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Rosales-Muñoz GJ, Souza-Arroyo V, Bucio-Ortiz L, Miranda-Labra RU, Gomez-Quiroz LE, Gutiérrez-Ruiz MC. Acute pancreatitis experimental models, advantages and disadvantages. J Physiol Biochem 2025:10.1007/s13105-025-01091-w. [PMID: 40380027 DOI: 10.1007/s13105-025-01091-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Accepted: 05/05/2025] [Indexed: 05/19/2025]
Abstract
Acute pancreatitis represents a severe health problem, not only because of the number of people affected but also because of the severity of its clinical presentation that can eventually lead to the death of patients. The study of the disease is complex, and we lack optimized models that can approach the clinical presentation in patients, in addition to the significant vulnerability of the organ itself. In the present work, we undertook the task of reviewing and analyzing the experimental methods most currently used for the induction of acute pancreatitis, emphasizing the advantages and disadvantages of each model and their delimitation based on experimental objectives. We aimed to provide an actual and quick-access guide for researchers interested in experimental acute pancreatitis.
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Affiliation(s)
- Genaro J Rosales-Muñoz
- Posgrado en Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico
- Departamento de Ciencias de La Salud, Área de Medicina Experimental y Traslacional, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico
| | - Verónica Souza-Arroyo
- Departamento de Ciencias de La Salud, Área de Medicina Experimental y Traslacional, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico
- Laboratorio de Medicina Experimental, Unidad de Medicina Traslacional IIB/UNAM, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
| | - Leticia Bucio-Ortiz
- Departamento de Ciencias de La Salud, Área de Medicina Experimental y Traslacional, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico
- Laboratorio de Medicina Experimental, Unidad de Medicina Traslacional IIB/UNAM, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
| | - Roxana U Miranda-Labra
- Departamento de Ciencias de La Salud, Área de Medicina Experimental y Traslacional, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico
- Laboratorio de Medicina Experimental, Unidad de Medicina Traslacional IIB/UNAM, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
| | - Luis E Gomez-Quiroz
- Departamento de Ciencias de La Salud, Área de Medicina Experimental y Traslacional, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico
- Laboratorio de Medicina Experimental, Unidad de Medicina Traslacional IIB/UNAM, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
| | - María Concepción Gutiérrez-Ruiz
- Departamento de Ciencias de La Salud, Área de Medicina Experimental y Traslacional, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico.
- Laboratorio de Medicina Experimental, Unidad de Medicina Traslacional IIB/UNAM, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico.
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2
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Gukovskaya AS, Lerch MM, Mayerle J, Sendler M, Ji B, Saluja AK, Gorelick FS, Gukovsky I. Trypsin in pancreatitis: The culprit, a mediator, or epiphenomenon? World J Gastroenterol 2024; 30:4417-4438. [PMID: 39534420 PMCID: PMC11551668 DOI: 10.3748/wjg.v30.i41.4417] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Revised: 06/19/2024] [Accepted: 07/16/2024] [Indexed: 10/23/2024] Open
Abstract
Pancreatitis is a common, life-threatening inflammatory disease of the exocrine pancreas. Its pathogenesis remains obscure, and no specific or effective treatment is available. Gallstones and alcohol excess are major etiologies of pancreatitis; in a small portion of patients the disease is hereditary. Pancreatitis is believed to be initiated by injured acinar cells (the main exocrine pancreas cell type), leading to parenchymal necrosis and local and systemic inflammation. The primary function of these cells is to produce, store, and secrete a variety of enzymes that break down all categories of nutrients. Most digestive enzymes, including all proteases, are secreted by acinar cells as inactive proforms (zymogens) and in physiological conditions are only activated when reaching the intestine. The generation of trypsin from inactive trypsinogen in the intestine plays a critical role in physiological activation of other zymogens. It was proposed that pancreatitis results from proteolytic autodigestion of the gland, mediated by premature/inappropriate trypsinogen activation within acinar cells. The intra-acinar trypsinogen activation is observed in experimental models of acute and chronic pancreatitis, and in human disease. On the basis of these observations, it has been considered the central pathogenic mechanism of pancreatitis - a concept with a century-old history. This review summarizes the data on trypsinogen activation in experimental and genetic rodent models of pancreatitis, particularly the more recent genetically engineered mouse models that mimic mutations associated with hereditary pancreatitis; analyzes the mechanisms mediating trypsinogen activation and protecting the pancreas against its' damaging effects; discusses the gaps in our knowledge, potential therapeutic approaches, and directions for future research. We conclude that trypsin is not the culprit in the disease pathogenesis but, at most, a mediator of some pancreatitis responses. Therefore, the search for effective therapies should focus on approaches to prevent or normalize other intra-acinar pathologic processes, such as defective autophagy leading to parenchymal cell death and unrelenting inflammation.
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Affiliation(s)
- Anna S Gukovskaya
- Department of Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90073, United States
- Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, United States
| | - Markus M Lerch
- Department of Medicine, Ludwig Maximilian University Hospital, Munich 81377, Germany
| | - Julia Mayerle
- Department of Medicine II, Ludwig Maximilian University of Munich, Munich 81377, Germany
| | - Matthias Sendler
- Department of Medicine A, University of Greifswald, Greifswald 17475, Germany
| | - Baoan Ji
- Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32224, United States
| | - Ashok K Saluja
- Department of Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, United States
| | - Fred S Gorelick
- Departments of Cell Biology and Internal Medicine, Yale University School of Medicine and VA West Haven, New Haven, CT 06519, United States
| | - Ilya Gukovsky
- Department of Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90073, United States
- Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, United States
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3
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Pečar Fonović U, Kos J, Mitrović A. Compensational role between cathepsins. Biochimie 2024; 226:62-76. [PMID: 38663456 DOI: 10.1016/j.biochi.2024.04.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2024] [Revised: 04/03/2024] [Accepted: 04/22/2024] [Indexed: 04/30/2024]
Abstract
Cathepsins, a family of lysosomal peptidases, play a crucial role in maintaining cellular homeostasis by regulating protein turnover and degradation as well as many specific regulatory actions that are important for proper cell function and human health. Alterations in the activity and expression of cathepsins have been observed in many diseases such as cancer, inflammation, neurodegenerative disorders, bone remodelling-related conditions and others. These changes are not exclusively harmful, but rather appear to be a compensatory response on the lack of one cathepsin in order to maintain tissue integrity. The upregulation of specific cathepsins in response to the inhibition or dysfunction of other cathepsins suggests a fine-tuned system of proteolytic balance and understanding the compensatory role of cathepsins may improve therapeutic potential of cathepsin's inhibitors. Selectively targeting one cathepsin or modulating their activity could offer new treatment strategies for a number of diseases. This review emphasises the need for comprehensive research into cathepsin biology in the context of disease. The identification of the specific cathepsins involved in compensatory responses, the elucidation of the underlying molecular mechanisms and the development of targeted interventions could lead to innovative therapeutic approaches.
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Affiliation(s)
- Urša Pečar Fonović
- Faculty of Pharmacy, University of Ljubljana, Aškerčeva Cesta 7, 1000, Ljubljana, Slovenia.
| | - Janko Kos
- Faculty of Pharmacy, University of Ljubljana, Aškerčeva Cesta 7, 1000, Ljubljana, Slovenia; Department of Biotechnology, Jožef Stefan Institute, Jamova 39, 1000, Ljubljana, Slovenia.
| | - Ana Mitrović
- Faculty of Pharmacy, University of Ljubljana, Aškerčeva Cesta 7, 1000, Ljubljana, Slovenia; Department of Biotechnology, Jožef Stefan Institute, Jamova 39, 1000, Ljubljana, Slovenia.
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4
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Saroha B, Kumar G, Arya P, Raghav N, Kumar S. Some morpholine tethered novel aurones: Design, synthesis, biological, kinetic and molecular docking studies. Bioorg Chem 2023; 140:106805. [PMID: 37634269 DOI: 10.1016/j.bioorg.2023.106805] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Revised: 08/17/2023] [Accepted: 08/21/2023] [Indexed: 08/29/2023]
Abstract
Enzymes are the biological macromolecules that have emerged as an important drug target as their upregulation/imbalance leads to various pathological conditions, such as inflammation, parasitic infection, Alzheimer's, cancer, and many others. Here, we designed and synthesized some morpholine tethered novel aurones and evaluated them as potential inhibitors for CTSB, α-amylase, lipase and activator for trypsin. All the newly synthesized compounds were fully characterized by various spectroscopic techniques (1H NMR, 13C NMR, HRMS) and the Z-configuration to them was assigned based on single crystal XRD data and 1H NMR chemical shift values. Further, the hybrids were evaluated for their intracellular (cathepsin B) and extracellular (trypsin, lipase, amylase) enzyme inhibition potencies. The in-vitro inhibition screening against cathepsin B revealed that most of the synthesized compounds are good competitive inhibitors (% inhibition = 22.91-75.04), with 6q (% inhibition = 75.04) and 6r (% inhibition = 71.13) as the eminent inhibitors of the series. At the same time, they exhibited weak to moderate inhibition towards amylase (% inhibition = 7.22-22.48) and lipase (% inhibition = 16.29-54.83). A significant trypsin activation (% activation = 107.42-196.47) was observed even at the micromolar concentration of the compounds. Furthermore, the drug-modeling studies showed a good correlation between the in-vitro experimental results and the calculated binding affinity of the screened compounds with all the tested enzymes. These findings are expected to provide a new lead in drug development for different pathological disorders wherever these enzymes are involved.
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Affiliation(s)
- Bhavna Saroha
- Department of Chemistry, Kurukshetra University, Kurukshetra, Haryana, 136119, India
| | - Gourav Kumar
- Department of Chemistry, Kurukshetra University, Kurukshetra, Haryana, 136119, India; Department of Biomedical Engineering, Oregon Health & Science University (OHSU), 2730 S Moody Ave., Portland, OR 97201
| | - Priyanka Arya
- Department of Chemistry, Kurukshetra University, Kurukshetra, Haryana, 136119, India
| | - Neera Raghav
- Department of Chemistry, Kurukshetra University, Kurukshetra, Haryana, 136119, India
| | - Suresh Kumar
- Department of Chemistry, Kurukshetra University, Kurukshetra, Haryana, 136119, India.
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Xie Z, Zhao M, Yan C, Kong W, Lan F, Zhao S, Yang Q, Bai Z, Qing H, Ni J. Cathepsin B in programmed cell death machinery: mechanisms of execution and regulatory pathways. Cell Death Dis 2023; 14:255. [PMID: 37031185 PMCID: PMC10082344 DOI: 10.1038/s41419-023-05786-0] [Citation(s) in RCA: 71] [Impact Index Per Article: 35.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Revised: 03/27/2023] [Accepted: 03/29/2023] [Indexed: 04/10/2023]
Abstract
Cathepsin B (CatB), a cysteine protease, is primarily localized within subcellular endosomal and lysosomal compartments. It is involved in the turnover of intracellular and extracellular proteins. Interest is growing in CatB due to its diverse roles in physiological and pathological processes. In functional defective tissues, programmed cell death (PCD) is one of the regulable fundamental mechanisms mediated by CatB, including apoptosis, pyroptosis, ferroptosis, necroptosis, and autophagic cell death. However, CatB-mediated PCD is responsible for disease progression under pathological conditions. In this review, we provide an overview of the critical roles and regulatory pathways of CatB in different types of PCD, and discuss the possibility of CatB as an attractive target in multiple diseases. We also summarize current gaps in the understanding of the involvement of CatB in PCD to highlight future avenues for research.
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Affiliation(s)
- Zhen Xie
- Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, 100081, Beijing, China
| | - Mengyuan Zhao
- Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, 100081, Beijing, China
| | - Chengxiang Yan
- Research Center for Resource Peptide Drugs, Shaanxi Engineering and Technological Research Center for Conversation and Utilization of Regional Biological Resources, Yan'an University, Yan'an, 716000, China
| | - Wei Kong
- Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, 100081, Beijing, China
| | - Fei Lan
- Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, 100081, Beijing, China
| | - Shuxuan Zhao
- Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, 100081, Beijing, China
| | - Qinghu Yang
- Research Center for Resource Peptide Drugs, Shaanxi Engineering and Technological Research Center for Conversation and Utilization of Regional Biological Resources, Yan'an University, Yan'an, 716000, China
| | - Zhantao Bai
- Research Center for Resource Peptide Drugs, Shaanxi Engineering and Technological Research Center for Conversation and Utilization of Regional Biological Resources, Yan'an University, Yan'an, 716000, China.
- Yan'an Key Laboratory for Neural Immuno-Tumor and Stem Cell and Engineering and Technological Research Center for Natural Peptide Drugs, Yan'an, 716000, China.
| | - Hong Qing
- Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, 100081, Beijing, China.
| | - Junjun Ni
- Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology, School of Life Science, Beijing Institute of Technology, 100081, Beijing, China.
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Zhu CS, Qiang X, Chen W, Li J, Lan X, Yang H, Gong J, Becker L, Wang P, Tracey KJ, Wang H. Identification of procathepsin L (pCTS-L)-neutralizing monoclonal antibodies to treat potentially lethal sepsis. SCIENCE ADVANCES 2023; 9:eadf4313. [PMID: 36735789 PMCID: PMC9897667 DOI: 10.1126/sciadv.adf4313] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 01/05/2023] [Indexed: 06/01/2023]
Abstract
Antibody-based strategies have been attempted to antagonize early cytokines of sepsis, but not yet been tried to target inducible late-acting mediators. Here, we report that the expression and secretion of procathepsin-L (pCTS-L) was induced by serum amyloid A (SAA) in innate immune cells, contributing to its late and systemic accumulation in experimental and clinical sepsis. Recombinant pCTS-L induced interleukin-6 (IL-6), IL-8, GRO-α/KC, GRO-β/MIP-2, and MCP-1 release in innate immune cells and moderately correlated with blood concentrations of these cytokines/chemokines in clinical sepsis. Mechanistically, pCTS-L interacted with Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) to induce cytokines/chemokines. Pharmacological suppression of pCTS-L with neutralizing polyclonal and monoclonal antibodies attenuated pCTS-L-mediated inflammation by impairing its interaction with TLR4 and RAGE receptors, and consequently rescued animals from lethal sepsis. Our findings have suggested a possibility of developing antibody strategies to prevent dysregulated immune responses mediated by late-acting cytokines.
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Affiliation(s)
- Cassie Shu Zhu
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
| | - Xiaoling Qiang
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
| | - Weiqiang Chen
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
| | - Jianhua Li
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
| | - Xiqian Lan
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
| | - Huan Yang
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
| | - Jonathan Gong
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
| | - Lance Becker
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
| | - Ping Wang
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
| | - Kevin J. Tracey
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
| | - Haichao Wang
- The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA
- Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA
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Biasizzo M, Javoršek U, Vidak E, Zarić M, Turk B. Cysteine cathepsins: A long and winding road towards clinics. Mol Aspects Med 2022; 88:101150. [PMID: 36283280 DOI: 10.1016/j.mam.2022.101150] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 10/13/2022] [Accepted: 10/14/2022] [Indexed: 12/03/2022]
Abstract
Biomedical research often focuses on properties that differentiate between diseased and healthy tissue; one of the current focuses is elevated expression and altered localisation of proteases. Among these proteases, dysregulation of cysteine cathepsins can frequently be observed in inflammation-associated diseases, which tips the functional balance from normal physiological to pathological manifestations. Their overexpression and secretion regularly exhibit a strong correlation with the development and progression of such diseases, making them attractive pharmacological targets. But beyond their mostly detrimental role in inflammation-associated diseases, cysteine cathepsins are physiologically highly important enzymes involved in various biological processes crucial for maintaining homeostasis and responding to different stimuli. Consequently, several challenges have emerged during the efforts made to translate basic research data into clinical applications. In this review, we present both physiological and pathological roles of cysteine cathepsins and discuss the clinical potential of cysteine cathepsin-targeting strategies for disease management and diagnosis.
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Affiliation(s)
- Monika Biasizzo
- Jozef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Jamova 39, SI-1000, Ljubljana, Slovenia; International Postgraduate School Jozef Stefan, Jamova 39, SI-1000, Ljubljana, Slovenia
| | - Urban Javoršek
- Jozef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Jamova 39, SI-1000, Ljubljana, Slovenia; International Postgraduate School Jozef Stefan, Jamova 39, SI-1000, Ljubljana, Slovenia
| | - Eva Vidak
- Jozef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Jamova 39, SI-1000, Ljubljana, Slovenia; International Postgraduate School Jozef Stefan, Jamova 39, SI-1000, Ljubljana, Slovenia
| | - Miki Zarić
- Jozef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Jamova 39, SI-1000, Ljubljana, Slovenia; International Postgraduate School Jozef Stefan, Jamova 39, SI-1000, Ljubljana, Slovenia
| | - Boris Turk
- Jozef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Jamova 39, SI-1000, Ljubljana, Slovenia; Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot 113, SI-1000, Ljubljana, Slovenia.
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8
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Smyth P, Sasiwachirangkul J, Williams R, Scott CJ. Cathepsin S (CTSS) activity in health and disease - A treasure trove of untapped clinical potential. Mol Aspects Med 2022; 88:101106. [PMID: 35868042 DOI: 10.1016/j.mam.2022.101106] [Citation(s) in RCA: 60] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 06/24/2022] [Accepted: 07/11/2022] [Indexed: 12/14/2022]
Abstract
Amongst the lysosomal cysteine cathepsin family of proteases, cathepsin S (CTSS) holds particular interest due to distinctive properties including a normal restricted expression profile, inducible upregulation and activity at a broad pH range. Consequently, while CTSS is well-established as a member of the proteolytic cocktail within the lysosome, degrading unwanted and damaged proteins, it has increasingly been shown to mediate a number of distinct, more selective roles including antigen processing and antigen presentation, and cleavage of substrates both intra and extracellularly. Increasingly, aberrant CTSS expression has been demonstrated in a variety of conditions and disease states, marking it out as both a biomarker and potential therapeutic target. This review seeks to contextualise CTSS within the cysteine cathepsin family before providing an overview of the broad range of pathologies in which roles for CTSS have been identified. Additionally, current clinical progress towards specific inhibitors is detailed, updating the position of the field in exploiting this most unique of proteases.
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Affiliation(s)
- Peter Smyth
- The Patrick G Johnston Centre for Cancer Research, Queen's University, 97 Lisburn Road, Belfast, BT9 7AE, UK
| | - Jutharat Sasiwachirangkul
- The Patrick G Johnston Centre for Cancer Research, Queen's University, 97 Lisburn Road, Belfast, BT9 7AE, UK
| | - Rich Williams
- The Patrick G Johnston Centre for Cancer Research, Queen's University, 97 Lisburn Road, Belfast, BT9 7AE, UK
| | - Christopher J Scott
- The Patrick G Johnston Centre for Cancer Research, Queen's University, 97 Lisburn Road, Belfast, BT9 7AE, UK.
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9
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Hagen CM, Roth E, Graf TR, Verrey F, Graf R, Gupta A, Pellegrini G, Poncet N, Camargo SMR. Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis. World J Gastroenterol 2022; 28:1024-1054. [PMID: 35431492 PMCID: PMC8968515 DOI: 10.3748/wjg.v28.i10.1024] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2021] [Revised: 10/08/2021] [Accepted: 01/26/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The expression of amino acid transporters is known to vary during acute pancreatitis (AP) except for LAT1 (slc7a5), the expression of which remains stable. LAT1 supports cell growth by importing leucine and thereby stimulates mammalian target of rapamycin (mTOR) activity, a phenomenon often observed in cancer cells. The mechanisms by which LAT1 influences physiological and pathophysiological processes and affects disease progression in the pancreas are not yet known.
AIM To evaluate the role of LAT1 in the development of and recovery from AP.
METHODS AP was induced with caerulein (cae) injections in female and male mice expressing LAT1 or after its knockout (LAT1 Cre/LoxP). The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight, assessing microscopical tissue architecture, mRNA and protein expression, protein synthesis, and enzyme activity levels, as well as by testing the recruitment of immune cells by FACS and ELISA.
RESULTS The initial injury, evaluated by measurements of plasma amylase, lipase, and trypsin activity, as well as the gene expression of dedifferentiation markers, did not differ between the groups. However, early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice. Especially in females, we observed less mTOR reactivation and dysfunctional autophagy. The later regeneration phase was clearly delayed in female LAT1 knockout mice, which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein (rbpjl) and basic helix-loop-helix family member A15 (mist1). Amylase mRNA and protein levels remained lower, and, strikingly, female LAT1 knockout mice presented signs of fibrosis lasting until day seven. In contrast, pancreas morphology had returned to normal in wild-type littermates.
CONCLUSION LAT1 supports the regeneration of acinar cells after AP. Female mice lacking LAT1 exhibited more pronounced alterations than male mice, indicating a sexual dimorphism of amino acid metabolism.
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Affiliation(s)
- Cristina M Hagen
- Institute of Physiology, University of Zurich, Zurich 8057, ZH, Switzerland
| | - Eva Roth
- Institute of Physiology, University of Zurich, Zurich 8057, ZH, Switzerland
| | - Theresia Reding Graf
- Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, Zurich University Hospital, Zurich 8091, ZH, Switzerland
| | - François Verrey
- Institute of Physiology, University of Zurich, Zurich 8057, ZH, Switzerland
| | - Rolf Graf
- Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, Zurich University Hospital, Zurich 8091, ZH, Switzerland
| | - Anurag Gupta
- Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, Zurich University Hospital, Zurich 8091, ZH, Switzerland
| | - Giovanni Pellegrini
- Institute of Veterinary Pathology, University of Zurich, Zurich 8057, ZH, Switzerland
| | - Nadège Poncet
- Institute of Physiology, University of Zurich, Zurich 8057, ZH, Switzerland
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Xia W, Lu Z, Chen W, Zhou J, Zhao Y. Excess fatty acids induce pancreatic acinar cell pyroptosis through macrophage M1 polarization. BMC Gastroenterol 2022; 22:72. [PMID: 35183119 PMCID: PMC8858517 DOI: 10.1186/s12876-022-02146-8] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/22/2021] [Accepted: 02/01/2022] [Indexed: 12/24/2022] Open
Abstract
AbstractFree fatty acid derived from hyperlipidemia contributes to the development of inflammation in the pancreas. Here we explore the molecular mechanisms of fatty acid-induced pancreatitis through cellular experiments and the construction of a mouse model of hyperlipidemic pancreatitis. We found that palmitic acid stimulation leads to M1 polarization of macrophage, which secretes cathepsin S via exosomes to pancreatic acinar cells and leads to activation of the caspase1-mediated classical pyrolysis pathway, resulting in inflammation and pancreatic tissue damage. In vivo experiments have also demonstrated that the high levels of fatty acids induced by hyperlipidaemia exacerbate the development of pancreatitis, and that cathepsin S inhibitors significantly alleviate hyperlipidemic pancreatitis. Therefore, cathepsin S may be a new target for the clinical treatment of hyperlipidemic pancreatitis.
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11
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Tu NH, Inoue K, Chen E, Anderson BM, Sawicki CM, Scheff NN, Tran HD, Kim DH, Alemu RG, Yang L, Dolan JC, Liu CZ, Janal MN, Latorre R, Jensen DD, Bunnett NW, Edgington-Mitchell LE, Schmidt BL. Cathepsin S Evokes PAR 2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models. Cancers (Basel) 2021; 13:4697. [PMID: 34572924 PMCID: PMC8466361 DOI: 10.3390/cancers13184697] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2021] [Accepted: 09/13/2021] [Indexed: 01/06/2023] Open
Abstract
Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.
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Affiliation(s)
- Nguyen Huu Tu
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Kenji Inoue
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Elyssa Chen
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Bethany M. Anderson
- Department of Biochemistry and Pharmacology, Bio21 Institute, University of Melbourne, Parkville, VIC 3052, Australia;
| | - Caroline M. Sawicki
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Nicole N. Scheff
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
- Hillman Cancer Research Center, Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15232, USA
| | - Hung D. Tran
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Dong H. Kim
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Robel G. Alemu
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Lei Yang
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - John C. Dolan
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
| | - Cheng Z. Liu
- Pathology Department, New York University (NYU) Langone Health, New York, NY 10016, USA;
| | - Malvin N. Janal
- Department of Epidemiology and Health Promotion, New York University (NYU) College of Dentistry, New York, NY 10010, USA;
| | - Rocco Latorre
- Department of Molecular Pathobiology, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (R.L.); (N.W.B.)
| | - Dane D. Jensen
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
- Department of Molecular Pathobiology, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (R.L.); (N.W.B.)
| | - Nigel W. Bunnett
- Department of Molecular Pathobiology, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (R.L.); (N.W.B.)
- Department of Neuroscience and Physiology, Neuroscience Institute, New York University (NYU) Langone Health, New York, NY 10016, USA
| | - Laura E. Edgington-Mitchell
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
- Department of Biochemistry and Pharmacology, Bio21 Institute, University of Melbourne, Parkville, VIC 3052, Australia;
- Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
| | - Brian L. Schmidt
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University (NYU) College of Dentistry, New York, NY 10010, USA; (N.H.T.); (K.I.); (E.C.); (C.M.S.); (N.N.S.); (H.D.T.); (D.H.K.); (R.G.A.); (L.Y.); (J.C.D.); (D.D.J.)
- Department of Neuroscience and Physiology, Neuroscience Institute, New York University (NYU) Langone Health, New York, NY 10016, USA
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12
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Li Y, Mei T, Han S, Han T, Sun Y, Zhang H, An F. Cathepsin B-responsive nanodrug delivery systems for precise diagnosis and targeted therapy of malignant tumors. CHINESE CHEM LETT 2020. [DOI: 10.1016/j.cclet.2020.05.027] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
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13
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Ren YC, Zhao Q, He Y, Li B, Wu Z, Dai J, Wen L, Wang X, Hu G. Legumain promotes fibrogenesis in chronic pancreatitis via activation of transforming growth factor β1. J Mol Med (Berl) 2020; 98:863-874. [PMID: 32415356 DOI: 10.1007/s00109-020-01911-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 04/01/2020] [Accepted: 04/06/2020] [Indexed: 01/09/2023]
Abstract
Chronic pancreatitis (CP) is a major risk factor for pancreatic cancer; however, little is known about the pathogenic mechanisms underlying the development of CP. Legumain (Lgmn) has been linked to some chronic inflammatory diseases. The present study investigated the role of legumain in pancreatic fibrogenesis. We induced CP in wild type C57BL6 (WT), Lgmn-deficient (Lgmn-/-), Lgmnflox/flox and Lgmnflox/flox × LysMCre mice by intraperitoneal injection of caerulein for 4 weeks. Pancreata were collected and analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and histology. Pancreatic stellate cells and macrophages were isolated and studied using immunofluorescence, gelatin zymography, and enzyme-linked immunosorbent assay. The effects of inhibition of legumain were investigated in vivo by administration of the specific legumain inhibitor, RR-11a. Legumain was found to be upregulated in the serum and pancreatic tissues of mice with caerulein-induced CP. Mice with global and macrophage-specific legumain deficiency exhibited significantly reduced development of pancreatic fibrosis compared with control mice, based on pancreas size, histology, and expression of fibrosis-associated genes. Our results indicate that legumain promotes activation of pancreatic stellate cells and increases synthesis of extracellular matrix proteins via activation of matrix metalloproteinase-2(MMP-2), which hydrolyzes the transforming growth factor-β1 (TGF-β1) precursor to form active TGF-β1. Administration of RR-11a markedly attenuated pancreatic fibrosis in mice with CP. Deficiency or inhibition of legumain significantly reduces the severity of pancreatic fibrosis by suppressing activation of the TGF-β1 precursor. Our results highlight the potential of legumain as a novel therapeutic target for CP. KEY MESSAGES: • Legumain expression was markedly upregulated in CP mice. • Deletion of legumain attenuated pancreatic fibrosis in CP mice. • Legumain promotes fibrosis via MMP-2 activation, which hydrolyzed the TGF-β1 precursor to the active form. • Legumain is a potential therapeutic target for the management of CP.
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Affiliation(s)
- Ying-Chun Ren
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China
| | - Qiuyan Zhao
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China
| | - Yan He
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China
| | - Bin Li
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China
| | - Zengkai Wu
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China
| | - Juanjuan Dai
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China
| | - Li Wen
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China
| | - Xingpeng Wang
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China.
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China.
| | - Guoyong Hu
- Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai, 200080, China.
- Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China.
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14
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Hook V, Yoon M, Mosier C, Ito G, Podvin S, Head BP, Rissman R, O'Donoghue AJ, Hook G. Cathepsin B in neurodegeneration of Alzheimer's disease, traumatic brain injury, and related brain disorders. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2020; 1868:140428. [PMID: 32305689 DOI: 10.1016/j.bbapap.2020.140428] [Citation(s) in RCA: 95] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/11/2020] [Revised: 03/31/2020] [Accepted: 04/08/2020] [Indexed: 12/21/2022]
Abstract
Investigations of Alzheimer's disease (AD), traumatic brain injury (TBI), and related brain disorders have provided extensive evidence for involvement of cathepsin B, a lysosomal cysteine protease, in mediating the behavioral deficits and neuropathology of these neurodegenerative diseases. This review integrates findings of cathepsin B regulation in clinical biomarker studies, animal model genetic and inhibitor evaluations, structural studies, and lysosomal cell biological mechanisms in AD, TBI, and related brain disorders. The results together indicate the role of cathepsin B in the behavioral deficits and neuropathology of these disorders. Lysosomal leakage occurs in AD and TBI, and related neurodegeneration, which leads to the hypothesis that cathepsin B is redistributed from the lysosome to the cytosol where it initiates cell death and inflammation processes associated with neurodegeneration. These results together implicate cathepsin B as a major contributor to these neuropathological changes and behavioral deficits. These findings support the investigation of cathepsin B as a potential drug target for therapeutic discovery and treatment of AD, TBI, and TBI-related brain disorders.
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Affiliation(s)
- Vivian Hook
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States of America; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, United States of America; Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA, United States of America.
| | - Michael Yoon
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States of America; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, United States of America
| | - Charles Mosier
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States of America
| | - Gen Ito
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States of America
| | - Sonia Podvin
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States of America
| | - Brian P Head
- VA San Diego Healthcare System, La Jolla, CA, United States of America; Department of Anesthesia, University of California San Diego, La Jolla, CA, United States of America
| | - Robert Rissman
- Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA, United States of America; VA San Diego Healthcare System, La Jolla, CA, United States of America
| | - Anthony J O'Donoghue
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, United States of America
| | - Gregory Hook
- American Life Sciences Pharmaceuticals, Inc., La Jolla, CA, United States of America
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15
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Walker E, Liu Y, Kim I, Biro M, Iyer SR, Ezaldein H, Scott J, Merati M, Mistur R, Zhou B, Straight B, Yim JJ, Bogyo M, Mann M, Wilson DL, Basilion JP, Popkin DL. A Protease-Activated Fluorescent Probe Allows Rapid Visualization of Keratinocyte Carcinoma during Excision. Cancer Res 2020; 80:2045-2055. [PMID: 32132111 DOI: 10.1158/0008-5472.can-19-3067] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Revised: 01/10/2020] [Accepted: 02/24/2020] [Indexed: 12/26/2022]
Abstract
Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.
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Affiliation(s)
- Ethan Walker
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio
| | - Yiqiao Liu
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio
| | - InYoung Kim
- Department of Dermatology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio.,Department of Dermatology, Case Western Reserve University, Cleveland, Ohio
| | - Mark Biro
- Case Western Reserve University School of Medicine, Cleveland, Ohio
| | - Sukanya Raj Iyer
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio
| | - Harib Ezaldein
- Department of Dermatology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio.,Department of Dermatology, Case Western Reserve University, Cleveland, Ohio
| | - Jeffrey Scott
- Department of Dermatology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio.,Department of Dermatology, Case Western Reserve University, Cleveland, Ohio
| | - Miesha Merati
- Department of Dermatology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio.,Department of Dermatology, Case Western Reserve University, Cleveland, Ohio
| | - Rachel Mistur
- Department of Dermatology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio.,Department of Dermatology, Case Western Reserve University, Cleveland, Ohio
| | - Bo Zhou
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio
| | | | - Joshua J Yim
- Chemical and Systems Biology, Stanford University, Palo Alto, California
| | - Matthew Bogyo
- Chemical and Systems Biology, Stanford University, Palo Alto, California.,Department of Pathology, Stanford University, Palo Alto, California.,Department of Microbiology and Immunology, Stanford University, Palo Alto, California
| | - Margaret Mann
- Department of Dermatology, Case Western Reserve University, Cleveland, Ohio
| | - David L Wilson
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio.,Department of Radiology, Case Western Reserve University, Cleveland, Ohio
| | - James P Basilion
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio. .,Department of Radiology, Case Western Reserve University, Cleveland, Ohio.,Fellow, National Foundation for Cancer Research, Case Western Reserve University, Cleveland, Ohio
| | - Daniel L Popkin
- Department of Dermatology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio. .,Department of Dermatology, Case Western Reserve University, Cleveland, Ohio
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16
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Sun Z, Li L, Qu J, Li H, Chen H. Proteomic analysis of therapeutic effects of Qingyi pellet on rodent severe acute pancreatitis-associated lung injury. Biomed Pharmacother 2019; 118:109300. [DOI: 10.1016/j.biopha.2019.109300] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2019] [Revised: 07/25/2019] [Accepted: 07/31/2019] [Indexed: 12/13/2022] Open
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17
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Kaphalia BS. Early Biomarkers of Acute and Chronic Pancreatitis. BIOMARKERS IN TOXICOLOGY 2019:341-353. [DOI: 10.1016/b978-0-12-814655-2.00019-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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18
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Quilles Junior JC, Carlos FDRR, Montanari A, Leitão A, Mignone VW, Arruda MA, Turyanska L, Bradshaw TD. Apoferritin encapsulation of cysteine protease inhibitors for cathepsin L inhibition in cancer cells. RSC Adv 2019; 9:36699-36706. [PMID: 35539052 PMCID: PMC9075514 DOI: 10.1039/c9ra07161j] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Accepted: 11/04/2019] [Indexed: 11/21/2022] Open
Abstract
Cysteine proteases play a key role in tumorigenesis causing protein degradation and promoting invasive tumour growth. Cathepsin L is overexpressed in cancer cells and could provide a specific target for delivery of anticancer agents. We encapsulated novel dipeptidyl nitrile based cysteine protease inhibitors (Neq0551, Neq0554 and Neq0568) into biocompatible apoferritin (AFt) protein nanocages to achieve specific delivery to tumours and pH-induced drug release. AFt-encapsulated Neq0554 demonstrated ∼3-fold enhanced in vitro activity (GI50 = 79 μM) compared to naked agent against MiaPaCa-2 pancreatic carcinoma cells. Selectivity for cancer cells was confirmed by comparing their activity to non-tumourigenic human fibroblasts (GI50 > 200 μM). Transferrin receptor (TfR-1) expression, detected only in lysates prepared from carcinoma cells, may contribute to the cancer-selectivity. The G1 cell cycle arrest caused by AFt-Neq0554 resulting in cytostasis was corroborated by clonogenic assays. Superior and more persistent inhibition of cathepsin L up to 80% was achieved with AFt-encapsulated agent in HCT-116 cells following 6 h exposure to 50 μM agent. The selective anticancer activity of AFt-encapsulated cysteine protease inhibitor Neq0554 reported here warrants further preclinical in vivo evaluation. Novel apoferritin encapsulated cysteine protease inhibitors are developed with enhanced and selective uptake by cancer cells, and sustained pH-induced release of the agent. The persistent inhibition of cathepsin L is demonstrated in vitro.![]()
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Affiliation(s)
- José C. Quilles Junior
- Centre for Biomolecular Sciences
- School of Pharmacy
- University of Nottingham
- UK
- Medicinal Chemistry Group (NEQUIMED)
| | | | - A. Montanari
- Medicinal Chemistry Group (NEQUIMED)
- São Carlos Institute of Chemistry (IQSC)
- University of São Paulo
- Brazil
| | - Andrei Leitão
- Medicinal Chemistry Group (NEQUIMED)
- São Carlos Institute of Chemistry (IQSC)
- University of São Paulo
- Brazil
| | | | | | | | - Tracey D. Bradshaw
- Centre for Biomolecular Sciences
- School of Pharmacy
- University of Nottingham
- UK
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19
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P2X7R Blockade Prevents NLRP3 Inflammasome Activation and Pancreatic Fibrosis in a Mouse Model of Chronic Pancreatitis. Pancreas 2017; 46:1327-1335. [PMID: 28930866 DOI: 10.1097/mpa.0000000000000928] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
OBJECTIVES The aim of this study was to investigate the role of P2X7R (purinergic 2X7 receptor) and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome activation in the process of pancreatic fibrosis in a mouse model of chronic pancreatitis (CP). METHODS Chronic pancreatitis was induced by repeated intraperitoneal injections of 50 μg/kg cerulein for 6 weeks in mice. P2X7R antagonist oxidized ATP (OxATP) or brilliant blue G (BBG) was administered after the last cerulein injection for 2 weeks. Pancreatic chronic inflammation and fibrosis were evaluated by histological score, Sirius red staining, and alpha-smooth muscle actin immunohistochemical staining. We further determined pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, interleukin 1β (IL-1β), and IL-18. RESULTS The pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, IL-1β, and IL-18 were all reduced significantly in both the OxATP and BBG groups (P < 0.05). The pancreatic chronic inflammation and the fibrosis indices were all remarkably attenuated (P < 0.05). CONCLUSIONS P2X7R antagonist OxATP and BBG significantly decreased pancreatic chronic inflammation and fibrosis in a mouse CP model and suggested that blockade of P2X7R-NLRP3 inflammasome signaling pathway may represent a novel therapeutic strategy for CP and its fibrotic process.
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20
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Deu E. Proteases as antimalarial targets: strategies for genetic, chemical, and therapeutic validation. FEBS J 2017; 284:2604-2628. [PMID: 28599096 PMCID: PMC5575534 DOI: 10.1111/febs.14130] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2017] [Revised: 04/29/2017] [Accepted: 06/06/2017] [Indexed: 01/17/2023]
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.
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Affiliation(s)
- Edgar Deu
- Chemical Biology Approaches to Malaria LaboratoryThe Francis Crick InstituteLondonUK
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21
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Edgington-Mitchell LE, Barlow N, Aurelio L, Samha A, Szabo M, Graham B, Bunnett N. Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation. Bioorg Med Chem Lett 2017; 27:254-260. [PMID: 27923620 PMCID: PMC10069441 DOI: 10.1016/j.bmcl.2016.11.064] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2016] [Revised: 11/21/2016] [Accepted: 11/22/2016] [Indexed: 12/29/2022]
Abstract
Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.
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Affiliation(s)
| | - Nicholas Barlow
- Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
| | - Luigi Aurelio
- Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
| | - Aminath Samha
- Drug Discovery Biology, Monash University, Parkville, VIC, Australia
| | - Monika Szabo
- Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
| | - Bim Graham
- Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
| | - Nigel Bunnett
- Drug Discovery Biology, Monash University, Parkville, VIC, Australia; Department of Pharmacology and Therapeutics, University of Melbourne, Parkville, VIC, Australia.
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22
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Live Cell Imaging and Profiling of Cysteine Cathepsin Activity Using a Quenched Activity-Based Probe. Methods Mol Biol 2017; 1491:145-159. [PMID: 27778287 DOI: 10.1007/978-1-4939-6439-0_11] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Since protease activity is highly regulated by structural and environmental influences, the abundance of a protease often does not directly correlate with its activity. Because in most of the cases it is the activity of a protease that gives rise to its biological relevance, tools to report on this activity are of great value to the research community. Activity-based probes (ABPs) are small molecule tools that allow for the monitoring and profiling of protease activities in complex biological systems. The class of fluorescent quenched ABPs (qABPs), being intrinsically "dark" and only emitting fluorescence after reaction with the target protease, are ideally suited for imaging techniques such as small animal noninvasive fluorescence imaging and live cell fluorescence microscopy. An additional powerful characteristic of qABPs is their covalent and irreversible modification of the labeled protease, enabling in-depth target characterization. Here we describe the synthesis of a pan-cysteine cathepsin qABP BMV109 and the application of this probe to live cell fluorescence imaging and fluorescent SDS-PAGE cysteine cathepsin activity profiling.
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23
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O'Donoghue AJ, Ivry SL, Chaudhury C, Hostetter DR, Hanahan D, Craik CS. Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors. Biol Chem 2016; 397:871-81. [PMID: 27149201 PMCID: PMC5712230 DOI: 10.1515/hsz-2016-0138] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2016] [Accepted: 04/20/2016] [Indexed: 12/31/2022]
Abstract
The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.
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24
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Edgington-Mitchell LE, Wartmann T, Fleming AK, Gocheva V, van der Linden WA, Withana NP, Verdoes M, Aurelio L, Edgington-Mitchell D, Lieu T, Parker BS, Graham B, Reinheckel T, Furness JB, Joyce JA, Storz P, Halangk W, Bogyo M, Bunnett NW. Legumain is activated in macrophages during pancreatitis. Am J Physiol Gastrointest Liver Physiol 2016; 311:G548-60. [PMID: 27514475 PMCID: PMC5075999 DOI: 10.1152/ajpgi.00047.2016] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Accepted: 07/18/2016] [Indexed: 01/31/2023]
Abstract
Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.
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Affiliation(s)
| | - Thomas Wartmann
- Department of Surgery, Division of Experimental Surgery, Otto-von-Guericke University, Magdeburg, Germany
| | - Alicia K Fleming
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, Florida
| | - Vasilena Gocheva
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York
| | | | - Nimali P Withana
- Department of Pathology, Stanford University School of Medicine, Stanford, California
| | - Martijn Verdoes
- Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud UMC, Nijmegen, The Netherlands
| | - Luigi Aurelio
- Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
| | - Daniel Edgington-Mitchell
- Laboratory for Turbulence Research in Aerospace and Combustion, Department of Mechanical and Aerospace Engineering, Monash University, Melbourne, Victoria, Australia
| | - TinaMarie Lieu
- Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
| | - Belinda S Parker
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
| | - Bim Graham
- Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
| | - Thomas Reinheckel
- Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - John B Furness
- Department of Anatomy and Neuroscience, University of Melbourne and Florey Institute of Neuroscience and Mental Health, Parkville, Victoria Australia
| | - Johanna A Joyce
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York
| | - Peter Storz
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, Florida
| | - Walter Halangk
- Department of Surgery, Division of Experimental Surgery, Otto-von-Guericke University, Magdeburg, Germany
| | - Matthew Bogyo
- Department of Pathology, Stanford University School of Medicine, Stanford, California
| | - Nigel W Bunnett
- Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia; Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia; and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria, Australia
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Edgington-Mitchell LE. Pathophysiological roles of proteases in gastrointestinal disease. Am J Physiol Gastrointest Liver Physiol 2016; 310:G234-9. [PMID: 26702140 DOI: 10.1152/ajpgi.00393.2015] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/05/2015] [Accepted: 12/21/2015] [Indexed: 01/31/2023]
Abstract
Gastrointestinal diseases, such as irritable bowel syndrome, inflammatory bowel disease, and colorectal cancer, affect a large proportion of the population and are associated with many unpleasant symptoms. Although the causes of these diseases remain largely unknown, there is increasing evidence to suggest that dysregulated protease activity may be a contributing factor. Proteases are enzymes that cleave other proteins, and their activity is normally very tightly regulated. During disease, however, the balance between proteases and their inhibitors is often shifted, leading to altered spatial and temporal control of substrate cleavage. Evaluating protease levels in normal physiology and disease has relied heavily on the use of chemical tools. Although these tools have greatly advanced the field, they are not without caveats. This review provides an introduction to these tools, their application in the gut, and a summary of the current knowledge on the contribution of protease activity to gastrointestinal disease.
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26
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Withana NP, Ma X, McGuire HM, Verdoes M, van der Linden WA, Ofori LO, Zhang R, Li H, Sanman LE, Wei K, Yao S, Wu P, Li F, Huang H, Xu Z, Wolters PJ, Rosen GD, Collard HR, Zhu Z, Cheng Z, Bogyo M. Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes. Sci Rep 2016; 6:19755. [PMID: 26797565 DOI: 10.1038/srep19755.sci] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Accepted: 12/17/2015] [Indexed: 08/02/2024] Open
Abstract
Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.
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Affiliation(s)
- Nimali P Withana
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Xiaowei Ma
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Helen M McGuire
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Martijn Verdoes
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | | | - Leslie O Ofori
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Ruiping Zhang
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Hao Li
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Laura E Sanman
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Ke Wei
- Department of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Shaobo Yao
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Peilin Wu
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Fang Li
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Hui Huang
- Respiratory Disease, Peking Union Medical College Hospital, Chinese Academy of Medical Science &Peking Union Medical College, Beijing, 100730, China
| | - Zuojun Xu
- Respiratory Disease, Peking Union Medical College Hospital, Chinese Academy of Medical Science &Peking Union Medical College, Beijing, 100730, China
| | - Paul J Wolters
- Department of Medicine, University of California San Francisco, San Francisco, CA 94143 USA
| | - Glenn D Rosen
- Department of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Harold R Collard
- Department of Medicine, University of California San Francisco, San Francisco, CA 94143 USA
| | - Zhaohui Zhu
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Zhen Cheng
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Matthew Bogyo
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305 USA
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27
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Withana NP, Ma X, McGuire HM, Verdoes M, van der Linden WA, Ofori LO, Zhang R, Li H, Sanman LE, Wei K, Yao S, Wu P, Li F, Huang H, Xu Z, Wolters PJ, Rosen GD, Collard HR, Zhu Z, Cheng Z, Bogyo M. Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes. Sci Rep 2016; 6:19755. [PMID: 26797565 PMCID: PMC4726431 DOI: 10.1038/srep19755] [Citation(s) in RCA: 83] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Accepted: 12/17/2015] [Indexed: 12/19/2022] Open
Abstract
Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.
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Affiliation(s)
- Nimali P. Withana
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Xiaowei Ma
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Helen M. McGuire
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Martijn Verdoes
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | | | - Leslie O. Ofori
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Ruiping Zhang
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Hao Li
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Laura E. Sanman
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Ke Wei
- Department of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Shaobo Yao
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Peilin Wu
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Fang Li
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Hui Huang
- Respiratory Disease, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, 100730, China
| | - Zuojun Xu
- Respiratory Disease, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, 100730, China
| | - Paul J. Wolters
- Department of Medicine, University of California San Francisco, San Francisco, CA 94143 USA
| | - Glenn D. Rosen
- Department of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Harold R. Collard
- Department of Medicine, University of California San Francisco, San Francisco, CA 94143 USA
| | - Zhaohui Zhu
- Department of Nuclear Medicine, Beijing, 100730, China
| | - Zhen Cheng
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Matthew Bogyo
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
- Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 USA
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305 USA
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Hook G, Jacobsen JS, Grabstein K, Kindy M, Hook V. Cathepsin B is a New Drug Target for Traumatic Brain Injury Therapeutics: Evidence for E64d as a Promising Lead Drug Candidate. Front Neurol 2015; 6:178. [PMID: 26388830 PMCID: PMC4557097 DOI: 10.3389/fneur.2015.00178] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2015] [Accepted: 07/31/2015] [Indexed: 12/22/2022] Open
Abstract
There is currently no therapeutic drug treatment for traumatic brain injury (TBI) despite decades of experimental clinical trials. This may be because the mechanistic pathways for improving TBI outcomes have yet to be identified and exploited. As such, there remains a need to seek out new molecular targets and their drug candidates to find new treatments for TBI. This review presents supporting evidence for cathepsin B, a cysteine protease, as a potentially important drug target for TBI. Cathepsin B expression is greatly up-regulated in TBI animal models, as well as in trauma patients. Importantly, knockout of the cathepsin B gene in TBI mice results in substantial improvements of TBI-caused deficits in behavior, pathology, and biomarkers, as well as improvements in related injury models. During the process of TBI-induced injury, cathepsin B likely escapes the lysosome, its normal subcellular location, into the cytoplasm or extracellular matrix (ECM) where the unleashed proteolytic power causes destruction via necrotic, apoptotic, autophagic, and activated glia-induced cell death, together with ECM breakdown and inflammation. Significantly, chemical inhibitors of cathepsin B are effective for improving deficits in TBI and related injuries including ischemia, cerebral bleeding, cerebral aneurysm, edema, pain, infection, rheumatoid arthritis, epilepsy, Huntington's disease, multiple sclerosis, and Alzheimer's disease. The inhibitor E64d is unique among cathepsin B inhibitors in being the only compound to have demonstrated oral efficacy in a TBI model and prior safe use in man and as such it is an excellent tool compound for preclinical testing and clinical compound development. These data support the conclusion that drug development of cathepsin B inhibitors for TBI treatment should be accelerated.
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Affiliation(s)
- Gregory Hook
- American Life Science Pharmaceuticals, Inc. , San Diego, CA , USA
| | | | - Kenneth Grabstein
- Department of Chemical Engineering, University of Washington , Seattle, WA , USA
| | - Mark Kindy
- Department of Neurosciences, Medical University of South Carolina , Charleston, SC , USA ; Ralph H. Johnson Veterans Administration Medical Center , Charleston, SC , USA
| | - Vivian Hook
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego , La Jolla, CA , USA ; Department of Neurosciences, Department of Pharmacology, University of California San Diego , La Jolla, CA , USA
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29
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Fukuo Y, Yamashina S, Sonoue H, Arakawa A, Nakadera E, Aoyama T, Uchiyama A, Kon K, Ikejima K, Watanabe S. Abnormality of autophagic function and cathepsin expression in the liver from patients with non-alcoholic fatty liver disease. Hepatol Res 2014; 44:1026-36. [PMID: 24299564 DOI: 10.1111/hepr.12282] [Citation(s) in RCA: 139] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/11/2013] [Revised: 11/25/2013] [Accepted: 11/26/2013] [Indexed: 02/08/2023]
Abstract
AIM Recent evidences indicate that hepatic steatosis suppresses autophagic proteolysis. The present study evaluated the correlation between autophagic function and cathepsin expression in the liver from patients with non-alcoholic fatty liver disease (NAFLD). METHODS Liver biopsy specimens were obtained from patients with chronic liver diseases (chronic hepatitis C [CHC; n = 20], chronic hepatitis B [CHB; n = 16], primary biliary cirrhosis [PBC; n = 23], NAFLD [n = 22] and control [n = 14]). The number of autophagic vesicles in hepatocytes was counted by using transmission electron microscopy. Expression of cathepsin B, D, L and p62 in the liver section was analyzed by immunohistochemical staining. The histological severity of NAFLD is assessed by NAFLD activity score (NAS). RESULTS The number of autophagic vesicles in hepatocytes was significantly increased in both CHC and NAFLD groups, but not CHB and PBC, more than control. Although hepatocytes with aggregation of p62 were observed in less than 15% of CHC, p62 aggregation was detected in approximately 65% of NAFLD. Cathepsin B, D and L expression was significantly suppressed in the liver from NAFLD patients. Suppression of cathepsin B, D and L expression was not observed in CHB, CHC and PBC. In NAFLD patients, p62 aggregation was correlated with serum alanine aminotransferase value and inflammatory activity by NAS. CONCLUSION These results indicate that a decrease in hepatic cathepsin expression in NAFLD is associated with autophagic dysfunction. Hepatic inflammation correlates with autophagic dysfunction in NAFLD. These findings indicate that the suppression of autophagic proteolysis by hepatic steatosis is involved in the pathogenesis of NAFLD.
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Affiliation(s)
- Yuka Fukuo
- Department of Gastroenterology, Juntendo University School of Medicine, Tokyo, Japan
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McComb S, Shutinoski B, Thurston S, Cessford E, Kumar K, Sad S. Cathepsins limit macrophage necroptosis through cleavage of Rip1 kinase. THE JOURNAL OF IMMUNOLOGY 2014; 192:5671-8. [PMID: 24799565 DOI: 10.4049/jimmunol.1303380] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells.
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Affiliation(s)
- Scott McComb
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada
| | - Bojan Shutinoski
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada
| | - Susan Thurston
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada
| | - Erin Cessford
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada
| | - Kriti Kumar
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada
| | - Subash Sad
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada
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32
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Lee GM, Balouch E, Goetz DH, Lazic A, McKerrow JH, Craik CS. Mapping inhibitor binding modes on an active cysteine protease via nuclear magnetic resonance spectroscopy. Biochemistry 2012. [PMID: 23181936 DOI: 10.1021/bi301305k] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.
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Affiliation(s)
- Gregory M Lee
- Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2280, USA
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