28 results on '"Edgar Deu"'
Search Results
2. Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.
- Author
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Michele S Y Tan, Dara Davison, Mateo I Sanchez, Bethany M Anderson, Stephen Howell, Ambrosius Snijders, Laura E Edgington-Mitchell, and Edgar Deu
- Subjects
Medicine ,Science - 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.
- Published
- 2020
- Full Text
- View/download PDF
3. Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.
- Author
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Mateo I Sanchez, Laura E de Vries, Christine Lehmann, Jeong T Lee, Kenny K Ang, Christopher Wilson, Steven Chen, Michelle R Arkin, Matthew Bogyo, and Edgar Deu
- Subjects
Medicine ,Science - 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.
- Published
- 2019
- Full Text
- View/download PDF
4. Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum.
- Author
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James A Thomas, Christine R Collins, Sujaan Das, Fiona Hackett, Arnault Graindorge, Donald Bell, Edgar Deu, and Michael J Blackman
- Subjects
Medicine ,Science - 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.
- Published
- 2016
- Full Text
- View/download PDF
5. Molecular mechanisms of bortezomib resistant adenocarcinoma cells.
- Author
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Erika Suzuki, Susan Demo, Edgar Deu, Jonathan Keats, Shirin Arastu-Kapur, P Leif Bergsagel, Mark K Bennett, and Christopher J Kirk
- Subjects
Medicine ,Science - 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.
- Published
- 2011
- Full Text
- View/download PDF
6. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.
- Author
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Edgar Deu, Zhimou Yang, Flora Wang, Michael Klemba, and Matthew Bogyo
- Subjects
Medicine ,Science - Abstract
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.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.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
7. The Aspartyl Protease Ddi1 Is Essential for Erythrocyte Invasion by the Malaria Parasite
- Author
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Steven Howell, Edgar Deu, Snijder A, Lucy M. Collinson, A. P. Patel, Anne Weston, Powell D, Jesper Q. Svejstrup, Bingham R, Christine Lehmann, Dirac-Svejstrup Ab, and S. Ridewood
- Subjects
Protease ,biology ,medicine.medical_treatment ,biology.organism_classification ,medicine.disease ,Plasmodium ,Cell biology ,chemistry.chemical_compound ,Aspartate protease ,Proteasome ,chemistry ,medicine ,Parasite hosting ,Blood stream ,DNA ,Malaria - Abstract
Malaria pathology is caused by the exponential replication of Plasmodium parasites in the blood stream. The bottleneck of the parasite life cycle is the invasion of erythrocytes immediately after parasites egress from infected red blood cells. DNA damage-inducible protein 1 (Ddi1) is a conserved eukaryotic proteasome shuttle protein containing an internal retroviral-like protease domain. Using conditional genetics, we now show that the proteolytic activity of the P. falciparum homologue, PfDdi1, is critically required for invasion of red blood cells. Furthermore, PfDdi1 disruption results in the accumulation of highly polyubiquitinated proteins that can be processed by purified PfDdi1 or distant eukaryotic homologues. We also show that PfDdi1 interacts with multiple components of the ubiquitin-proteasome system and that parasites lacking PfDdi1 are more sensitive to proteasome inhibition. Overall, this study establishes PfDdi1 as a key component of the eukaryotic ubiquitin-proteasome system and as a promising antimalarial target.
- Published
- 2021
8. High Content and High Throughout Phenotypic Assay for the Hourly Resolution of the Malaria Parasite Erythrocytic Cycle
- Author
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A. P. Patel, D. Bell, Edgar Deu, S. Ridewood, S. H. Lee, and Young-Tae Chang
- Subjects
medicine.diagnostic_test ,Drug resistance ,Computational biology ,Biology ,medicine.disease ,Genome ,Phenotype ,Genetically modified organism ,Flow cytometry ,parasitic diseases ,medicine ,Parasite hosting ,Malaria ,Genetic screen - Abstract
Over the last 20 years increased funding for malaria research has resulted in very significant technical advances to study the biology ofPlasmodiumspecies. High throughput phenotypic assays have been developed to screen millions of compounds and identify small molecules with antiparasitic activity. At the same time, advances in malaria genetic have greatly facilitated the generation of genetically modified parasites, and whole genome genetic screens are now feasible inPlasmodiumspecies. Finally, there has been an increased interest to study malaria parasites at the population level, in particular in the area of drug resistance. Drug resistant field isolates have been collected around the world, and drug resistant strains are routinely generated in the lab to study the mechanisms of drug resistance. As a result, one of the current bottlenecks in malaria research is our ability to quickly characterize the phenotype associated with compound treatment or genetic modification, or to quickly compare differences in intracellular development between strains. Here, we present a high content/high throughput phenotypic assay that combines highly selective RNA, DNA, and RBC membrane dyes to provide hourly resolution of the full erythrocytic cycle for bothP. falciparumandP. knowlesi. A flow cytometry assay allows the analysis of samples in a 384-well format and a quick way to determine the parasite developmental stage. On the other hand, the fluorescence microscopy format allows for a detailed visualization of parasite morphology. Finally, using open source software we have developed protocols for the automated cluster analysis of microscopy images. This assay can be applied to anyPlasmodiumspecies, requires very little amount of sample, is performed with fixed cells, and is easily scalable. Overall, we believe this assay will be a great tool for the malaria community to studyPlasmodiumspecies.
- Published
- 2021
9. Novel broad-spectrum activity-based probes to profile malarial cysteine proteases
- Author
-
Mateo I. Sánchez, Edgar Deu, Dara Davison, Ambrosius P. Snijders, Bethany M. Anderson, Michele S. Y. Tan, Laura E. Edgington-Mitchell, and Stephen Howell
- Subjects
0301 basic medicine ,Plasmodium ,Cell Membrane Permeability ,Cell ,Biochemistry ,Broad spectrum ,0302 clinical medicine ,Cysteine Proteases ,Medicine and Health Sciences ,Sulfones ,Amino Acids ,media_common ,Protozoans ,Chemical Biology & High Throughput ,Multidisciplinary ,biology ,Chemistry ,Organic Compounds ,Tryptophan ,Malarial Parasites ,Eukaryota ,Proteases ,3. Good health ,Enzymes ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Physical Sciences ,Medicine ,Research Article ,Drug ,Model organisms ,media_common.quotation_subject ,Science ,Plasmodium falciparum ,Infectious Disease ,Cysteine Proteinase Inhibitors ,Biochemistry & Proteomics ,Antimalarials ,03 medical and health sciences ,Parasite Groups ,medicine ,Parasitic Diseases ,Animals ,Humans ,Sulfur Containing Amino Acids ,Cysteine ,Computational & Systems Biology ,Cathepsin ,Merozoites ,Organic Chemistry ,Organisms ,Chemical Compounds ,Correction ,Biology and Life Sciences ,Proteins ,Cell Biology ,biology.organism_classification ,Tropical Diseases ,Parasitic Protozoans ,Malaria ,Protein profiling ,030104 developmental biology ,Molecular Probes ,Enzymology ,Parasitology ,Apicomplexa - 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.
- Published
- 2020
- Full Text
- View/download PDF
10. Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening
- Author
-
Kenny K. H. Ang, Michelle R. Arkin, Matthew Bogyo, Edgar Deu, Steven Chen, Laura E. de Vries, Mateo I. Sánchez, Christine Lehmann, Jeong T. Lee, and Christopher W. Wilson
- Subjects
0301 basic medicine ,Plasmodium ,Peptidomimetic ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Druggability ,Drug Evaluation, Preclinical ,Protozoan Proteins ,Biochemistry ,Cathepsin C ,0302 clinical medicine ,Cysteine Proteases ,Medicine and Health Sciences ,Enzyme Inhibitors ,Cells, Cultured ,Protozoans ,Multidisciplinary ,Cultured ,biology ,Malarial Parasites ,Eukaryota ,Drugs ,Proteases ,Preclinical ,3. Good health ,Enzymes ,Drug development ,030220 oncology & carcinogenesis ,Medicine ,Research Article ,General Science & Technology ,Cells ,Science ,Allosteric regulation ,Plasmodium falciparum ,Cysteine Proteinase Inhibitors ,Parasite Replication ,03 medical and health sciences ,Antimalarials ,All institutes and research themes of the Radboud University Medical Center ,Parasite Groups ,Parasitic Diseases ,Humans ,Dipeptidyl-Peptidases and Tripeptidyl-Peptidases ,Cathepsin ,Pharmacology ,Organisms ,Biology and Life Sciences ,Proteins ,biology.organism_classification ,Tropical Diseases ,Parasitic Protozoans ,Malaria ,030104 developmental biology ,Enzymology ,Drug Evaluation ,Parasitology ,Apicomplexa - 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.
- Published
- 2019
11. Proteases as antimalarial targets: strategies for genetic, chemical, and therapeutic validation
- Author
-
Edgar Deu
- Subjects
0301 basic medicine ,Drug ,Proteases ,Plasmodium ,media_common.quotation_subject ,Population ,Chemical biology ,malaria ,Protozoan Proteins ,Computational biology ,Review Article ,Biochemistry ,Models, Biological ,03 medical and health sciences ,Antimalarials ,parasitic diseases ,medicine ,Animals ,Humans ,Artemisinin ,education ,Molecular Biology ,Review Articles ,media_common ,education.field_of_study ,030102 biochemistry & molecular biology ,biology ,business.industry ,protease ,Cell Biology ,biology.organism_classification ,medicine.disease ,3. Good health ,Biotechnology ,030104 developmental biology ,Drug development ,target validation ,business ,Malaria ,medicine.drug ,Peptide Hydrolases - 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. This article is protected by copyright. All rights reserved.
- Published
- 2017
12. Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and covalent inhibitors
- Author
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Marcin Drag, Fang Yuan, Katarzyna Groborz, Martin Horn, Edgar Deu, Chrislaine Withers-Martinez, Laurie Kuppens, Neysa Nevins, Shirin Arastu-Kapur, Matthew Bogyo, Michael Mareš, Mateo I. Sánchez, Christine Lehmann, David Jonathan Hirst, Marcin Poreba, and Laura E. de Vries
- Subjects
Models, Molecular ,0301 basic medicine ,Proteases ,Erythrocytes ,Protein Conformation ,medicine.medical_treatment ,Plasmodium falciparum ,malaria ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,specificity ,Peptide ,Biochemistry ,Aminopeptidase ,Substrate Specificity ,Cathepsin C ,03 medical and health sciences ,0302 clinical medicine ,All institutes and research themes of the Radboud University Medical Center ,positional scanning ,medicine ,Humans ,Protease Inhibitors ,Amino Acids ,Malaria, Falciparum ,Dipeptidyl-Peptidases and Tripeptidyl-Peptidases ,Molecular Biology ,chemistry.chemical_classification ,Protease ,Molecular Structure ,Chemistry ,Original Articles ,Cell Biology ,Peptide Fragments ,3. Good health ,Amino acid ,030104 developmental biology ,Enzyme ,Docking (molecular) ,030220 oncology & carcinogenesis ,Original Article ,proteases ,dipeptidyl aminopeptidase - 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., We have used peptide‐based libraries of substrates and covalent inhibitors to characterize the specificity of Plasmodium dipeptidyl aminopeptidase 3, a protease important for red blood cell invasion. Interestingly, we observed significant differences in amino acid preference between substrates and inhibitors, and showed that highly potent and specific inhibitors can be developed based on the sequences of nonoptimal peptide substrates.
- Published
- 2019
13. Plasmodium falciparum dipeptidyl aminopeptidase 3 activity is important for efficient erythrocyte invasion by the malaria parasite
- Author
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Christine Lehmann, Michele S. Y. Tan, Daniel E. Goldberg, Ilaria Russo, Edgar Deu, Laura E. de Vries, and Mateo I. Sánchez
- Subjects
0301 basic medicine ,Plasmodium ,Erythrocytes ,Molecular biology ,Mutant ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Protozoan Proteins ,Artificial Gene Amplification and Extension ,Aminopeptidase ,Polymerase Chain Reaction ,Cysteine Proteases ,Parasite hosting ,Biology (General) ,Malaria, Falciparum ,Staining ,Protozoans ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Malarial Parasites ,Eukaryota ,Cysteine protease ,Specimen preparation and treatment ,3. Good health ,Cell biology ,medicine.anatomical_structure ,Research Article ,Proteases ,QH301-705.5 ,Immunoelectron microscopy ,Immunology ,Plasmodium falciparum ,DNA construction ,Microbiology ,Parasite Replication ,Host-Parasite Interactions ,QH301 ,03 medical and health sciences ,All institutes and research themes of the Radboud University Medical Center ,Virology ,Organelle ,Parasite Groups ,medicine ,Genetics ,Animals ,Dipeptidyl-Peptidases and Tripeptidyl-Peptidases ,Molecular Biology ,030304 developmental biology ,Organelles ,Rhoptry ,Merozoites ,QH ,Intracellular parasite ,Wild type ,DAPI staining ,Organisms ,Biology and Life Sciences ,RC581-607 ,biology.organism_classification ,Parasitic Protozoans ,QR ,Research and analysis methods ,Red blood cell ,030104 developmental biology ,Molecular biology techniques ,Genetic Loci ,Proteolysis ,Nuclear staining ,Plasmid Construction ,Parasitology ,Immunologic diseases. Allergy ,Apicomplexa ,Peptide Hydrolases - 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., Author summary Malaria remains one of the most devastating infectious diseases and its clinical manifestation is caused by the exponential multiplication of parasites in patients. This asexual replication cycle consists of red blood cell invasion, intracellular parasite multiplication, and release (also known as egress) of daughter parasites for further red blood cell invasion. Host cell invasion is therefore essential for parasite replication and the only moment in this cycle when parasites are exposed to the immune system. Understanding the molecular mechanisms that control red blood cell invasion might not only lead to the identification of novel antimalarial targets but also to the development of better invasion blocking vaccines. DPAP3 is a druggable cysteine protease that was previously believed to be essential for parasite egress. In this study, we show that parasites lacking DPAP3 activity are unable to efficiently invade red blood cells but escape the confines of the host cell normally. Overall, this study increases our understanding of the proteolytic pathways that govern host cell invasion by the malaria parasite.
- Published
- 2017
14. A Coupled Protein and Probe Engineering Approach for Selective Inhibition and Activity-Based Probe Labeling of the Caspases
- Author
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Aaron W. Puri, Montse Morell, Junpeng Xiao, Petr Broz, Denise M. Monack, Edgar Deu, and Matthew Bogyo
- Subjects
0303 health sciences ,Proteases ,Protease ,biology ,Chemistry ,Effector ,medicine.medical_treatment ,General Chemistry ,Protein engineering ,010402 general chemistry ,Caspase 8 ,01 natural sciences ,Biochemistry ,Catalysis ,0104 chemical sciences ,3. Good health ,03 medical and health sciences ,Colloid and Surface Chemistry ,Apoptosis ,medicine ,biology.protein ,Caspase ,030304 developmental biology ,Cysteine - 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.
- Published
- 2013
15. Coupling Protein Engineering with Probe Design To Inhibit and Image Matrix Metalloproteinases with Controlled Specificity
- Author
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Stephen J. Weiss, Thinh Nguyen Duc, Jason R. Jessen, Jiyoun Lee, Montse Morell, Amanda L. Willis, Yang Deng, Matthew Bogyo, Salahuddin Syed, Junpeng Xiao, Edgar Deu, and Benjamin E. Turk
- Subjects
Models, Molecular ,Proteases ,medicine.medical_treatment ,Molecular Sequence Data ,Molecular Probe Techniques ,Sequence alignment ,Matrix metalloproteinase ,Protein Engineering ,Biochemistry ,Article ,Catalysis ,Cell Line ,Mice ,Colloid and Surface Chemistry ,Matrix Metalloproteinase 12 ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Cysteine ,Zebrafish ,Protease ,Chemistry ,Optical Imaging ,General Chemistry ,Protein engineering ,Matrix Metalloproteinase 1 ,Sequence Alignment ,Function (biology) - 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).
- Published
- 2013
16. Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum
- Author
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Donald M. Bell, Arnault Graindorge, Christine R. Collins, Michael J. Blackman, Edgar Deu, James A. Thomas, Sujaan Das, and Fiona Hackett
- Subjects
0301 basic medicine ,Plasmodium ,Erythrocytes ,lcsh:Medicine ,Animal Cells ,Red Blood Cells ,Medicine and Health Sciences ,Parasite hosting ,Malaria, Falciparum ,lcsh:Science ,Pathogen ,Merozoite Surface Protein 1 ,Virus quantification ,Protozoans ,Multidisciplinary ,biology ,Malarial Parasites ,3. Good health ,Phenotype ,Cellular Types ,Research Article ,030106 microbiology ,Plasmodium falciparum ,Virulence ,Hemolytic Plaque Technique ,Research and Analysis Methods ,Parasite Replication ,03 medical and health sciences ,parasitic diseases ,Parasite Groups ,medicine ,Parasitic Diseases ,Animals ,Humans ,Parasites ,Molecular Biology Techniques ,Molecular Biology ,Life Cycle Stages ,Blood Cells ,Obligate ,Merozoites ,lcsh:R ,Organisms ,Biology and Life Sciences ,Cell Biology ,biology.organism_classification ,medicine.disease ,Tropical Diseases ,Virology ,Parasitic Protozoans ,Malaria ,030104 developmental biology ,Mutation ,lcsh:Q ,Parasitology ,Apicomplexa ,Cloning - 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.
- Published
- 2016
17. Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study
- Author
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Edgar Deu and Jack F. Kirsch
- Subjects
Stereochemistry ,Allosteric regulation ,Tyrosine Transaminase ,Protein Engineering ,medicine.disease_cause ,Biochemistry ,Article ,Tyrosine aminotransferase ,Allosteric Regulation ,Catalytic Domain ,Escherichia coli ,medicine ,Aspartate Aminotransferases ,Molecular Biology ,Transaminases ,chemistry.chemical_classification ,biology ,Binding properties ,Active site ,Protein engineering ,Enzyme ,chemistry ,biology.protein ,Protein Multimerization - 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.
- Published
- 2011
18. Functional Characterization of a SUMO Deconjugating Protease of Plasmodium falciparum Using Newly Identified Small Molecule Inhibitors
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Amy J. Campbell, Victoria E. Albrow, James C. Powers, Elizabeth L. Ponder, Junpeng Xiao, Miklós Békés, Edgar Deu, Urša Pečar Fonović, Marcin Drag, Jowita Mikolajczyk, Brittany A. Leader, Matthew Bogyo, Aimee Shen, and Guy S. Salvesen
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medicine.medical_treatment ,Molecular Sequence Data ,Plasmodium falciparum ,Clinical Biochemistry ,Phthalic Acids ,Protozoan Proteins ,SUMO protein ,SUMO enzymes ,Biochemistry ,Article ,Substrate Specificity ,03 medical and health sciences ,Transcription (biology) ,Small Ubiquitin-Related Modifier Proteins ,Catalytic Domain ,Endopeptidases ,parasitic diseases ,Drug Discovery ,medicine ,Humans ,Protease Inhibitors ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Protease ,biology ,030302 biochemistry & molecular biology ,General Medicine ,biology.organism_classification ,Small molecule ,Recombinant Proteins ,3. Good health ,Cysteine Endopeptidases ,Hydrazines ,Molecular Medicine - Abstract
SummarySmall 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.
- Published
- 2011
19. A Fragmenting Hybrid Approach for Targeted Delivery of Multiple Therapeutic Agents to the Malaria Parasite
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Edgar Deu, Erica M. W. Lauterwasser, Jonathan A. Ellman, Matthew Bogyo, Melissa J. Leyva, Sumit Mahajan, and Adam R. Renslo
- Subjects
Drug ,media_common.quotation_subject ,Iron ,malaria ,Drug resistance ,Biology ,Pharmacology ,Bioinformatics ,01 natural sciences ,Biochemistry ,Cathepsin C ,03 medical and health sciences ,Antimalarials ,Heterocyclic Compounds, 1-Ring ,prodrugs ,parasitic diseases ,Drug Discovery ,medicine ,Animals ,Protease Inhibitors ,Spiro Compounds ,General Pharmacology, Toxicology and Pharmaceutics ,Artemisinin ,030304 developmental biology ,media_common ,0303 health sciences ,Drug Carriers ,010405 organic chemistry ,Organic Chemistry ,Hybrid approach ,medicine.disease ,Antiparasitic agent ,Communications ,0104 chemical sciences ,3. Good health ,Peroxides ,hybrid drugs ,Drug delivery ,drug delivery ,Molecular Medicine ,antiparasitic agents ,Carbamates ,Drug carrier ,Malaria ,medicine.drug - Abstract
Artemisinin combination therapies (ACT) represent the current standard of care in the treatment of uncomplicated malaria. The widespread adoption of ACT has been motivated by a desire to minimize the emergence of drug resistance and to address the problem of recrudescence associated with artemisinin monotherapy.1–4 We set out to explore a single-molecule ‘fragmenting hybrid’ strategy in which an artemisinin-like peroxide is employed to deliver a partner drug, only upon activation by ferrous iron in the parasite. In principle, iron(II)-dependent drug delivery from a fragmenting hybrid could alleviate unwanted off-target bioactivities of the partner drug, which would be inactive in its hybrid form.
- Published
- 2011
20. Functional Studies of Plasmodium falciparum Dipeptidyl Aminopeptidase I Using Small Molecule Inhibitors and Active Site Probes
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Jonathan A. Ellman, Matthew Bogyo, Edgar Deu, Mark J. Rice, Melissa J. Leyva, and Victoria E. Albrow
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Drug ,MICROBIO ,media_common.quotation_subject ,Clinical Biochemistry ,Biology ,Pharmacology ,01 natural sciences ,Biochemistry ,Cathepsin C ,03 medical and health sciences ,Drug Discovery ,medicine ,Parasite hosting ,Molecular Biology ,030304 developmental biology ,media_common ,chemistry.chemical_classification ,0303 health sciences ,010405 organic chemistry ,Plasmodium falciparum ,General Medicine ,biology.organism_classification ,medicine.disease ,Small molecule ,Antiparasitic agent ,3. Good health ,0104 chemical sciences ,CHEMBIO ,Enzyme ,chemistry ,Molecular Medicine ,Malaria - Abstract
SummaryThe 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.
- Published
- 2010
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21. 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
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Edgar Deu and Jack F. Kirsch
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Protein Denaturation ,Protein Folding ,Circular dichroism ,Stereochemistry ,Protein Renaturation ,medicine.disease_cause ,Biochemistry ,Cofactor ,chemistry.chemical_compound ,Enzyme Stability ,Escherichia coli ,medicine ,Urea ,Denaturation (biochemistry) ,Aspartate Aminotransferases ,Pyridoxal phosphate ,Protein Structure, Quaternary ,Protein secondary structure ,Guanidine ,chemistry.chemical_classification ,biology ,Circular Dichroism ,Active site ,Crystallography ,Spectrometry, Fluorescence ,Enzyme ,chemistry ,Pyridoxal Phosphate ,biology.protein ,Thermodynamics ,Holoenzymes ,Pyridoxamine - 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 DMRASR in the apoenzyme to ASRDMR in PPL-eAATase) and results in a reordering of the eAATase denaturation pathway.
- Published
- 2007
22. The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: Multiple forced covariant amino acid substitutions in natural variants
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Jack F. Kirsch, Edgar Deu, and Keith A. Koch
- Subjects
Stereochemistry ,Molecular Sequence Data ,Mutant ,Kinetics ,Sequence alignment ,Biology ,medicine.disease_cause ,Models, Biological ,Biochemistry ,Article ,Substrate Specificity ,Protein structure ,medicine ,Amino Acid Sequence ,Aspartate Aminotransferases ,Protein Structure, Quaternary ,Molecular Biology ,Escherichia coli ,Peptide sequence ,Phylogeny ,chemistry.chemical_classification ,Amino acid ,Enzyme ,Amino Acid Substitution ,chemistry ,Sequence Alignment - 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
23. Plasmodium dipeptidyl aminopeptidases as malaria transmission-blocking drug targets
- Author
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Omar Ali, Kim C. Williamson, Michael J. Ashburne, Amreena Suri, Matthew Bogyo, Takeshi Tanaka, Sandhya Kortagere, Edgar Deu, and Alvaro Molina-Cruz
- Subjects
Male ,Plasmodium falciparum ,Biology ,Aminopeptidase ,Aminopeptidases ,Antimalarials ,Mice ,Transcription (biology) ,parasitic diseases ,Gametocyte ,Homologous chromosome ,medicine ,Parasite hosting ,Animals ,Pharmacology (medical) ,Experimental Therapeutics ,Pharmacology ,Infectivity ,Reverse Transcriptase Polymerase Chain Reaction ,medicine.disease ,biology.organism_classification ,Virology ,Infectious Diseases ,Female ,Malaria - 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 ( Pbdpap2 Δ) line was generated. The Pbdpap2 Δ 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 Pbdpap2 Δ clonal lines, possibly compensating for the loss of Pbdpap2 . The role of DPAP1 and -3 in the dpap2 Δ parasites was then evaluated using a DPAP inhibitor, ML4118S. When ML4118S was added to the Pbdpap2 Δ 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
24. New approaches for dissecting protease functions to improve probe development and drug discovery
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Matthew Bogyo, Martijn Verdoes, and Edgar Deu
- Subjects
Drug ,Proteomics ,Proteases ,medicine.medical_treatment ,media_common.quotation_subject ,Computational biology ,Biology ,Article ,Structural Biology ,In vivo ,Drug Discovery ,medicine ,Animals ,Humans ,Screening tool ,Protease Inhibitors ,Molecular Biology ,media_common ,Protease ,Molecular Structure ,Mechanism (biology) ,Drug discovery ,Extramural ,Biochemistry ,Models, Chemical ,Proteolysis ,Biocatalysis ,Peptide Hydrolases - Abstract
Proteases have long been considered targets for pharmaceutical development because of our deep understanding of their enzymatic mechanism and their regulatory roles in many pathologies. However, despite our ability to develop potent inhibitors, many clinical lead compounds have failed due either to a lack of specificity or a limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to first understand protease function and second, to expand the platform of inhibitor development beyond active site-directed design and in vitro optimization. Several newly developed technologies will enable much broader assessment of drug selectivity in living cells and in animal models, allowing for lead optimization to focus on compounds that show high specificity and minimal side effects in vivo. In this perspective, we highlight the current advances in the development of new chemical probes, proteomic methods, and screening tools that we feel will help facilitate this paradigm shift in drug discovery methods.
- Published
- 2012
25. Molecular mechanisms of bortezomib resistant adenocarcinoma cells
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Susan Demo, Shirin Arastu-Kapur, Jonathan J Keats, P. Leif Bergsagel, Erika Suzuki, Christopher J. Kirk, Edgar Deu, and Mark K. Bennett
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Cancer Treatment ,lcsh:Medicine ,Biochemistry ,Plasma Cell Disorders ,Bortezomib ,chemistry.chemical_compound ,0302 clinical medicine ,hemic and lymphatic diseases ,Cytotoxic T cell ,lcsh:Science ,Multiple myeloma ,0303 health sciences ,Multidisciplinary ,Hematology ,Boronic Acids ,3. Good health ,Oncology ,030220 oncology & carcinogenesis ,Pyrazines ,Medicine ,Multiple Myeloma ,medicine.drug ,Research Article ,Biotechnology ,Drugs and Devices ,Drug Research and Development ,Blotting, Western ,Antineoplastic Agents ,Enzyme-Linked Immunosorbent Assay ,Cycloheximide ,Biology ,Adenocarcinoma ,03 medical and health sciences ,Cell Line, Tumor ,Chemical Biology ,medicine ,Humans ,030304 developmental biology ,lcsh:R ,Chemotherapy and Drug Treatment ,medicine.disease ,Carfilzomib ,Molecular biology ,chemistry ,Proteasome ,Cell culture ,Small Molecules ,Drug Resistance, Neoplasm ,Proteasome inhibitor ,Cancer research ,lcsh:Q - 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.
- Published
- 2011
26. The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant
- Author
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Jack F. Kirsch and Edgar Deu
- Subjects
Protein Denaturation ,Protein Folding ,Molecular mass ,Stereochemistry ,Hydrochloride ,Circular Dichroism ,Temperature ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Monomer ,Apoenzymes ,Spectrometry, Fluorescence ,chemistry ,medicine ,Escherichia coli ,Urea ,Denaturation (biochemistry) ,Aspartate Aminotransferases ,Guanidine ,Protein Structure, Quaternary - 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
27. Inside Cover: A Fragmenting Hybrid Approach for Targeted Delivery of Multiple Therapeutic Agents to the Malaria Parasite (ChemMedChem 3/2011)
- Author
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Matthew Bogyo, Jonathan A. Ellman, Adam R. Renslo, Melissa J. Leyva, Sumit Mahajan, Edgar Deu, and Erica M. W. Lauterwasser
- Subjects
Pharmacology ,Organic Chemistry ,Biology ,Hybrid approach ,medicine.disease ,Biochemistry ,Antiparasitic agent ,Drug Discovery ,Drug delivery ,medicine ,Molecular Medicine ,Parasite hosting ,Cover (algebra) ,General Pharmacology, Toxicology and Pharmaceutics ,Malaria - Published
- 2011
28. Rational Design of Inhibitors and Activity-Based Probes Targeting Clostridium difficile Virulence Factor TcdB
- Author
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Aaron W. Puri, K. Christopher Garcia, Victoria E. Albrow, Patrick J. Lupardus, Matthew Bogyo, Aimee Shen, and Edgar Deu
- Subjects
Phytic Acid ,medicine.medical_treatment ,Bacterial Toxins ,Clinical Biochemistry ,Virulence ,Cysteine Proteinase Inhibitors ,Biology ,Crystallography, X-Ray ,Biochemistry ,Virulence factor ,Microbiology ,Structure-Activity Relationship ,Allosteric Regulation ,Bacterial Proteins ,Cysteine Proteases ,Drug Discovery ,medicine ,Computer Simulation ,Molecular Biology ,Pharmacology ,Binding Sites ,Protease ,Clostridioides difficile ,Effector ,Rational design ,General Medicine ,Clostridium difficile ,Cysteine protease ,Anti-Bacterial Agents ,Protein Structure, Tertiary ,Drug Design ,Molecular Probes ,Molecular Medicine ,Protein Binding - Abstract
SummaryClostridium 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.
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