, Goldberg DE, and Deu E
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- Animals, Cysteine Proteases metabolism, Erythrocytes microbiology, Erythrocytes parasitology, Host-Parasite Interactions, Malaria, Falciparum metabolism, Malaria, Falciparum pathology, Merozoites metabolism, Merozoites physiology, Organelles metabolism, Peptide Hydrolases metabolism, Plasmodium falciparum genetics, Plasmodium falciparum metabolism, Plasmodium falciparum pathogenicity, Proteolysis, Protozoan Proteins metabolism, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases metabolism, Malaria, Falciparum parasitology, Plasmodium falciparum enzymology
- Abstract
Parasite egress from infected erythrocytes and invasion of new red blood cells are essential processes for the exponential asexual replication of the malaria parasite. These two tightly coordinated events take place in less than a minute and are in part regulated and mediated by proteases. Dipeptidyl aminopeptidases (DPAPs) are papain-fold cysteine proteases that cleave dipeptides from the N-terminus of protein substrates. DPAP3 was previously suggested to play an essential role in parasite egress. However, little is known about its enzymatic activity, intracellular localization, or biological function. In this study, we recombinantly expressed DPAP3 and demonstrate that it has indeed dipeptidyl aminopeptidase activity, but contrary to previously studied DPAPs, removal of its internal prodomain is not required for activation. By combining super resolution microscopy, time-lapse fluorescence microscopy, and immunoelectron microscopy, we show that Plasmodium falciparum DPAP3 localizes to apical organelles that are closely associated with the neck of the rhoptries, and from which DPAP3 is secreted immediately before parasite egress. Using a conditional knockout approach coupled to complementation studies with wild type or mutant DPAP3, we show that DPAP3 activity is important for parasite proliferation and critical for efficient red blood cell invasion. We also demonstrate that DPAP3 does not play a role in parasite egress, and that the block in egress phenotype previously reported for DPAP3 inhibitors is due to off target or toxicity effects. Finally, using a flow cytometry assay to differentiate intracellular parasites from extracellular parasites attached to the erythrocyte surface, we show that DPAP3 is involved in the initial attachment of parasites to the red blood cell surface. Overall, this study establishes the presence of a DPAP3-dependent invasion pathway in malaria parasites., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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14. Proteases as antimalarial targets: strategies for genetic, chemical, and therapeutic validation.
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Deu E
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- Animals, Humans, Malaria genetics, Models, Biological, Peptide Hydrolases genetics, Protozoan Proteins genetics, Protozoan Proteins metabolism, Antimalarials therapeutic use, Malaria drug therapy, Malaria enzymology, Peptide Hydrolases metabolism, Plasmodium physiology
- Abstract
Malaria is a devastating parasitic disease affecting half of the world's population. The rapid emergence of resistance against new antimalarial drugs, including artemisinin-based therapies, has made the development of drugs with novel mechanisms of action extremely urgent. Proteases are enzymes proven to be well suited for target-based drug development due to our knowledge of their enzymatic mechanisms and active site structures. More importantly, Plasmodium proteases have been shown to be involved in a variety of pathways that are essential for parasite survival. However, pharmacological rather than target-based approaches have dominated the field of antimalarial drug development, in part due to the challenge of robustly validating Plasmodium targets at the genetic level. Fortunately, over the last few years there has been significant progress in the development of efficient genetic methods to modify the parasite, including several conditional approaches. This progress is finally allowing us not only to validate essential genes genetically, but also to study their molecular functions. In this review, I present our current understanding of the biological role proteases play in the malaria parasite life cycle. I also discuss how the recent advances in Plasmodium genetics, the improvement of protease-oriented chemical biology approaches, and the development of malaria-focused pharmacological assays, can be combined to achieve a robust biological, chemical and therapeutic validation of Plasmodium proteases as viable drug targets., (© 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2017
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15. Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum.
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Thomas JA, Collins CR, Das S, Hackett F, Graindorge A, Bell D, Deu E, and Blackman MJ
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- Animals, Erythrocytes parasitology, Humans, Life Cycle Stages, Merozoite Surface Protein 1 metabolism, Mutation genetics, Phenotype, Plasmodium falciparum growth & development, Hemolytic Plaque Technique methods, Malaria, Falciparum parasitology, Parasites isolation & purification, Plasmodium falciparum isolation & purification
- Abstract
Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro.
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- 2016
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16. Plasmodium dipeptidyl aminopeptidases as malaria transmission-blocking drug targets.
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Tanaka TQ, Deu E, Molina-Cruz A, Ashburne MJ, Ali O, Suri A, Kortagere S, Bogyo M, and Williamson KC
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- Aminopeptidases genetics, Aminopeptidases metabolism, Animals, Female, Male, Mice, Plasmodium falciparum genetics, Reverse Transcriptase Polymerase Chain Reaction, Aminopeptidases antagonists & inhibitors, Antimalarials pharmacology, Plasmodium falciparum drug effects, Plasmodium falciparum enzymology
- Abstract
The Plasmodium falciparum and P. berghei genomes each contain three dipeptidyl aminopeptidase (dpap) homologs. dpap1 and -3 are critical for asexual growth, but the role of dpap2, the gametocyte-specific homolog, has not been tested. If DPAPs are essential for transmission as well as asexual growth, then a DPAP inhibitor could be used for treatment and to block transmission. To directly analyze the role of DPAP2, a dpap2-minus P. berghei (Pbdpap2Δ) line was generated. The Pbdpap2Δ parasites grew normally, differentiated into gametocytes, and generated sporozoites that were infectious to mice when fed to a mosquito. However, Pbdpap1 transcription was >2-fold upregulated in the Pbdpap2Δ clonal lines, possibly compensating for the loss of Pbdpap2. The role of DPAP1 and -3 in the dpap2Δ parasites was then evaluated using a DPAP inhibitor, ML4118S. When ML4118S was added to the Pbdpap2Δ parasites just before a mosquito membrane feed, mosquito infectivity was not affected. To assess longer exposures to ML4118S and further evaluate the role of DPAPs during gametocyte development in a parasite that causes human malaria, the dpap2 deletion was repeated in P. falciparum. Viable P. falciparum dpap2 (Pfdpap2)-minus parasites were obtained that produced morphologically normal gametocytes. Both wild-type and Pfdpap2-negative parasites were sensitive to ML4118S, indicating that, unlike many antimalarials, ML4118S has activity against parasites at both the asexual and sexual stages and that DPAP1 and -3 may be targets for a dual-stage drug that can treat patients and block malaria transmission.
- Published
- 2013
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17. A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases.
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Xiao J, Broz P, Puri AW, Deu E, Morell M, Monack DM, and Bogyo M
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- Binding Sites, Caspase 1 analysis, Caspase 8 analysis, Cell Line, Humans, Models, Molecular, Molecular Imaging, Mutation, Recombinant Proteins analysis, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Caspase 1 genetics, Caspase 1 metabolism, Caspase 8 genetics, Caspase 8 metabolism, Protein Engineering methods
- Abstract
Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation.
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- 2013
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18. Coupling protein engineering with probe design to inhibit and image matrix metalloproteinases with controlled specificity.
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Morell M, Nguyen Duc T, Willis AL, Syed S, Lee J, Deu E, Deng Y, Xiao J, Turk BE, Jessen JR, Weiss SJ, and Bogyo M
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- Amino Acid Sequence, Animals, Cell Line, Cysteine analysis, Cysteine genetics, Cysteine metabolism, Humans, Matrix Metalloproteinase 1 genetics, Matrix Metalloproteinase 1 metabolism, Matrix Metalloproteinase 12 genetics, Matrix Metalloproteinase 12 metabolism, Mice, Models, Molecular, Molecular Sequence Data, Optical Imaging, Sequence Alignment, Zebrafish, Matrix Metalloproteinase 1 analysis, Matrix Metalloproteinase 12 analysis, Molecular Probe Techniques, Protein Engineering methods
- Abstract
Matrix metalloproteinases (MMPs) are zinc endopeptidases that play roles in numerous pathophysiological processes and therefore are promising drug targets. However, the large size of this family and a lack of highly selective compounds that can be used for imaging or inhibition of specific MMPs members has limited efforts to better define their biological function. Here we describe a protein engineering strategy coupled with small-molecule probe design to selectively target individual members of the MMP family. Specifically, we introduce a cysteine residue near the active-site of a selected protease that does not alter its overall activity or function but allows direct covalent modification by a small-molecule probe containing a reactive electrophile. This specific engineered interaction between the probe and the target protease provides a means to both image and inhibit the modified protease with absolute specificity. Here we demonstrate the feasibility of the approach for two distinct MMP proteases, MMP-12 and MT1-MMP (or MMP-14).
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- 2013
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19. The antimalarial natural product symplostatin 4 is a nanomolar inhibitor of the food vacuole falcipains.
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Stolze SC, Deu E, Kaschani F, Li N, Florea BI, Richau KH, Colby T, van der Hoorn RA, Overkleeft HS, Bogyo M, and Kaiser M
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- Antimalarials chemistry, Antimalarials therapeutic use, Antimicrobial Cationic Peptides, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Enzyme Inhibitors therapeutic use, Hemoglobins metabolism, Host-Parasite Interactions drug effects, Humans, Malaria, Falciparum parasitology, Models, Molecular, Peptides chemistry, Peptides therapeutic use, Plasmodium falciparum drug effects, Plasmodium falciparum physiology, Antimalarials pharmacology, Cysteine Endopeptidases metabolism, Erythrocytes parasitology, Malaria, Falciparum drug therapy, Peptides pharmacology, Plasmodium falciparum enzymology
- Abstract
The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4's mode of action. Finally, we reveal a critical influence of the unusual methyl-methoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
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- 2012
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20. Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity.
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Li H, Ponder EL, Verdoes M, Asbjornsdottir KH, Deu E, Edgington LE, Lee JT, Kirk CJ, Demo SD, Williamson KC, and Bogyo M
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- Animals, Antimalarials chemistry, Antimalarials pharmacology, Antimalarials toxicity, Erythrocytes parasitology, Humans, Malaria parasitology, Mice, Mice, Inbred BALB C, Oligopeptides chemistry, Oligopeptides pharmacology, Oligopeptides therapeutic use, Oligopeptides toxicity, Plasmodium berghei drug effects, Plasmodium berghei enzymology, Proteasome Inhibitors chemistry, Proteasome Inhibitors pharmacology, Proteasome Inhibitors toxicity, Antimalarials therapeutic use, Malaria drug therapy, Plasmodium drug effects, Plasmodium enzymology, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors therapeutic use
- Abstract
The Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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21. New approaches for dissecting protease functions to improve probe development and drug discovery.
- Author
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Deu E, Verdoes M, and Bogyo M
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- Animals, Biocatalysis drug effects, Humans, Models, Chemical, Molecular Structure, Peptide Hydrolases genetics, Proteolysis drug effects, Proteomics methods, Drug Discovery methods, Peptide Hydrolases chemistry, Peptide Hydrolases metabolism, Protease Inhibitors chemistry, Protease Inhibitors pharmacology
- Abstract
Proteases are well-established targets for pharmaceutical development because of their known enzymatic mechanism and their regulatory roles in many pathologies. However, many potent clinical lead compounds have been unsuccessful either because of a lack of specificity or because of our limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to understand protease functions and to expand the platform of inhibitor development beyond active site-directed design and in vitro optimization. Several newly developed technologies will enhance assessment of drug selectivity in living cells and animal models, allowing researchers to focus on compounds with high specificity and minimal side effects in vivo. In this review, we highlight advances in the development of chemical probes, proteomic methods and screening tools that we feel will help facilitate this paradigm shift in drug discovery.
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- 2012
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22. Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case study.
- Author
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Deu E and Kirsch JF
- Subjects
- Allosteric Regulation, Aspartate Aminotransferases chemistry, Aspartate Aminotransferases genetics, Aspartate Aminotransferases metabolism, Catalytic Domain, Escherichia coli chemistry, Escherichia coli metabolism, Protein Multimerization, Transaminases chemistry, Tyrosine Transaminase chemistry, Tyrosine Transaminase genetics, Tyrosine Transaminase metabolism, Escherichia coli enzymology, Escherichia coli genetics, Protein Engineering methods, Transaminases genetics, Transaminases metabolism
- Abstract
The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes., (Copyright © 2011 The Protein Society.)
- Published
- 2011
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23. Development of small molecule inhibitors and probes of human SUMO deconjugating proteases.
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Albrow VE, Ponder EL, Fasci D, Békés M, Deu E, Salvesen GS, and Bogyo M
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- Aza Compounds chemistry, Catalytic Domain, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Endopeptidases genetics, Endopeptidases metabolism, Epoxy Compounds chemistry, Epoxy Compounds pharmacology, Humans, Ketones chemistry, Ketones pharmacology, Peptide Hydrolases chemistry, Peptide Hydrolases genetics, Peptide Hydrolases metabolism, Protease Inhibitors pharmacology, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins genetics, Recombinant Proteins metabolism, Small Ubiquitin-Related Modifier Proteins metabolism, Structure-Activity Relationship, Drug Design, Endopeptidases chemistry, Protease Inhibitors chemistry
- Abstract
Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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24. Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors.
- Author
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Ponder EL, Albrow VE, Leader BA, Békés M, Mikolajczyk J, Fonović UP, Shen A, Drag M, Xiao J, Deu E, Campbell AJ, Powers JC, Salvesen GS, and Bogyo M
- Subjects
- Amino Acid Sequence, Catalytic Domain, Cysteine Endopeptidases, Endopeptidases metabolism, Humans, Hydrazines chemistry, Molecular Sequence Data, Phthalic Acids chemistry, Protease Inhibitors chemistry, Protozoan Proteins classification, Protozoan Proteins genetics, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Hydrazines pharmacology, Phthalic Acids pharmacology, Plasmodium falciparum enzymology, Protease Inhibitors pharmacology, Protozoan Proteins chemistry, Small Ubiquitin-Related Modifier Proteins metabolism
- Abstract
Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
25. A fragmenting hybrid approach for targeted delivery of multiple therapeutic agents to the malaria parasite.
- Author
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Mahajan SS, Deu E, Lauterwasser EM, Leyva MJ, Ellman JA, Bogyo M, and Renslo AR
- Subjects
- Animals, Antimalarials administration & dosage, Carbamates chemistry, Cathepsin C antagonists & inhibitors, Cathepsin C metabolism, Heterocyclic Compounds, 1-Ring chemistry, Iron chemistry, Peroxides chemistry, Protease Inhibitors administration & dosage, Protease Inhibitors chemistry, Spiro Compounds chemistry, Antimalarials chemistry, Drug Carriers chemistry, Malaria drug therapy
- Published
- 2011
- Full Text
- View/download PDF
26. Molecular mechanisms of bortezomib resistant adenocarcinoma cells.
- Author
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Suzuki E, Demo S, Deu E, Keats J, Arastu-Kapur S, Bergsagel PL, Bennett MK, and Kirk CJ
- Subjects
- Adenocarcinoma pathology, Blotting, Western, Bortezomib, Cell Line, Tumor, Drug Resistance, Neoplasm, Enzyme-Linked Immunosorbent Assay, Humans, Adenocarcinoma drug therapy, Antineoplastic Agents therapeutic use, Boronic Acids therapeutic use, Pyrazines therapeutic use
- Abstract
Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ~30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response., (© 2011 Suzuki et al.)
- Published
- 2011
- Full Text
- View/download PDF
27. Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.
- Author
-
Wang F, Krai P, Deu E, Bibb B, Lauritzen C, Pedersen J, Bogyo M, and Klemba M
- Subjects
- Amino Acids metabolism, Cathepsin C antagonists & inhibitors, Cathepsin C isolation & purification, Chlorides metabolism, Enzyme Activators metabolism, Fluorescent Dyes metabolism, Hemoglobins metabolism, Humans, Hydrogen-Ion Concentration, Kinetics, Protease Inhibitors metabolism, Protein Multimerization, Protozoan Proteins antagonists & inhibitors, Protozoan Proteins isolation & purification, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Substrate Specificity, Cathepsin C metabolism, Plasmodium falciparum enzymology, Protozoan Proteins metabolism
- Abstract
Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
28. Rational design of inhibitors and activity-based probes targeting Clostridium difficile virulence factor TcdB.
- Author
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Puri AW, Lupardus PJ, Deu E, Albrow VE, Garcia KC, Bogyo M, and Shen A
- Subjects
- Allosteric Regulation, Anti-Bacterial Agents pharmacology, Bacterial Proteins metabolism, Bacterial Toxins metabolism, Binding Sites, Clostridioides difficile enzymology, Computer Simulation, Crystallography, X-Ray, Cysteine Proteases chemistry, Cysteine Proteinase Inhibitors pharmacology, Drug Design, Molecular Probes chemistry, Molecular Probes pharmacology, Phytic Acid chemistry, Protein Binding, Protein Structure, Tertiary, Structure-Activity Relationship, Anti-Bacterial Agents chemistry, Bacterial Proteins antagonists & inhibitors, Bacterial Toxins antagonists & inhibitors, Cysteine Proteinase Inhibitors chemistry
- Abstract
Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
29. Functional studies of Plasmodium falciparum dipeptidyl aminopeptidase I using small molecule inhibitors and active site probes.
- Author
-
Deu E, Leyva MJ, Albrow VE, Rice MJ, Ellman JA, and Bogyo M
- Subjects
- Animals, Antiparasitic Agents blood, Antiparasitic Agents chemistry, Antiparasitic Agents pharmacology, Antiparasitic Agents therapeutic use, Cathepsin C chemistry, Cell Line, Dose-Response Relationship, Drug, Drug Design, Drug Stability, Female, Malaria drug therapy, Mice, Plasmodium falciparum drug effects, Plasmodium falciparum physiology, Protease Inhibitors blood, Protease Inhibitors therapeutic use, Trophozoites drug effects, Catalytic Domain, Cathepsin C antagonists & inhibitors, Cathepsin C metabolism, Computational Biology, Plasmodium falciparum enzymology, Protease Inhibitors chemistry, Protease Inhibitors pharmacology
- Abstract
The widespread resistance of malaria parasites to all affordable drugs has made the identification of new targets urgent. Dipeptidyl aminopeptidases (DPAPs) represent potentially valuable new targets that are involved in hemoglobin degradation (DPAP1) and parasite egress (DPAP3). Here we use activity-based probes to demonstrate that specific inhibition of DPAP1 by a small molecule results in the formation of an immature trophozoite that leads to parasite death. Using computational methods, we designed stable, nonpeptidic covalent inhibitors that kill Plasmodium falciparum at low nanomolar concentrations. These compounds show signs of slowing parasite growth in a murine model of malaria, which suggests that DPAP1 might be a viable antimalarial target. Interestingly, we found that resynthesis and activation of DPAP1 after inhibition is rapid, suggesting that effective drugs would need to sustain DPAP1 inhibition for a period of 2-3 hr., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
30. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.
- Author
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Deu E, Yang Z, Wang F, Klemba M, and Bogyo M
- Subjects
- Animals, Cathepsin C antagonists & inhibitors, Cathepsin C metabolism, Humans, Liver cytology, Plasmodium falciparum cytology, Protease Inhibitors pharmacology, Rats, Small Molecule Libraries pharmacology, Substrate Specificity, Cell Extracts, Drug Evaluation, Preclinical methods, High-Throughput Screening Assays methods, Molecular Probes metabolism
- Abstract
Background: High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities., Methodology and Principal Findings: Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates., Conclusions: We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.
- Published
- 2010
- Full Text
- View/download PDF
31. The partially folded homodimeric intermediate of Escherichia coli aspartate aminotransferase contains a "molten interface" structure.
- Author
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Deu E, Dhoot J, and Kirsch JF
- Subjects
- Alanine metabolism, Amino Acid Sequence, Amino Acid Substitution, Aspartate Aminotransferases genetics, Aspartate Aminotransferases metabolism, Binding Sites, Circular Dichroism, Enzyme Stability, Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Hydrogen Bonding, Kinetics, Models, Biological, Molecular Sequence Data, Molecular Weight, Protein Denaturation, Protein Folding, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Static Electricity, Thermodynamics, Urea pharmacology, Aspartate Aminotransferases chemistry, Escherichia coli enzymology, Escherichia coli Proteins chemistry
- Abstract
The role of intersubunit side chain-side chain interactions in the stability of the Escherichia coli aspartate aminotransferase (eAATase) homodimer was investigated by directed mutagenesis at 10 different interface contacts. The urea-mediated unfolding pathway of this enzyme proceeds through the formation of a dimeric intermediate, D*, that retains only 40% of the native enzyme secondary structure as judged by circular dichroism. Disruption of any single intersubunit interaction results in a >2.6 kcal mol(-1) decrease in native state stability, independent of its location or nature. However, the stability of D* with respect to U, the unfolded monomer, is the same for all mutants. The stability of the eAATase interface cannot be ascribed to the contribution of a few hot spots, or to the accumulation of a large number of weak interactions, but only to the presence of multiple important and interconnected interactions. It is proposed that a "molten interface" structure, flexible enough to accommodate point mutations, accounts for the stability of D*. Nuclei of tertiary structure, which are not involved in native intersubunit contacts, likely provide a scaffold for the unstructured interface of D*. Such a scaffold would account for the cooperative unfolding of the intermediate.
- Published
- 2009
- Full Text
- View/download PDF
32. Cofactor-directed reversible denaturation pathways: the cofactor-stabilized Escherichia coli aspartate aminotransferase homodimer unfolds through a pathway that differs from that of the apoenzyme.
- Author
-
Deu E and Kirsch JF
- Subjects
- Circular Dichroism, Enzyme Stability, Escherichia coli enzymology, Guanidine pharmacology, Holoenzymes chemistry, Protein Folding, Protein Renaturation drug effects, Protein Structure, Quaternary, Pyridoxal Phosphate pharmacology, Pyridoxamine analogs & derivatives, Pyridoxamine chemistry, Spectrometry, Fluorescence, Thermodynamics, Urea pharmacology, Aspartate Aminotransferases chemistry, Protein Denaturation drug effects, Pyridoxal Phosphate chemistry
- Abstract
While the urea-mediated unfolding pathway of the Escherichia coli aspartate aminotransferase (eAATase) homodimer proceeds through a reversible three-state process with a partially folded dimeric intermediate, D D* 2U (E. Deu and J. F. Kirsch, accompanying paper), that of a cofactor-stabilized form differs. Pyridoxal phosphate, which binds at the intersubunit active sites, stabilizes the native form by 6 kcal mol-1 and dissociates during the D <==> D* transition. Reductive trapping of the cofactor to a nondissociable derivative (PPL-eAATase) precludes the formation of D*. A novel monomeric intermediate (M'-PPL) with 70% of the native secondary structure (circular dichroism) was identified in the unfolding pathway of PPL-eAATase: D-PPL2 <==> 2M'-PPL <==> 2U-PPL. The combined results define two structural regions with distinct stabilities: the active site region (ASR) and the generally more stable, dimerization region (DMR). The DMR includes the key intersubunit contacts. It is responsible for the multimeric nature of D*, and its disorder leads to dimer dissociation. Selective strengthening of the ASR-cofactor interactions by cofactor trapping reverses the relative stabilities of the two regions (from DMR > ASR in the apoenzyme to ASR > DMR in PPL-eAATase) and results in a reordering of the eAATase denaturation pathway.
- Published
- 2007
- Full Text
- View/download PDF
33. The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant.
- Author
-
Deu E and Kirsch JF
- Subjects
- Circular Dichroism, Escherichia coli enzymology, Protein Folding, Protein Structure, Quaternary, Spectrometry, Fluorescence, Temperature, Apoenzymes chemistry, Aspartate Aminotransferases chemistry, Guanidine pharmacology, Protein Denaturation drug effects, Urea pharmacology
- Abstract
The guanidine hydrochloride (GdnHCl) mediated denaturation pathway for the apo form of homodimeric Escherichia coli aspartate aminotransferase (eAATase) (molecular mass = 43.5 kDa/monomer) includes a partially folded monomeric intermediate, M* [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913; Birolo, L., Dal Piaz, F., Pucci, P., and Marino, G. (2002) J. Biol. Chem. 277, 17428-17437]. The present investigation of the urea-mediated denaturation of eAATase finds no evidence for an M* species but uncovers a partially denatured dimeric form, D*, that is unpopulated in GdnHCl. Thus, the unfolding process is a function of the employed denaturant. D* retains less than 50% of the native secondary structure (circular dichroism), conserves significant quaternary and tertiary interactions, and unfolds cooperatively (mD*<==>U = 3.4 +/- 0.3 kcal mol-1 M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D <==> 2M 2M* <==> 2U in GdnHCl and D <==> D* <==> 2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D <==> 2U) is 36 +/- 3 kcal mol-1, while that for the D* 2U transition is 24 +/- 2 kcal mol-1, both at 1 M standard state and pH 7.5.
- Published
- 2007
- Full Text
- View/download PDF
34. The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: multiple forced covariant amino acid substitutions in natural variants.
- Author
-
Deu E, Koch KA, and Kirsch JF
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Aspartate Aminotransferases metabolism, Kinetics, Models, Biological, Molecular Sequence Data, Phylogeny, Protein Structure, Quaternary, Sequence Alignment, Substrate Specificity, Aspartate Aminotransferases chemistry
- Abstract
The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.
- Published
- 2002
- Full Text
- View/download PDF
35. Pyogenic liver abscess: studies of therapy and analysis of risk factors.
- Author
-
Mischinger HJ, Hauser H, Rabl H, Quehenberger F, Werkgartner G, Rubin R, and Deu E
- Subjects
- Adult, Aged, Aged, 80 and over, Female, Humans, Male, Middle Aged, Risk Factors, Suppuration, Treatment Outcome, Drainage methods, Liver Abscess surgery
- Abstract
Experience with 46 patients diagnosed with liver abscesses over a 13-year period was reviewed to ascertain the impact of percutaneous versus surgical drainage. In most of the cases the abscesses were diagnosed by sonography or computed tomography. The most common pathogenetic mechanism was ascending biliary tract infection. Of the 46 total patients, 27 were primarily treated surgically, whereas 19 underwent percutaneous drainage. In the surgical group five (18.5%) patients required reoperation. Percutaneous drainage failed in four patients (21.1%). Multivariate stepwise logistic regression analysis revealed that a high APACHE II score, low hemoglobin level, and high serum bilirubin level were significant predictors of a complicated clinical course. Death was related more closely to the overall condition of the patient, as expressed by a high APACHE II score, and the underlying disease (malignancy) than to the mode of therapy.
- Published
- 1994
- Full Text
- View/download PDF
36. Nodular focal fatty infiltration of the liver in acquired porphyria cutanea tarda.
- Author
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Flueckiger F, Steiner H, Leitinger G, Hoedl S, and Deu E
- Subjects
- Adult, Fatty Liver etiology, Humans, Liver diagnostic imaging, Male, Porphyrias complications, Radiography, Skin Diseases complications, Ultrasonography, Valproic Acid adverse effects, Fatty Liver diagnostic imaging, Porphyrias chemically induced, Skin Diseases chemically induced
- Abstract
A case is reported of acquired porphyria cutanea tarda (PCT) due to administered valproate acid in an epileptic patient. Sonography (US) and computed tomography (CT) showed multiple nodular, well-defined lesions of the liver. Biopsy confirmed the diagnosis of focal fatty infiltration and revealed additionally the signs of hepatic involvement in PCT. Focal fatty infiltration should be considered in patients with multiple liver lesions if there is a predisposing condition. PCT can be a further cause of this type of infiltration.
- Published
- 1991
- Full Text
- View/download PDF
37. [The limits of the differential diagnostic capacity of CT and sonography in cystic hepatic echinococcosis].
- Author
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Deu E, Steiner H, Flückiger F, Stünzner D, and Weybora W
- Subjects
- Animals, Antibodies, Helminth blood, Contrast Media, Diagnosis, Differential, Echinococcosis, Hepatic pathology, Echinococcus immunology, Evaluation Studies as Topic, Humans, Iohexol analogs & derivatives, Ultrasonography, Echinococcosis, Hepatic diagnosis, Liver diagnostic imaging, Tomography, X-Ray Computed
- Abstract
In regions of only sporadic occurrence of hydatid disease, the heterogenic appearance of the cysts often causes a diagnostic dilemma. In 28 patients the reports of sonography and/or computed tomography, without and during bolus injection of 100ml anionic contrast material, were compared to the results of the indirect haemagglutination test (IHA). In six cases additionally the enzyme-linked immunosorbent assay (ELISA), and in nine the histologic specimen were evaluated. The authors discuss the value of CT and US reporting on their experience and the difficulties in establishing the diagnosis in 14 histologically and/or serologically proven cases and another 14 cases with "typical" appearance but negative serology and/or histology.
- Published
- 1991
- Full Text
- View/download PDF
38. [Color-coded duplex sonography and the resistive index in dysfunctional kidney transplants].
- Author
-
Flückiger F, Steiner H, Horn S, Ratschek M, and Deu E
- Subjects
- Adult, Aged, Color, Female, Humans, Kidney blood supply, Male, Middle Aged, Postoperative Complications etiology, Postoperative Complications physiopathology, Transplantation, Homologous, Ultrasonography instrumentation, Vascular Resistance physiology, Graft Rejection physiology, Kidney diagnostic imaging, Kidney Transplantation physiology, Postoperative Complications diagnostic imaging
- Abstract
In a prospective analysis we performed 182 colour duplex US studies in 60 patients with renal transplants to determine the effects of different causes of graft dysfunction on the resistive index (RI) and the colour imaging pattern of the transplant arteries. Allografts with normal function, cyclosporin toxicity, cytomegaly infection, acute tubular necrosis, glomerulonephritis and postoperative functional nephrotoxicity showed normal RI (less than 0.7) and normal pattern of the arteries in colour duplex. Grafts with acute and chronic rejection, obstructive uropathy and arteriolosclerosis showed significant elevated RI and in colour duplex always a characteristic blinking of the arteries and a numerical reduction of the peripheral arteries. It seems that the assessment of an increase of vascular impedance is possible also with colour duplex US.
- Published
- 1990
- Full Text
- View/download PDF
39. [Venous compression syndrome of the lower extremity: comparison of phlebography and color coded duplex sonography].
- Author
-
Lederer A, Steiner H, Rabl H, Deu E, and Wildling R
- Subjects
- Constriction, Pathologic etiology, Diagnosis, Differential, Humans, Thrombophlebitis diagnosis, Phlebography, Ultrasonography, Venous Insufficiency etiology
- Published
- 1990
40. [Pseudothrombosis of the inferior vena cava in CT-cavography. Value of duplex sonography in diagnostic assessment].
- Author
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Rabl H, Deu E, Lederer A, and Steiner H
- Subjects
- Adult, Diagnosis, Differential, Humans, Male, Thrombosis diagnosis, Tomography, X-Ray Computed, Ultrasonography, Vena Cava, Inferior diagnostic imaging
- Published
- 1990
41. [Tuberculosis of the duodenum].
- Author
-
Flückiger F, Deu E, and Hausegger K
- Subjects
- Adult, Duodenal Diseases etiology, Duodenum diagnostic imaging, Humans, Male, Radiography, Tuberculosis, Gastrointestinal etiology, Tuberculosis, Pulmonary complications, Duodenal Diseases diagnostic imaging, Tuberculosis, Gastrointestinal diagnostic imaging
- Published
- 1989
- Full Text
- View/download PDF
42. [Tuberculous colitis--Crohn disease--a clinico-radiologic-endoscopic approach].
- Author
-
Deu E, Ortner R, Kratochvil P, Brandstätter G, and Ratschek M
- Subjects
- Adult, Diagnosis, Differential, Female, Humans, Intestinal Obstruction diagnosis, Colonic Diseases diagnosis, Colonoscopy, Crohn Disease diagnosis, Tuberculosis, Gastrointestinal diagnosis
- Abstract
The authors report on a case of multifocal stenosing tuberculosis of the stomach, pointing out the difficulties in differential diagnosis especially against Crohn's disease. The importance of x-ray examination is discussed.
- Published
- 1986
43. [Endoscopic palliative drainage of the bile ducts].
- Author
-
Brandstätter G, Kratochvil P, Stupnicki T, and Deu E
- Subjects
- Aged, Common Bile Duct surgery, Common Bile Duct Neoplasms surgery, Gallbladder Neoplasms surgery, Humans, Palliative Care, Pancreatic Neoplasms surgery, Prostheses and Implants, Bile Ducts surgery, Drainage methods, Endoscopy
- Abstract
Initial results of palliative endoscopic retrograde drainage of the common bile duct are reported. In 12 out of 19 patients the treatment was successful (63% cases). In every case the serum bilirubin level quickly decreased to normal values. The life span of the endoprostheses lasted on average for 3 months. No fatal complications occurred in connexion with prosthesis implantation. Endoscopic drainage of the common bile duct in patients with an inoperable tumour of the biliary tract represents an alternative procedure to palliative surgery in the management of malignant occlusion icterus.
- Published
- 1985
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