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Rumer I, Tóth L, Wohlert A, Adorján A, Jerzsele Á, Lange RW, Steinmetzer T, Gere-Pászti E. Repurposing of Furin Inhibitors to Reduce Pathogenic E. coli- and Shigella flexneri-Induced Cytotoxicity, Oxidative Stress and Inflammation in Mammalian Epithelial Cells. Antibiotics (Basel) 2025; 14:431. [PMID: 40426498 PMCID: PMC12108508 DOI: 10.3390/antibiotics14050431] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2025] [Revised: 04/11/2025] [Accepted: 04/18/2025] [Indexed: 05/29/2025] Open
Abstract
BACKGROUND/OBJECTIVES Enterobacteriaceae, including pathogenic Shigella (S.) flexneri and Escherichia (E.) coli, cause severe gastrointestinal infections through toxins like Shiga and Shiga-like toxins. Antibiotic use is often discouraged due to its potential to increase toxin effects or contribute to the development of resistance. The host protease furin is capable of activating several viral glycoproteins and bacterial toxins, thus enhancing pathogen infectivity. METHODS To assess the therapeutic potential of furin inhibitors, cultured epithelial cell models (IPEC-J2 and MDCK) were used. The effects of MI-1851 and MI-2415 were evaluated after short-term (2 h) and long-term (6 h) exposure to S. flexneri, enterohemorrhagic E. coli (EHEC), and enteropathogenic E. coli (EPEC). Cytotoxicity was determined using the CCK-8 assay, and the inflammatory response was assessed by measuring interleukin (IL)-6 and IL-8 levels. Additionally, extracellular hydrogen peroxide production was monitored in IPEC-J2 cells to evaluate the potential alterations in redox status. RESULTS Infections with EHEC, EPEC, and S. flexneri significantly reduced the viability of epithelial cells after 6 h of incubation. Furin inhibitors MI-1851 and MI-2415 decreased cytotoxicity and compensated for IL-6 and IL-8 overproduction in cells during infection with EHEC and S. flexneri, but not in cells exposed to EPEC. In addition, they alleviated oxidative stress, particularly during S. flexneri addition. CONCLUSIONS The development of new antimicrobial drugs that act via alternative mechanisms and effectively manage life-threatening enterobacterial infections is of key importance. Targeting furin with inhibitors MI-1851 and MI-2415, thus blocking toxin activation, could prevent the development of antimicrobial resistance, reduce the need for antibiotics and enhance overall treatment outcomes.
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Affiliation(s)
- Isabella Rumer
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
| | - Lilla Tóth
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
| | - Annelie Wohlert
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
| | - András Adorján
- Department of Microbiology and Infectious Diseases, University of Veterinary Medicine, Hungária krt. 23-25, H-1143 Budapest, Hungary
- National Laboratory of Infectious Animal Diseases, Antimicrobial Resistance, Veterinary Public Health and Food Chain Safety, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
| | - Ákos Jerzsele
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
- National Laboratory of Infectious Animal Diseases, Antimicrobial Resistance, Veterinary Public Health and Food Chain Safety, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
| | - Roman W. Lange
- Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg, Germany
| | - Torsten Steinmetzer
- Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg, Germany
| | - Erzsébet Gere-Pászti
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
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Schaale D, Laspa Z, Balmes A, Sigle M, Dicenta-Baunach V, Hochuli R, Fu X, Serafimov K, Castor T, Harm T, Müller KAL, Rohlfing AK, Laufer S, Schäffer TE, Lämmerhofer M, Gawaz M. Hemin promotes platelet activation and plasma membrane disintegration regulated by the subtilisin-like proprotein convertase furin. FASEB J 2024; 38:e70155. [PMID: 39530531 DOI: 10.1096/fj.202400863rr] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2024] [Revised: 10/14/2024] [Accepted: 10/23/2024] [Indexed: 11/16/2024]
Abstract
Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the in vitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, ex vivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis.
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Affiliation(s)
- David Schaale
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Zoi Laspa
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Aylin Balmes
- Institute of Applied Physics, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Manuel Sigle
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Valerie Dicenta-Baunach
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Ravi Hochuli
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Xiaoqing Fu
- Institute of Pharmaceutical Sciences, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Kristian Serafimov
- Institute of Pharmaceutical Sciences, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Tatsiana Castor
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Tobias Harm
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Karin Anne Lydia Müller
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Anne-Katrin Rohlfing
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Stefan Laufer
- Institute of Pharmaceutical Sciences, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Tilman E Schäffer
- Institute of Applied Physics, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Michael Lämmerhofer
- Institute of Pharmaceutical Sciences, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Meinrad Gawaz
- Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University of Tübingen, Tübingen, Germany
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Maluck S, Bobrovsky R, Poór M, Lange RW, Steinmetzer T, Jerzsele Á, Adorján A, Bajusz D, Rácz A, Pászti-Gere E. In Vitro Evaluation of Antipseudomonal Activity and Safety Profile of Peptidomimetic Furin Inhibitors. Biomedicines 2024; 12:2075. [PMID: 39335588 PMCID: PMC11444200 DOI: 10.3390/biomedicines12092075] [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: 08/09/2024] [Revised: 09/06/2024] [Accepted: 09/10/2024] [Indexed: 09/30/2024] Open
Abstract
Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α1-acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin-Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4.
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Affiliation(s)
- Sara Maluck
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, Hungary István utca 2, H-1078 Budapest, Hungary
| | - Rivka Bobrovsky
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, Hungary István utca 2, H-1078 Budapest, Hungary
| | - Miklós Poór
- Department of Laboratory Medicine, Medical School, University of Pécs, Ifjúság útja 13, H-7624 Pécs, Hungary
- Molecular Medicine Research Group, János Szentágothai Research Centre, University of Pécs, Ifjúság útja 20, H-7624 Pécs, Hungary
| | - Roman W Lange
- Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg, Germany
| | - Torsten Steinmetzer
- Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg, Germany
| | - Ákos Jerzsele
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, Hungary István utca 2, H-1078 Budapest, Hungary
- National Laboratory of Infectious Animal Diseases, Antimicrobial Resistance, Veterinary Public Health and Food Chain Safety, University of Veterinary Medicine, István utca 2, H-1078 Budapest, Hungary
| | - András Adorján
- Department of Microbiology and Infectious Diseases, University of Veterinary Medicine, Hungária krt. 23-25, H-1143 Budapest, Hungary
| | - Dávid Bajusz
- Medicinal Chemistry Research Group and Drug Innovation Centre, HUN-REN Research Centre for Natural Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary
| | - Anita Rácz
- Plasma Chemistry Research Group, HUN-REN Research Centre for Natural Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary
| | - Erzsébet Pászti-Gere
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, Hungary István utca 2, H-1078 Budapest, Hungary
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Ivachtchenko AV, Khvat AV, Shkil DO. Development and Prospects of Furin Inhibitors for Therapeutic Applications. Int J Mol Sci 2024; 25:9199. [PMID: 39273149 PMCID: PMC11394684 DOI: 10.3390/ijms25179199] [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: 07/22/2024] [Revised: 08/17/2024] [Accepted: 08/22/2024] [Indexed: 09/15/2024] Open
Abstract
Furin, a serine protease enzyme located in the Golgi apparatus of animal cells, plays a crucial role in cleaving precursor proteins into their mature, active forms. It is ubiquitously expressed across various tissues, including the brain, lungs, gastrointestinal tract, liver, pancreas, and reproductive organs. Since its discovery in 1990, furin has been recognized as a significant therapeutic target, leading to the active development of furin inhibitors for potential use in antiviral, antibacterial, anticancer, and other therapeutic applications. This review provides a comprehensive overview of the progress in the development and characterization of furin inhibitors, encompassing peptides, linear and macrocyclic peptidomimetics, and non-peptide compounds, highlighting their potential in the treatment of both infectious and non-infectious diseases.
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Li Y, Lu SM, Wang JL, Yao HP, Liang LG. Progress in SARS-CoV-2, diagnostic and clinical treatment of COVID-19. Heliyon 2024; 10:e33179. [PMID: 39021908 PMCID: PMC11253070 DOI: 10.1016/j.heliyon.2024.e33179] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Revised: 06/13/2024] [Accepted: 06/15/2024] [Indexed: 07/20/2024] Open
Abstract
Background Corona Virus Disease 2019(COVID-19)is a global pandemic novel coronavirus infection disease caused by Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Although rapid, large-scale testing plays an important role in patient management and slowing the spread of the disease. However, there has been no good and widely used drug treatment for infection and transmission of SARS-CoV-2. Key findings Therefore, this review updates the body of knowledge on viral structure, infection routes, detection methods, and clinical treatment, with the aim of responding to the large-section caused by SARS-CoV-2. This paper focuses on the structure of SARS-CoV-2 viral protease, RNA polymerase, serine protease and main proteinase-like protease as well as targeted antiviral drugs. Conclusion In vitro or clinical trials have been carried out to provide deeper thinking for the pathogenesis, clinical diagnosis, vaccine development and treatment of SARS-CoV-2.
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Affiliation(s)
- Yang Li
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Si-Ming Lu
- Department of Laboratory Medicine, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Zhejiang Key Laboratory of Clinical in Vitro Diagnostic Techniques, Hangzhou, China
- Institute of Laboratory Medicine, Zhejiang University, Hangzhou, China
| | - Jia-Long Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Hang-Ping Yao
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Li-Guo Liang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Centre for Clinical Laboratory, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
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Zhang Y, Gao X, Bai X, Yao S, Chang YZ, Gao G. The emerging role of furin in neurodegenerative and neuropsychiatric diseases. Transl Neurodegener 2022; 11:39. [PMID: 35996194 PMCID: PMC9395820 DOI: 10.1186/s40035-022-00313-1] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 08/10/2022] [Indexed: 12/02/2022] Open
Abstract
Furin is an important mammalian proprotein convertase that catalyzes the proteolytic maturation of a variety of prohormones and proproteins in the secretory pathway. In the brain, the substrates of furin include the proproteins of growth factors, receptors and enzymes. Emerging evidence, such as reduced FURIN mRNA expression in the brains of Alzheimer's disease patients or schizophrenia patients, has implicated a crucial role of furin in the pathophysiology of neurodegenerative and neuropsychiatric diseases. Currently, compared to cancer and infectious diseases, the aberrant expression of furin and its pharmaceutical potentials in neurological diseases remain poorly understood. In this article, we provide an overview on the physiological roles of furin and its substrates in the brain, summarize the deregulation of furin expression and its effects in neurodegenerative and neuropsychiatric disorders, and discuss the implications and current approaches that target furin for therapeutic interventions. This review may expedite future studies to clarify the molecular mechanisms of furin deregulation and involvement in the pathogenesis of neurodegenerative and neuropsychiatric diseases, and to develop new diagnosis and treatment strategies for these diseases.
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Affiliation(s)
- Yi Zhang
- Ministry of Education Key Laboratory of Molecular and Cellular Biology, Laboratory of Molecular Iron Metabolism, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China
| | - Xiaoqin Gao
- Shijiazhuang People's Hospital, Hebei Medical University, Shijiazhuang, 050027, China
| | - Xue Bai
- Ministry of Education Key Laboratory of Molecular and Cellular Biology, Laboratory of Molecular Iron Metabolism, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China
| | - Shanshan Yao
- Ministry of Education Key Laboratory of Molecular and Cellular Biology, Laboratory of Molecular Iron Metabolism, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China
| | - Yan-Zhong Chang
- Ministry of Education Key Laboratory of Molecular and Cellular Biology, Laboratory of Molecular Iron Metabolism, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
| | - Guofen Gao
- Ministry of Education Key Laboratory of Molecular and Cellular Biology, Laboratory of Molecular Iron Metabolism, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
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Peiter GC, de Souza CDBT, de Oliveira LM, Pagliarin LG, Dos Anjos VNF, da Silva FAF, de Melo FF, Teixeira KN. COVID-19 liver and gastroenterology findings: An in silico analysis of SARS-CoV-2 interactions with liver molecules. World J Hepatol 2022; 14:1131-1141. [PMID: 35978663 PMCID: PMC9258260 DOI: 10.4254/wjh.v14.i6.1131] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 02/22/2022] [Accepted: 05/16/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Coronavirus disease 19 (COVID-19) has not only been shown to affect the respiratory system, but has also demonstrated variable clinical presentations including gastrointestinal tract disorders. In addition, abnormalities in liver enzymes have been reported indicating hepatic injury. It is known that severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) might infect cells via the viral receptor angiotensin-converting enzyme 2 (ACE2) which is expressed in several organs including the liver. The viral Spike glycoprotein binds to ACE2 and must be cleaved by Furin and Type 2 Serine Protease to enter the cells. After that, the Akt/mTOR signaling pathway is activated and several COVID-19 changes are triggered. AIM To analyze liver and gastrointestinal symptoms and cell signaling pathways triggered by SARS-CoV-2 infection due to virus-liver interactions in silico. METHODS In this in silico study, the three-dimensional structures of the Akt, mTORC1 and Furin (receptors) were selected from the Protein Data Bank (PDB) and the structures of inhibitors (ligands) MK-2206, CC-223 and Naphthofluorescein were selected from PubChem and ZINC databases. Ligand files were downloaded as 2D structures and converted to optimized 3D structures using ViewerLite 4.2 software. Marvin Sketch® software was used to calculate prediction of the protonated form of inhibitors in a physiological environment (pH 7.4). AutoDock Tools (ADT) software was used to calculate and delimit the Grid box used in the molecular docking of each structure selected in the PDB. In addition, protonated ligands were prepared for molecular docking using ADT software. Molecular docking was performed using ADT software tools connected to Vina software. Analysis of the amino acid residues involved in ligand interactions, as well as ligand twists, the atoms involved in interactions, bond type and strength of interactions were performed using PyMol® and Discovery Studio® (BIOVIA) software. RESULTS Molecular docking analysis showed that the mTORC1/CC-223 complex had affinity energy between the receptor and ligand of -7.7 kcal/moL with interactions ranging from 2.7 to 4.99 Å. There were four significant chemical bonds which involved two of five polypeptide chains that formed the FKBP12-Rapamycin-Binding (FRB) domain. The strongest was a hydrogen bond, the only polar interaction, and Van der Waals interactions shown to be present in 12 residues of mTORC1's FRB domain. With regard to the Akt/MK-2206 complex there were three Van der Waals interactions and 12 chemical bonds in which seven residues of Akt were involved with all five rings of the MK-2206 structure. In this way, both ASP 388 and GLN 391 bind to the same MK-2206 ring, the smaller one. However, LYS 386 had four chemical bonds with the inhibitor, one with each structure ring, while LYS 387 binds two distinct rings. One of the MK-2206 inhibitor's rings which binds to LYS 387 also binds simultaneously to ILE 367 and LEU 385 residues, and the fifth ring of the structure was involved in a bond with the ALA 382 residue. The hydrogen bonds were the shortest bonds in the complex (2.61 and 3.08 Å) and all interactions had an affinity energy of -8.8 kcal/moL. The affinity energy in the Furin/Naphhofluorescein complex was -9.8 kcal/moL and involved six interactions ranging from 2.57 to 4.98 Å. Among them, two were polar and the others were non-polar, in addition to twelve more Van der Waals interactions. Two distinct hydrogen bonds were formed between Furin and its inhibitor involving GLN 388 and ALA 532 residues. ALA 532 also binds to two distinct rings of Naphthofluorescein, while TRP 531 residue has two simultaneous bonds with the inhibitor. CONCLUSION Liver infection and signaling pathways altered by SARS-CoV-2 can be modulated by inhibitors that demonstrate significant interaction affinity with human proteins, which could prevent the development of infection and symptoms.
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Affiliation(s)
| | | | | | | | | | | | - Fabrício Freire de Melo
- Universidade Federal da Bahia, Campus Anísio Teixeira, Vitória da Conquista 45029-094, Bahia, Brazil
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He Z, Khatib AM, Creemers JWM. The proprotein convertase furin in cancer: more than an oncogene. Oncogene 2022; 41:1252-1262. [PMID: 34997216 DOI: 10.1038/s41388-021-02175-9] [Citation(s) in RCA: 35] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 12/13/2021] [Accepted: 12/30/2021] [Indexed: 02/01/2023]
Abstract
Furin is the first discovered proprotein convertase member and is present in almost all mammalian cells. Therefore, by regulating the maturation of a wide range of proproteins, Furin expression and/or activity is involved in various physiological and pathophysiological processes ranging from embryonic development to carcinogenesis. Since many of these protein precursors are involved in initiating and maintaining the hallmarks of cancer, Furin has been proposed as a potential target for treating several human cancers. In contrast, other studies have revealed that some types of cancer do not benefit from Furin inhibition. Therefore, understanding the heterogeneous functions of Furin in cancer will provide important insights into the design of effective strategies targeting Furin in cancer treatment. Here, we present recent advances in understanding how Furin expression and activity are regulated in cancer cells and their influences on the activity of Furin substrates in carcinogenesis. Furthermore, we discuss how Furin represses tumorigenic properties of several cancer cells and why Furin inhibition leads to aggressive phenotypes in other tumors. Finally, we summarize the clinical applications of Furin inhibition in treating human cancers.
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Affiliation(s)
- Zongsheng He
- Department of Gastroenterology, Daping Hospital, Army Medical University, Chongqing, China
- Laboratory of Biochemical Neuroendocrinology, Department of Human Genetics, KU Leuven, Leuven, Belgium
| | - Abdel-Majid Khatib
- INSERM, LAMC, UMR 1029, Allée Geoffroy St Hilaire, Pessac, France.
- Institut Bergoinié, Bordeaux, France.
| | - John W M Creemers
- Laboratory of Biochemical Neuroendocrinology, Department of Human Genetics, KU Leuven, Leuven, Belgium.
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Tuft S, Somerville TF, Li JPO, Neal T, De S, Horsburgh MJ, Fothergill JL, Foulkes D, Kaye S. Bacterial keratitis: identifying the areas of clinical uncertainty. Prog Retin Eye Res 2021; 89:101031. [PMID: 34915112 DOI: 10.1016/j.preteyeres.2021.101031] [Citation(s) in RCA: 46] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2021] [Revised: 11/24/2021] [Accepted: 11/29/2021] [Indexed: 12/12/2022]
Abstract
Bacterial keratitis is a common corneal infection that is treated with topical antimicrobials. By the time of presentation there may already be severe visual loss from corneal ulceration and opacity, which may persist despite treatment. There are significant differences in the associated risk factors and the bacterial isolates between high income and low- or middle-income countries, so that general management guidelines may not be appropriate. Although the diagnosis of bacterial keratitis may seem intuitive there are multiple uncertainties about the criteria that are used, which impacts the interpretation of investigations and recruitment to clinical studies. Importantly, the concept that bacterial keratitis can only be confirmed by culture ignores the approximately 50% of cases clinically consistent with bacterial keratitis in which investigations are negative. The aetiology of these culture-negative cases is unknown. Currently, the estimation of bacterial susceptibility to antimicrobials is based on data from systemic administration and achievable serum or tissue concentrations, rather than relevant corneal concentrations and biological activity in the cornea. The provision to the clinician of minimum inhibitory concentrations of the antimicrobials for the isolated bacteria would be an important step forward. An increase in the prevalence of antimicrobial resistance is a concern, but the effect this has on disease outcomes is yet unclear. Virulence factors are not routinely assessed although they may affect the pathogenicity of bacteria within species and affect outcomes. New technologies have been developed to detect and kill bacteria, and their application to bacterial keratitis is discussed. In this review we present the multiple areas of clinical uncertainty that hamper research and the clinical management of bacterial keratitis, and we address some of the assumptions and dogma that have become established in the literature.
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Affiliation(s)
- Stephen Tuft
- Moorfields Eye Hospital NHS Foundation Trust, 162 City Road, London, EC1V 2PD, UK.
| | - Tobi F Somerville
- Department of Eye and Vision Sciences, University of Liverpool, 6 West Derby Street, Liverpool, L7 8TX, UK.
| | - Ji-Peng Olivia Li
- Moorfields Eye Hospital NHS Foundation Trust, 162 City Road, London, EC1V 2PD, UK.
| | - Timothy Neal
- Department of Clinical Microbiology, Liverpool Clinical Laboratories, Liverpool University Hospital NHS Foundation Trust, Prescot Street, Liverpool, L7 8XP, UK.
| | - Surjo De
- Department of Clinical Microbiology, University College London Hospitals NHS Foundation Trust, 250 Euston Road, London, NW1 2PG, UK.
| | - Malcolm J Horsburgh
- Department of Infection and Microbiomes, University of Liverpool, Crown Street, Liverpool, L69 7BX, UK.
| | - Joanne L Fothergill
- Department of Eye and Vision Sciences, University of Liverpool, 6 West Derby Street, Liverpool, L7 8TX, UK.
| | - Daniel Foulkes
- Department of Eye and Vision Sciences, University of Liverpool, 6 West Derby Street, Liverpool, L7 8TX, UK.
| | - Stephen Kaye
- Department of Eye and Vision Sciences, University of Liverpool, 6 West Derby Street, Liverpool, L7 8TX, UK.
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Rahbar Saadat Y, Hosseiniyan Khatibi SM, Zununi Vahed S, Ardalan M. Host Serine Proteases: A Potential Targeted Therapy for COVID-19 and Influenza. Front Mol Biosci 2021; 8:725528. [PMID: 34527703 PMCID: PMC8435734 DOI: 10.3389/fmolb.2021.725528] [Citation(s) in RCA: 45] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Accepted: 08/11/2021] [Indexed: 12/23/2022] Open
Abstract
The ongoing pandemic illustrates limited therapeutic options for controlling SARS-CoV-2 infections, calling a need for additional therapeutic targets. The viral spike S glycoprotein binds to the human receptor angiotensin-converting enzyme 2 (ACE2) and then is activated by the host proteases. Based on the accessibility of the cellular proteases needed for SARS-S activation, SARS-CoV-2 entrance and activation can be mediated by endosomal (such as cathepsin L) and non-endosomal pathways. Evidence indicates that in the non-endosomal pathway, the viral S protein is cleaved by the furin enzyme in infected host cells. To help the virus enter efficiently, the S protein is further activated by the serine protease 2 (TMPRSS2), provided that the S has been cleaved by furin previously. In this review, important roles for host proteases within host cells will be outlined in SARS-CoV-2 infection and antiviral therapeutic strategies will be highlighted. Although there are at least five highly effective vaccines at this time, the appearance of the new viral mutations demands the development of therapeutic agents. Targeted inhibition of host proteases can be used as a therapeutic approach for viral infection.
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11
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Kakarla V, Kaneko N, Nour M, Khatibi K, Elahi F, Liebeskind DS, Hinman JD. Pathophysiologic mechanisms of cerebral endotheliopathy and stroke due to Sars-CoV-2. J Cereb Blood Flow Metab 2021; 41:1179-1192. [PMID: 33530831 PMCID: PMC8142132 DOI: 10.1177/0271678x20985666] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Revised: 11/19/2020] [Accepted: 12/05/2020] [Indexed: 12/15/2022]
Abstract
Cerebrovascular events have emerged as a central feature of the clinical syndrome associated with Sars-CoV-2 infection. This increase in infection-related strokes is marked by atypical presentations including stroke in younger patients and a high rate of hemorrhagic transformation after ischemia. A variety of pathogenic mechanisms may underlie this connection. Efforts to identify synergism in the pathophysiology underlying stroke and Sars-CoV-2 infection can inform the understanding of both conditions in novel ways. In this review, the molecular cascades connected to Sars-CoV-2 infection are placed in the context of the cerebral vasculature and in relationship to pathways known to be associated with stroke. Cytokine-mediated promotion of systemic hypercoagulability is suggested while direct Sars-CoV-2 infection of cerebral endothelial cells may also contribute. Endotheliopathy resulting from direct Sars-CoV-2 infection of the cerebral vasculature can modulate ACE2/AT1R/MasR signaling pathways, trigger direct viral activation of the complement cascade, and activate feed-forward cytokine cascades that impact the blood-brain barrier. All of these pathways are already implicated as independent mechanisms driving stroke and cerebrovascular injury irrespective of Sars-CoV-2. Recognizing the overlap of molecular pathways triggered by Sars-CoV-2 infection with those implicated in the pathogenesis of stroke provides an opportunity to identify future therapeutics targeting both Sars-CoV-2 and stroke thereby reducing the impact of the global pandemic.
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Affiliation(s)
- Visesha Kakarla
- School of Medicine, University of California San Diego, San Diego, CA, USA
| | - Naoki Kaneko
- Department of Radiological Sciences, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
| | - May Nour
- Department of Radiological Sciences, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
- Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
| | - Kasra Khatibi
- Department of Radiological Sciences, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
| | - Fanny Elahi
- Memory and Aging Center, Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA
| | - David S Liebeskind
- Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
| | - Jason D Hinman
- Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
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12
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Abstract
The kexin-like proprotein convertases perform the initial proteolytic cleavages that ultimately generate a variety of different mature peptide and proteins, ranging from brain neuropeptides to endocrine peptide hormones, to structural proteins, among others. In this review, we present a general introduction to proprotein convertase structure and biochemistry, followed by a comprehensive discussion of each member of the kexin-like subfamily of proprotein convertases. We summarize current knowledge of human proprotein convertase insufficiency syndromes, including genome-wide analyses of convertase polymorphisms, and compare these to convertase null and mutant mouse models. These mouse models have illuminated our understanding of the roles specific convertases play in human disease and have led to the identification of convertase-specific substrates; for example, the identification of procorin as a specific PACE4 substrate in the heart. We also discuss the limitations of mouse null models in interpreting human disease, such as differential precursor cleavage due to species-specific sequence differences, and the challenges presented by functional redundancy among convertases in attempting to assign specific cleavages and/or physiological roles. However, in most cases, knockout mouse models have added substantively both to our knowledge of diseases caused by human proprotein convertase insufficiency and to our appreciation of their normal physiological roles, as clearly seen in the case of the furin, proprotein convertase 1/3, and proprotein convertase 5/6 mouse models. The creation of more sophisticated mouse models with tissue- or temporally-restricted expression of specific convertases will improve our understanding of human proprotein convertase insufficiency and potentially provide support for the emerging concept of therapeutic inhibition of convertases.
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Affiliation(s)
- Manita Shakya
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Iris Lindberg
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, USA
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13
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Santopolo S, Riccio A, Santoro MG. The biogenesis of SARS-CoV-2 spike glycoprotein: multiple targets for host-directed antiviral therapy. Biochem Biophys Res Commun 2021; 538:80-87. [PMID: 33303190 PMCID: PMC7698684 DOI: 10.1016/j.bbrc.2020.10.080] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Accepted: 10/28/2020] [Indexed: 12/15/2022]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease-19), represents a far more serious threat to public health than SARS and MERS coronaviruses, due to its ability to spread more efficiently than its predecessors. Currently, there is no worldwide-approved effective treatment for COVID-19, urging the scientific community to intense efforts to accelerate the discovery and development of prophylactic and therapeutic solutions against SARS-CoV-2 infection. In particular, effective antiviral drugs are urgently needed. With few exceptions, therapeutic approaches to combat viral infections have traditionally focused on targeting unique viral components or enzymes; however, it has now become evident that this strategy often fails due to the rapid emergence of drug-resistant viruses. Targeting host factors that are essential for the virus life cycle, but are dispensable for the host, has recently received increasing attention. The spike glycoprotein, a component of the viral envelope that decorates the virion surface as a distinctive crown ("corona") and is essential for SARS-CoV-2 entry into host cells, represents a key target for developing therapeutics capable of blocking virus invasion. This review highlights aspects of the SARS-CoV-2 spike biogenesis that may be amenable to host-directed antiviral targeting.
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Affiliation(s)
- Silvia Santopolo
- Department of Biology, University of Rome Tor Vergata, Rome, Italy
| | - Anna Riccio
- Department of Biology, University of Rome Tor Vergata, Rome, Italy
| | - M Gabriella Santoro
- Department of Biology, University of Rome Tor Vergata, Rome, Italy; Institute of Translational Pharmacology, CNR, Rome, Italy.
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14
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Bhullar KS, Drews SJ, Wu J. Translating bioactive peptides for COVID-19 therapy. Eur J Pharmacol 2021; 890:173661. [PMID: 33098835 PMCID: PMC7577279 DOI: 10.1016/j.ejphar.2020.173661] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2020] [Accepted: 10/20/2020] [Indexed: 02/07/2023]
Abstract
COVID-19 (Coronavirus disease 2019) is a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. This virus has emerged as a threat to global health, social stability, and the global economy. This pandemic continues to cause rampant mortality worldwide with the dire urgency to develop novel therapeutic agents. To meet this task, this article discusses advances in the research and potential application of bioactive peptides for possible mitigation of infection by SARS-CoV-2. Growing insight into the molecular biology of SARS-CoV-2 has revealed potential druggable targets for bioactive peptides. Bioactive peptides with unique amino acid sequences can mitigate such targets including, type II transmembrane serine proteases (TMPRSS2) inhibition, furin cleavage, and renin-angiotensin-aldosterone system (RAAS) members. Based on current evidence and structure-function analysis, multiple bioactive peptides present potency to neutralize the virus. To date, no SARS-CoV-2-explicit drug has been reported, but we here introduce bioactive peptides in the perspective of their potential activity against SARS-CoV-2 infection.
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Affiliation(s)
- Khushwant S Bhullar
- Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada; Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada
| | - Steven J Drews
- Canadian Blood Services, Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada
| | - Jianping Wu
- Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada.
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15
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Abstract
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide since its first incidence in Wuhan, China, in December 2019. Although the case fatality rate of COVID-19 appears to be lower than that of SARS and Middle East respiratory syndrome (MERS), the higher transmissibility of SARS-CoV-2 has caused the total fatality to surpass other viral diseases, reaching more than 1 million globally as of October 6, 2020. The rate at which the disease is spreading calls for a therapy that is useful for treating a large population. Multiple intersecting viral and host factor targets involved in the life cycle of the virus are being explored. Because of the frequent mutations, many coronaviruses gain zoonotic potential, which is dependent on the presence of cell receptors and proteases, and therefore the targeting of the viral proteins has some drawbacks, as strain-specific drug resistance can occur. Moreover, the limited number of proteins in a virus makes the number of available targets small. Although SARS-CoV and SARS-CoV-2 share common mechanisms of entry and replication, there are substantial differences in viral proteins such as the spike (S) protein. In contrast, targeting cellular factors may result in a broader range of therapies, reducing the chances of developing drug resistance. In this Review, we discuss the role of primary host factors such as the cell receptor angiotensin-converting enzyme 2 (ACE2), cellular proteases of S protein priming, post-translational modifiers, kinases, inflammatory cells, and their pharmacological intervention in the infection of SARS-CoV-2 and related viruses.
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Affiliation(s)
- Anil Mathew Tharappel
- Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, NY 12208, USA
| | - Subodh Kumar Samrat
- Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, NY 12208, USA
| | - Zhong Li
- Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, NY 12208, USA
| | - Hongmin Li
- Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, NY 12208, USA
- Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12201, USA
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16
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Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep 2020; 33:108254. [PMID: 33007239 PMCID: PMC7510585 DOI: 10.1016/j.celrep.2020.108254] [Citation(s) in RCA: 194] [Impact Index Per Article: 38.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Revised: 08/31/2020] [Accepted: 09/18/2020] [Indexed: 12/17/2022] Open
Abstract
Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus production and cytopathic effects. Further analysis reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.
The furin cleavage site in the SARS-CoV-2 spike protein mediates syncytium formation The SARS-CoV-2 spike-mediated syncytium is suppressed by specific furin inhibitors Furin inhibitors block SARS-CoV-2 virus entry and virus replication Furin inhibitors are potential antiviral agents for SARS-CoV-2 infection and pathogenesis
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Affiliation(s)
- Ya-Wen Cheng
- Department of Microbiology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Tai-Ling Chao
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Chiao-Ling Li
- Department of Microbiology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Mu-Fan Chiu
- Department of Microbiology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Han-Chieh Kao
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Sheng-Han Wang
- Hepatitis Research Center, National Taiwan University Hospital, Taipei 100, Taiwan
| | - Yu-Hao Pang
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Chih-Hui Lin
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Ya-Min Tsai
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Wen-Hau Lee
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Mi-Hua Tao
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
| | - Tung-Ching Ho
- Department of Microbiology, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Ping-Yi Wu
- Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
| | - Li-Ting Jang
- Biomedical Resource Core at the First Core Labs, Branch Office of Research and Development, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Pei-Jer Chen
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei 100, Taiwan; Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan; National Taiwan University Center for Genomic Medicine, National Taiwan University College of Medicine, Taipei 100, Taiwan
| | - Sui-Yuan Chang
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei 100, Taiwan.
| | - Shiou-Hwei Yeh
- Department of Microbiology, National Taiwan University College of Medicine, Taipei 100, Taiwan; Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan; National Taiwan University Center for Genomic Medicine, National Taiwan University College of Medicine, Taipei 100, Taiwan.
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17
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Bestle D, Heindl MR, Limburg H, Van Lam van T, Pilgram O, Moulton H, Stein DA, Hardes K, Eickmann M, Dolnik O, Rohde C, Klenk HD, Garten W, Steinmetzer T, Böttcher-Friebertshäuser E. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Sci Alliance 2020; 3:3/9/e202000786. [PMID: 32703818 PMCID: PMC7383062 DOI: 10.26508/lsa.202000786] [Citation(s) in RCA: 583] [Impact Index Per Article: 116.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 07/15/2020] [Accepted: 07/15/2020] [Indexed: 11/24/2022] Open
Abstract
The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.
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Affiliation(s)
- Dorothea Bestle
- Institute of Virology, Philipps-University, Marburg, Germany
| | | | - Hannah Limburg
- Institute of Virology, Philipps-University, Marburg, Germany
| | - Thuy Van Lam van
- Institute of Pharmaceutical Chemistry, Philipps-University, Marburg, Germany
| | - Oliver Pilgram
- Institute of Pharmaceutical Chemistry, Philipps-University, Marburg, Germany
| | - Hong Moulton
- Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA
| | - David A Stein
- Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA
| | - Kornelia Hardes
- Institute of Pharmaceutical Chemistry, Philipps-University, Marburg, Germany.,Fraunhofer Institute for Molecular Biology and Applied Ecology, Gießen, Germany
| | - Markus Eickmann
- Institute of Virology, Philipps-University, Marburg, Germany.,German Center for Infection Research (DZIF), Marburg-Gießen-Langen Site, Emerging Infections Unit, Philipps-University, Marburg, Germany
| | - Olga Dolnik
- Institute of Virology, Philipps-University, Marburg, Germany.,German Center for Infection Research (DZIF), Marburg-Gießen-Langen Site, Emerging Infections Unit, Philipps-University, Marburg, Germany
| | - Cornelius Rohde
- Institute of Virology, Philipps-University, Marburg, Germany.,German Center for Infection Research (DZIF), Marburg-Gießen-Langen Site, Emerging Infections Unit, Philipps-University, Marburg, Germany
| | | | - Wolfgang Garten
- Institute of Virology, Philipps-University, Marburg, Germany
| | - Torsten Steinmetzer
- Institute of Pharmaceutical Chemistry, Philipps-University, Marburg, Germany
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18
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Hoffmann M, Kleine-Weber H, Pöhlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell 2020; 78:779-784.e5. [PMID: 32362314 PMCID: PMC7194065 DOI: 10.1016/j.molcel.2020.04.022] [Citation(s) in RCA: 1346] [Impact Index Per Article: 269.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2020] [Revised: 04/14/2020] [Accepted: 04/17/2020] [Indexed: 12/13/2022]
Abstract
The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
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Affiliation(s)
- Markus Hoffmann
- Deutsches Primatenzentrum - Leibniz Institut für Primatenforschung, Göttingen, Germany.
| | - Hannah Kleine-Weber
- Deutsches Primatenzentrum - Leibniz Institut für Primatenforschung, Göttingen, Germany; Faculty of Biology and Psychology, University Göttingen, Göttingen, Germany
| | - Stefan Pöhlmann
- Deutsches Primatenzentrum - Leibniz Institut für Primatenforschung, Göttingen, Germany; Faculty of Biology and Psychology, University Göttingen, Göttingen, Germany.
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19
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He Z, Thorrez L, Siegfried G, Meulemans S, Evrard S, Tejpar S, Khatib AM, Creemers JWM. The proprotein convertase furin is a pro-oncogenic driver in KRAS and BRAF driven colorectal cancer. Oncogene 2020; 39:3571-3587. [PMID: 32139876 DOI: 10.1038/s41388-020-1238-z] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Revised: 02/19/2020] [Accepted: 02/19/2020] [Indexed: 12/24/2022]
Abstract
Mutations in KRAS and/or BRAF that activate the ERK kinase are frequently found in colorectal cancer (CRC) and drive resistance to targeted therapies. Therefore, the identification of therapeutic targets that affect multiple signaling pathways simultaneously is crucial for improving the treatment of patients with KRAS or BRAF mutations. The proprotein convertase furin activates several oncogenic protein precursors involved in the ERK-MAPK pathway by endoproteolytic cleavage. Here we show that genetic inactivation of furin suppresses tumorigenic growth, proliferation, and migration in KRAS or BRAF mutant CRC cell lines but not in wild-type KRAS and BRAF cells. In a mouse xenograft model, these KRAS or BRAF mutant cells lacking furin displayed reduced growth and angiogenesis, and increased apoptosis. Mechanistically, furin inactivation prevents the processing of various protein pecursors including proIGF1R, proIR, proc-MET, proTGF-β1 and NOTCH1 leading to potent and durable ERK-MAPK pathway suppression in KRAS or BRAF mutant cells. Furthermore, we identified genes involved in activating the ERK-MAPK pathway, such as PTGS2, which are downregulated in the KRAS or BRAF mutant cells after furin inactivation but upregulated in wild-type KRAS and BRAF cells. Analysis of human colorectal tumor samples reveals a positive correlation between enhanced furin expression and KRAS or BRAF expression. These results indicate that furin plays an important role in KRAS or BRAF-associated ERK-MAPK pathway activation and tumorigenesis, providing a potential target for personalized treatment.
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Affiliation(s)
- Zongsheng He
- Laboratory of Biochemical Neuroendocrinology, Department of Human Genetics, KU Leuven, Leuven, Belgium
| | - Lieven Thorrez
- Interdisciplinary Research Facility, Department of Development and Regeneration, KU Leuven, Campus Kulak Kortrijk, Kortrijk, Belgium
| | | | - Sandra Meulemans
- Laboratory of Biochemical Neuroendocrinology, Department of Human Genetics, KU Leuven, Leuven, Belgium
| | - Serge Evrard
- INSERM, LAMC, UMR, Allée Geoffroy St Hilaire, 1029, Pessac, France.,Institut Bergonié, Bordeaux, France
| | - Sabine Tejpar
- Digestive Oncology Unit, Department of Oncology, University Hospitals Leuven, Leuven, Belgium
| | | | - John W M Creemers
- Laboratory of Biochemical Neuroendocrinology, Department of Human Genetics, KU Leuven, Leuven, Belgium.
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20
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Braun E, Sauter D. Furin-mediated protein processing in infectious diseases and cancer. Clin Transl Immunology 2019; 8:e1073. [PMID: 31406574 PMCID: PMC6682551 DOI: 10.1002/cti2.1073] [Citation(s) in RCA: 217] [Impact Index Per Article: 36.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2019] [Revised: 07/10/2019] [Accepted: 07/13/2019] [Indexed: 12/17/2022] Open
Abstract
Proteolytic cleavage regulates numerous processes in health and disease. One key player is the ubiquitously expressed serine protease furin, which cleaves a plethora of proteins at polybasic recognition motifs. Mammalian substrates of furin include cytokines, hormones, growth factors and receptors. Thus, it is not surprising that aberrant furin activity is associated with a variety of disorders including cancer. Furthermore, the enzymatic activity of furin is exploited by numerous viral and bacterial pathogens, thereby enhancing their virulence and spread. In this review, we describe the physiological and pathophysiological substrates of furin and discuss how dysregulation of a simple proteolytic cleavage event may promote infectious diseases and cancer. One major focus is the role of furin in viral glycoprotein maturation and pathogenicity. We also outline cellular mechanisms regulating the expression and activation of furin and summarise current approaches that target this protease for therapeutic intervention.
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Affiliation(s)
- Elisabeth Braun
- Institute of Molecular VirologyUlm University Medical CenterUlmGermany
| | - Daniel Sauter
- Institute of Molecular VirologyUlm University Medical CenterUlmGermany
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21
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Zhou M, Zhang Y, Wei H, He J, Wang D, Chen B, Zeng J, Gong A, Xu M. Furin inhibitor D6R suppresses epithelial-mesenchymal transition in SW1990 and PaTu8988 cells via the Hippo-YAP signaling pathway. Oncol Lett 2017; 15:3192-3196. [PMID: 29435056 DOI: 10.3892/ol.2017.7672] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2016] [Accepted: 10/03/2017] [Indexed: 02/07/2023] Open
Abstract
Hexa-D-arginine (D6R), an inhibitor of furin, has potential therapeutic applications in different types of human tumor. However, the function of D6R in targeting pancreatic cancer cells remains to be elucidated. In the present study, the proliferation, invasion and migration abilities of SW1990 and PaTu8988 cells were examined using a Cell Counting Kit-8, and Transwell and wound healing assays. Subsequently, the expression of proteins associated with epithelial-mesenchymal transition (EMT) and the Hippo-yes-associated protein (YAP) pathway were detected using western blot analysis. It was revealed that D6R significantly inhibited the proliferation, migration and invasion abilities of SW1990 and PaTu8988 cells. Additionally, D6R led to the upregulation of E-cadherin (an epithelial marker), and the downregulation of N-cadherin and vimentin (mesenchymal markers) in SW1990 and PaTu8988 cells. Furthermore, the results of the present study revealed that D6R significantly affected the YAP phosphorylation level and the total YAP protein level, indicating that D6R was functionally involved in the Hippo-YAP signaling pathway. It has been suggested that D6R-suppressed EMT in SW1990 and PaTu8988 cells may occur via the Hippo-YAP pathway and that it may be a feasible drug to ameliorate the malignant phenotype of SW1990 and PaTu8988 cells.
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Affiliation(s)
- Meng Zhou
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Youli Zhang
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Hong Wei
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Junbo He
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Dawei Wang
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Baoding Chen
- Department of Ultrasound Diagnosis, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Jian Zeng
- Department of Cell Biology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Aihua Gong
- Department of Cell Biology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
| | - Min Xu
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China
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Klein-Szanto AJ, Bassi DE. Proprotein convertase inhibition: Paralyzing the cell's master switches. Biochem Pharmacol 2017; 140:8-15. [PMID: 28456517 DOI: 10.1016/j.bcp.2017.04.027] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2017] [Accepted: 04/25/2017] [Indexed: 12/18/2022]
Abstract
Proprotein convertases are serine proteases responsible for the cleavage and subsequent activation of protein substrates, many of them relevant for the development of an ample variety of diseases. Seven of the PCs, including furin and PACE4, recognize and hydrolyze the C-terminal end of the general sequence RXRR/KXR, whereas PCSK-9 recognizes a series of non-basic amino acids. In some systems, PC-mediated substrate activation results in the development of pathological processes, such as cancer, endocrinopathies, and cardiovascular and infectious diseases. After establishing PCs as relevant contributors to disease processes, research efforts were directed towards the development of inhibition strategies, including small and large molecules, anti-sense therapies, and antibody-based therapies. Most of these inhibitors mimic the consensus sequence of PCs, blocking the active site in a competitive manner. The most promising inhibitors were designed as bioengineered proteins; however, some non-protein and peptidomimetic agents have also proved to be effective. These efforts led to the design of pre-clinical studies and clinical trials utilizing inhibitors to PCs. Although the initial studies were performed using non-selective PCs inhibitors, such as CMK, the search for more specific, and compartmentalized selective inhibitors resulted in specific activities ascribed to some, but not all of the PCs. For instance, PACE4 inhibitors were effective in decreasing prostate cancer cell proliferation, and neovascularization. Decreased metastatic ovarian cancer utilizing furin inhibitors represents one of the major endeavors, currently in a phase II trial stage. Antibodies targeting PCSK-9 decreased significantly the levels of HDL-cholesterol, in a phase III trial. The study of Proprotein convertases has reached a stage of maturity. New strategies based on the alteration of their activity at the cellular and clinical level represent a promising experimental pharmacology field. The development of allosteric inhibitors, or specific agents directed against individual PCs is one of the challenges to be unraveled in the future.
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Affiliation(s)
| | - Daniel E Bassi
- Fox Chase Cancer Center, 333 Cotman Ave, Philadelphia 19111, USA.
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Hardes K, Ivanova T, Thaa B, McInerney GM, Klokk TI, Sandvig K, Künzel S, Lindberg I, Steinmetzer T. Elongated and Shortened Peptidomimetic Inhibitors of the Proprotein Convertase Furin. ChemMedChem 2017; 12:613-620. [PMID: 28334511 PMCID: PMC5572662 DOI: 10.1002/cmdc.201700108] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2017] [Revised: 03/16/2017] [Indexed: 12/13/2022]
Abstract
Novel elongated and shortened derivatives of the peptidomimetic furin inhibitor phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide were synthesized. The most potent compounds, such as Nα (carbamidoyl)Arg-Arg-Val-Arg-4-amidinobenzylamide (Ki =6.2 pm), contain additional basic residues at the N terminus and inhibit furin in the low-picomolar range. Furthermore, to decrease the molecular weight of this inhibitor type, compounds that lack the P5 moiety were prepared. The best inhibitors of this series, 5-(guanidino)valeroyl-Val-Arg-4-amidinobenzylamide and its P3 tert-leucine analogue displayed Ki values of 2.50 and 1.26 nm, respectively. Selected inhibitors, together with our previously described 4-amidinobenzylamide derivatives as references, were tested in cell culture for their activity against furin-dependent infectious pathogens. The propagation of the alphaviruses Semliki Forest virus and chikungunya virus was strongly inhibited in the presence of selected derivatives. Moreover, a significant protective effect of the inhibitors against diphtheria toxin was observed. These results confirm that the inhibition of furin should be a promising approach for the short-term treatment of acute infectious diseases.
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Affiliation(s)
- Kornelia Hardes
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032 Marburg, Germany
| | - Teodora Ivanova
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032 Marburg, Germany
| | - Bastian Thaa
- Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, SE-171 77 Stockholm, Sweden
| | - Gerald M. McInerney
- Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, SE-171 77 Stockholm, Sweden
| | - Tove Irene Klokk
- Department of Molecular Cell Biology and Centre for Cancer Biomedicine, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, NO-0310 Oslo, Norway
| | - Kirsten Sandvig
- Department of Molecular Cell Biology and Centre for Cancer Biomedicine, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, NO-0310 Oslo, Norway
| | - Sebastian Künzel
- Faculty of Engineering Sciences, Hochschule Ansbach, Residenzstraße 8, D-91522 Ansbach, Germany
| | - Iris Lindberg
- Department of Anatomy and Neurobiology, University of Maryland Medical School, Baltimore, Maryland 21201
| | - Torsten Steinmetzer
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032 Marburg, Germany
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Kavaliauskiene S, Dyve Lingelem AB, Skotland T, Sandvig K. Protection against Shiga Toxins. Toxins (Basel) 2017; 9:E44. [PMID: 28165371 PMCID: PMC5331424 DOI: 10.3390/toxins9020044] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2016] [Revised: 01/18/2017] [Accepted: 01/19/2017] [Indexed: 12/12/2022] Open
Abstract
Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans.
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Affiliation(s)
- Simona Kavaliauskiene
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, N-0379 Oslo, Norway.
- Center for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, N-0379 Oslo, Norway.
| | - Anne Berit Dyve Lingelem
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, N-0379 Oslo, Norway.
- Center for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, N-0379 Oslo, Norway.
| | - Tore Skotland
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, N-0379 Oslo, Norway.
- Center for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, N-0379 Oslo, Norway.
| | - Kirsten Sandvig
- Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, N-0379 Oslo, Norway.
- Center for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, N-0379 Oslo, Norway.
- Department of Biosciences, University of Oslo, N-0316 Oslo, Norway.
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25
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Ramos-Molina B, Lick AN, Nasrolahi Shirazi A, Oh D, Tiwari R, El-Sayed NS, Parang K, Lindberg I. Cationic Cell-Penetrating Peptides Are Potent Furin Inhibitors. PLoS One 2015; 10:e0130417. [PMID: 26110264 PMCID: PMC4482483 DOI: 10.1371/journal.pone.0130417] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2015] [Accepted: 05/20/2015] [Indexed: 12/30/2022] Open
Abstract
Cationic cell-penetrating peptides have been widely used to enhance the intracellular delivery of various types of cargoes, such as drugs and proteins. These reagents are chemically similar to the multi-basic peptides that are known to be potent proprotein convertase inhibitors. Here, we report that both HIV-1 TAT47-57 peptide and the Chariot reagent are micromolar inhibitors of furin activity in vitro. In agreement, HIV-1 TAT47-57 reduced HT1080 cell migration, thought to be mediated by proprotein convertases, by 25%. In addition, cyclic polyarginine peptides containing hydrophobic moieties which have been previously used as transfection reagents also exhibited potent furin inhibition in vitro and also inhibited intracellular convertases. Our finding that cationic cell-penetrating peptides exert potent effects on cellular convertase activity should be taken into account when biological effects are assessed.
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Affiliation(s)
- Bruno Ramos-Molina
- Department of Anatomy and Neurobiology, School of Medicine, University of Maryland-Baltimore, Baltimore, Maryland, United States of America
| | - Adam N. Lick
- Department of Anatomy and Neurobiology, School of Medicine, University of Maryland-Baltimore, Baltimore, Maryland, United States of America
| | | | - Donghoon Oh
- Chapman University, School of Pharmacy, Irvine, California, United States of America
| | - Rakesh Tiwari
- Chapman University, School of Pharmacy, Irvine, California, United States of America
| | - Naglaa Salem El-Sayed
- Chapman University, School of Pharmacy, Irvine, California, United States of America
| | - Keykavous Parang
- Chapman University, School of Pharmacy, Irvine, California, United States of America
| | - Iris Lindberg
- Department of Anatomy and Neurobiology, School of Medicine, University of Maryland-Baltimore, Baltimore, Maryland, United States of America
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26
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Lu Y, Hardes K, Dahms SO, Böttcher-Friebertshäuser E, Steinmetzer T, Than ME, Klenk HD, Garten W. Peptidomimetic furin inhibitor MI-701 in combination with oseltamivir and ribavirin efficiently blocks propagation of highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in MDCK cells. Antiviral Res 2015; 120:89-100. [PMID: 26022200 DOI: 10.1016/j.antiviral.2015.05.006] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2015] [Revised: 05/12/2015] [Accepted: 05/20/2015] [Indexed: 12/30/2022]
Abstract
Antiviral medication is used for the treatment of severe influenza infections, of which the neuraminidase inhibitors (NAIs) are the most effective drugs, approved so far. Here, we investigated the antiviral efficacy of the peptidomimetic furin inhibitor MI-701 in combination with oseltamivir carboxylate and ribavirin against the infection of highly pathogenic avian influenza viruses (HPAIV) that are activated by the host protease furin. Cell cultures infected with the strains A/Thailand/1(KAN-1)/2004 (H5N1) and A/FPV/Rostock/1934 (H7N1) were treated with each agent alone, or in double and triple combinations. MI-701 alone achieved a concentration-dependent reduction of virus propagation. Double treatment of MI-701 with oseltamivir carboxylate and triple combination with ribavirin showed synergistic inhibition and a pronounced delay of virus propagation. MI-701 resistant mutants were not observed. Emergence of NA mutation H275Y conferring high oseltamivir resistance was significantly delayed in the presence of MI-701. Our data indicate that combination with a potent furin inhibitor significantly enhances the therapeutic efficacy of conventional antivirals drugs against HPAIV infection.
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Affiliation(s)
- Yinghui Lu
- Institute of Virology, Philipps University, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany
| | - Kornelia Hardes
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6-10, 35032 Marburg, Germany
| | - Sven O Dahms
- Protein Crystallography Group, Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany
| | | | - Torsten Steinmetzer
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6-10, 35032 Marburg, Germany
| | - Manuel E Than
- Protein Crystallography Group, Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany
| | - Hans-Dieter Klenk
- Institute of Virology, Philipps University, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany
| | - Wolfgang Garten
- Institute of Virology, Philipps University, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany.
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Ramos-Molina B, Lick AN, Blanco EH, Posada-Salgado JA, Martinez-Mayorga K, Johnson AT, Jiao GS, Lindberg I. Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays. Biochem Pharmacol 2015; 96:107-18. [PMID: 26003844 DOI: 10.1016/j.bcp.2015.05.008] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2015] [Accepted: 05/11/2015] [Indexed: 12/20/2022]
Abstract
The proprotein convertase furin is implicated in a variety of pathogenic processes such as bacterial toxin activation, viral propagation, and cancer. Several groups have identified non-peptide compounds with high inhibitory potency against furin in vitro, although their efficacy in various cell-based assays is largely unknown. In this study we show that certain guanidinylated 2,5-dideoxystreptamine derivatives exhibit interesting ex vivo properties. Compound 1b (1,1'-(4-((2,4-diguanidino-5-(4-guanidinophenoxy)cyclohexyl)oxy)-1,3-phenylene)diguanidine) is a potent and cell-permeable inhibitor of cellular furin, since it was able to retard tumor cell migration, block release of a Golgi reporter, and protect cells against Bacillus anthracis (anthrax) and Pseudomonas aeruginosa intoxication, with no evident cell toxicity. Other compounds based on the 2,5-dideoxystreptamine scaffold, such as compound 1g (1,1'-(4,6-bis(4-guanidinophenoxy)cyclohexane-1,3-diyl)diguanidine) also efficiently protected cells against anthrax, but displayed only moderate protection against Pseudomonas exotoxin A and did not inhibit cell migration, suggesting poor cell permeability. Certain bis-guanidinophenyl ether derivatives such as 2f (1,3-bis(2,4-diguanidinophenoxy) benzene) exhibited micromolar potency against furin in vitro, low cell toxicity, and highly efficient protection against anthrax toxin; this compound only slightly inhibited intracellular furin. Thus, compounds 1g and 2f both represent potent furin inhibitors at the cell surface with low intracellular inhibitory action, and these particular compounds might therefore be of preferred therapeutic interest in the treatment of certain bacterial and viral infections.
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Affiliation(s)
- Bruno Ramos-Molina
- Department of Anatomy and Neurobiology, School of Medicine, University of Maryland-Baltimore, MD, USA
| | - Adam N Lick
- Department of Anatomy and Neurobiology, School of Medicine, University of Maryland-Baltimore, MD, USA
| | - Elias H Blanco
- Department of Anatomy and Neurobiology, School of Medicine, University of Maryland-Baltimore, MD, USA
| | | | | | - Alan T Johnson
- Department of Chemistry, Hawaii Biotech, Inc., Aiea, HI, USA
| | - Guan-Sheng Jiao
- Department of Chemistry, Hawaii Biotech, Inc., Aiea, HI, USA.
| | - Iris Lindberg
- Department of Anatomy and Neurobiology, School of Medicine, University of Maryland-Baltimore, MD, USA.
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28
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Hardes K, Becker GL, Lu Y, Dahms SO, Köhler S, Beyer W, Sandvig K, Yamamoto H, Lindberg I, Walz L, von Messling V, Than ME, Garten W, Steinmetzer T. Novel Furin Inhibitors with Potent Anti-infectious Activity. ChemMedChem 2015; 10:1218-31. [PMID: 25974265 DOI: 10.1002/cmdc.201500103] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2015] [Indexed: 11/10/2022]
Abstract
New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4-guanidinomethyl-phenylacteyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148) inhibits furin with a Ki value of 5.5 pM. The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI-1148 was crystallized in complex with human furin. Its N-terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short-term treatment of acute infectious diseases.
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Affiliation(s)
- Kornelia Hardes
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg (Germany)
| | - Gero L Becker
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg (Germany)
| | - Yinghui Lu
- Institute of Virology, Philipps University, Hans-Meerwein-Strasse 2, Marburg (Germany)
| | - Sven O Dahms
- Protein Crystallography Group, Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)
| | - Susanne Köhler
- Institute of Environmental and Animal Hygiene, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart (Germany)
| | - Wolfgang Beyer
- Institute of Environmental and Animal Hygiene, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart (Germany)
| | - Kirsten Sandvig
- Department of Biochemistry and Centre for Cancer Biomedicine, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo (Norway)
| | - Hiroyuki Yamamoto
- Department of Anatomy and Neurobiology, University of Maryland, Baltimore, Maryland 21201 (USA)
| | - Iris Lindberg
- Department of Anatomy and Neurobiology, University of Maryland, Baltimore, Maryland 21201 (USA)
| | - Lisa Walz
- Veterinary Medicine Division, Paul-Ehrlich-Institute, Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany)
| | - Veronika von Messling
- Veterinary Medicine Division, Paul-Ehrlich-Institute, Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany)
| | - Manuel E Than
- Protein Crystallography Group, Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)
| | - Wolfgang Garten
- Institute of Virology, Philipps University, Hans-Meerwein-Strasse 2, Marburg (Germany)
| | - Torsten Steinmetzer
- Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, 35032 Marburg (Germany).
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Couture F, Kwiatkowska A, Dory YL, Day R. Therapeutic uses of furin and its inhibitors: a patent review. Expert Opin Ther Pat 2015; 25:379-96. [PMID: 25563687 DOI: 10.1517/13543776.2014.1000303] [Citation(s) in RCA: 70] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
INTRODUCTION Since the discovery of furin, numerous reports have studied its role in health and diseases, including cancer, inflammatory and infectious diseases. This interest has led to the development of both large protein- and peptide-based inhibitors aiming to control furin activity to treat these disorders. The most recent advances include the development of potent peptidomimetic furin inhibitors, considerably expanding the field of therapeutic applications. AREA COVERED In this review, the use of furin or its inhibitors for therapeutic conditions is described through the patent literature since 1994. Only compounds with biological efficacy or augmented properties demonstrated within the patent literature or the associated publications concerning their claimed uses are discussed. EXPERT OPINION Considering the diseases that may benefit from furin inhibition, several patents detail the use of the restricted number of furin inhibitors. However, there have been recent reports of new scaffolds, and even the use of furin itself, as a therapeutic agent. Despite considerable evidence of in vivo efficacy, limited confirmation from clinical trials supports or refutes the further use of these compounds in a therapeutic context. The most advanced application is the use of furin knockdown in the generation of an autologous cancer vaccine, which has initiated clinical trials.
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Affiliation(s)
- Frédéric Couture
- Université de Sherbrooke, Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé , 3001 12e Ave. Nord, Sherbrooke, Québec, J1H 5N4 , Canada +1 819 564 5428 ; +1 819 564 5400 ;
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Omotuyi IO. Ebola virus envelope glycoprotein derived peptide in human Furin-bound state: computational studies. J Biomol Struct Dyn 2014; 33:461-70. [PMID: 25347780 DOI: 10.1080/07391102.2014.981207] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Ebola virus (EboV) is currently ravaging West Africa with estimated case fatality rate of 52%. Currently, no drug treatment is available and immunoglobulin therapy is still at the rudimentary stage. For anti-EboV drug development, druggable viral and host protein targets, including human Furin are under intense investigation. Here, molecular dynamics simulation was performed on Apo-Furin, meta-guanidinomethyl-Phac-RVR-Amba-bound, and two EboV glycoprotein (GP) 494-TGGRRTRREA-503/Furin complexes (Accurate and one amino acid shift alignment). The results of the simulation established ligand-induced desolvation of Furin active site and structural compactness. Accurately aligned EboV-GP peptide exhibited a tighter binding mode with Furin and showed 1.5- and 3.0-fold MMPBSA binding free energy estimate compared with the displaced peptide and inhibitor, respectively. The difference in free energy was traced to the difference in contribution of threonine residues of the peptides. Furthermore, Furin subsites I conferred substrate specificity and ligand binding accuracy. Accurately aligned peptide trapped active site His194 side chain into gauche (-) (+60(o)) χ1-dihedral compared with gauche+ (-60(o)) in other biosystems while Asp153 is trapped in gauche+ (-60(o)) in ligand bound not Apo state. Ramachandran plot showed that the scissile Arg8 of the accurately aligned peptide showed β conformation distribution as apposed to 310R, αL, and 310L. Finally, the active site proximal Na(+) binding is dependent on substrate peptide occupancy of the active site but detaches in the absence of a ligand. In conclusion, Furin might represent candidate drug target for Ebola virus disease treatment via therapeutic target of the active site and Na(+) binding pocket.
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Affiliation(s)
- I Olaposi Omotuyi
- a Department of Pharmacology and Therapeutic Innovation , Graduate School of Biomedical Science, Nagasaki University , Nagasaki , Japan
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31
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Brandt CR. Peptide therapeutics for treating ocular surface infections. J Ocul Pharmacol Ther 2014; 30:691-9. [PMID: 25250986 DOI: 10.1089/jop.2014.0089] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Microbial pathogens-bacteria, viruses, fungi, and parasites-are significant causes of blindness, particularly in developing countries. For bacterial and some viral infections a number of antimicrobial drugs are available for therapy but there are fewer available for use in treating fungal and parasitic keratitis. There are also problems with current antimicrobials, such as limited efficacy and the presence of drug-resistant microbes. Thus, there is a need to develop additional drugs. Nature has given us an example of 1 potential source of new antimicrobials: antimicrobial peptides and proteins that are either present in bodily fluids and tissues constitutively or are induced upon infection. Given the nature of peptides, topical applications are the most likely use to be successful and this is ideal for treating keratitis. Such peptides would also be active against drug-resistant pathogens and might act synergistically if used in combination therapy. Hundreds of peptides with antimicrobial properties have been isolated or synthesized but only a handful have been tested against ocular pathogens and even fewer have been tested in animal models. This review summarizes the currently available information on the use of peptides to treat keratitis, outlines some of the problems that have been identified, and discusses future studies that will be needed. Most of the peptides that have been tested have shown activity at concentrations that do not warrant further development, but 1 or 2 have promising activity raising the possibility that peptides can be developed to treat keratitis.
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Affiliation(s)
- Curtis R Brandt
- Departments of Ophthalmology and Visual Sciences and Medical Microbiology and Immunology, McPherson Eye Research Institute, University of Wisconsin School of Medicine and Public Health , Madison, Wisconsin
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Yang HY, Zheng NQ, Li DM, Gu L, Peng XM. Entecavir combined with furin inhibitor simultaneously reduces hepatitis B virus replication and e antigen secretion. Virol J 2014; 11:165. [PMID: 25224377 PMCID: PMC4177756 DOI: 10.1186/1743-422x-11-165] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2014] [Accepted: 09/11/2014] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The antiviral therapy of chronic hepatitis B virus (HBV) infection pursues the dual goals, virological response (undetectable serum HBV DNA) and hepatitis B e antigen (HBeAg) serological response (serum HBeAg loss/seroconversion). It is relatively difficult, however, to realize the serological response, especially for nucleotide/nucleoside analogs. Furin, a proprotein convertase, is involved in HBeAg maturation. The suppression of furin using inhibitors accordingly reduces HBeAg secretion, but possibly enhances HBV replication. For these reasons, the strategy based on the combination of nucleoside analog entecavir (ETV) and furin inhibitors to inhibit HBV replication and HBeAg secretion simultaneously were studied here. METHODS The suppression of furin was performed using inhibitors decanoyl-RVKR-chloromethylketone (CMK) and hexa-D-arginine (D6R) or the expression of furin inhibitory prosegment. The influence of furin suppression on HBV replication and the effect of CMK combined with nucleoside analog entecavir (ETV) on HBV replication and HBeAg secretion was investigated in HepG2.2.15 cells. HBeAg level in media was detected using enzyme-linked immunosorbent assay. Intracellular viral antigens and HBV DNA were detected using Western and Southern blotting analyses, respectively. RESULTS CMK, D6R and the expression of inhibitory prosegment all significantly reduced HBeAg secretion, but only CMK enhance HBV replication. Concordantly, only CMK post-transcriptionally accumulated cytosolic HBV replication-essential hepatitis B core antigen (HBcAg). The HBcAg-accumulating effect of CMK was further found to be resulted from its redundant inhibitory effect on the trypsin-like activity of cellular proteasomes that are responsible for HBcAg degradation. Moreover, the viral replication-enhancing effect of CMK was abrogated by ETV and ETV combined with CMK reduced HBV replication and HBeAg secretion simultaneously. CONCLUSION The suppression of furin itself does not enhance HBV replication. Nucleotide/nucleoside analogs combined with furin inhibitors may be a potential easy way to realize the dual goals of the antiviral therapy for chronic hepatitis B in the future.
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Affiliation(s)
| | | | | | | | - Xiao M Peng
- Hepatology Laboratory, the Hospital for Liver Disease, Sun Yat-Sen University, 600 Tianhe Road, Guangzhou 510630, China.
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Young GH, Huang TM, Wu CH, Lai CF, Hou CC, Peng KY, Liang CJ, Lin SL, Chang SC, Tsai PR, Wu KD, Wu VC, Ko WJ. Hemojuvelin modulates iron stress during acute kidney injury: improved by furin inhibitor. Antioxid Redox Signal 2014; 20:1181-94. [PMID: 23901875 PMCID: PMC3934545 DOI: 10.1089/ars.2013.5366] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
AIMS Free iron plays an important role in the pathogenesis of acute kidney injury (AKI) via the formation of hydroxyl radicals. Systemic iron homeostasis is controlled by the hemojuvelin-hepcidin-ferroportin axis in the liver, but less is known about this role in AKI. RESULTS By proteomics, we identified a 42 kDa soluble hemojuvelin (sHJV), processed by furin protease from membrane-bound hemojuvelin (mHJV), in the urine during AKI after cardiac surgery. Biopsies from human and mouse specimens with AKI confirm that HJV is extensively increased in renal tubules. Iron overload enhanced the expression of hemojuvelin-hepcidin signaling pathway. The furin inhibitor (FI) decreases furin-mediated proteolytic cleavage of mHJV into sHJV and augments the mHJV/sHJV ratio after iron overload with hypoxia condition. The FI could reduce renal tubule apoptosis, stabilize hypoxic induced factor-1, prevent the accumulation of iron in the kidney, and further ameliorate ischemic-reperfusion injury. mHJV is associated with decreasing total kidney iron, secreting hepcidin, and promoting the degradation of ferroportin at AKI, whereas sHJV does the opposite. INNOVATION This study suggests the ratio of mHJV/sHJV affects the iron deposition during acute kidney injury and sHJV could be an early biomarker of AKI. CONCLUSION Our findings link endogenous HJV inextricably with renal iron homeostasis for the first time, add new significance to early predict AKI, and identify novel therapeutic targets to reduce the severity of AKI using the FI.
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Affiliation(s)
- Guang-Huar Young
- 1 Department of Surgery, National Taiwan University Hospital , Taipei, Taiwan
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Pang YJ, Tan XJ, Li DM, Zheng ZH, Lei RX, Peng XM. Therapeutic potential of furin inhibitors for the chronic infection of hepatitis B virus. Liver Int 2013; 33:1230-8. [PMID: 23617302 DOI: 10.1111/liv.12185] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/22/2012] [Accepted: 04/01/2013] [Indexed: 12/21/2022]
Abstract
BACKGROUND & AIMS Hepatitis B e antigen (HBeAg) is essential for the development of chronic hepatitis B virus (HBV) infection. Furin, a proprotein convertase, plays a key role in processing of HBeAg precursor into maturated HBeAg. For these reasons, the therapeutic potential of furin inhibition for chronic HBV infection was studied. METHODS The effects of furin inhibitor I (decanoyl-RVKR-chloromethylketone, CMK) and furin inhibitor II (hexa-D-arginine, D6R) on HBeAg secretion, the destination of unprocessed precursor and cellular secretory functions were comparatively investigated. RESULTS CMK and D6R significantly decreased the supernatant level of HBeAg and increased the intracellular level of HBeAg precursor in HepG2.2.15 cells in vitro. The accumulated HBeAg precursor was not found to be retro-transported into the cytosol to inhibit HBV replication as expected, but was found to be expressed on the cell surface, where it may be more convenient to mediate host immune responses. Furthermore, these inhibitors at effective concentrations were not found to interfere with the maturations of albumin and prothrombin. Compared with CMK, D6R was suboptimal in effectiveness; however, D6R neither enhanced HBV replication through the accumulation of cytosolic HBcAg nor did it cause severe cell damage in an elongated safety analyses. CONCLUSION Furin inhibitors CMK and D6R reduce HBeAg secretion and increase cell surface expression of the HBeAg precursor in HepG2.2.15 cells. Novel furin inhibitors or modified forms of D6R may promote the reduction of immune tolerance and the elimination of infected hepatocytes in patients with chronic HBV infection.
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Affiliation(s)
- Yan J Pang
- Hepatology Laboratory, The Hospital for Liver Disease, Sun Yat-Sen University, Guangzhou, China
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Abstract
The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFβ (transforming growth factor β) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.
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Becker GL, Lu Y, Hardes K, Strehlow B, Levesque C, Lindberg I, Sandvig K, Bakowsky U, Day R, Garten W, Steinmetzer T. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. J Biol Chem 2012; 287:21992-2003. [PMID: 22539349 DOI: 10.1074/jbc.m111.332643] [Citation(s) in RCA: 91] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compounds containing an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with K(i) values of 16 and 8 pM, respectively. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compounds with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivatives were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 24 (4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide) resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.
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Affiliation(s)
- Gero L Becker
- Institute of Pharmaceutical Chemistry, Philipps University, 35032 Marburg, Germany
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Rogers JV, Price JA, Wendling MQS, Long JP, Bresler HS. Preliminary microRNA analysis in lung tissue to identify potential therapeutic targets against H5N1 infection. Viral Immunol 2012; 25:3-11. [PMID: 22233254 DOI: 10.1089/vim.2011.0047] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Within the past decade, human infections with the highly pathogenic avian influenza H5N1 have resulted in approximately 60% mortality and increased the need for vaccines and therapeutics. Understanding the molecular events associated with pathology can aid this effort; therefore, this study was conducted to assess microRNA (miRNA) expression in mouse lungs infected with H5N1 A/Vietnam/1203/04. Intranasal administration of 1500 median tissue culture infectious dose of H5N1 promoted differences in the number and expression pattern of miRNA from lung tissue collected at 2, 4, 6, 24, and 96 h post-exposure that mapped to common biological functions. Informatics analysis identified miRNA-specific predicted genes known to be therapeutic drug targets in which Furin was common to all time periods. This study provides insight into the differential miRNA expression with respect to the host-pathogen relationship and identification of potential therapeutic drug targets.
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Affiliation(s)
- Andrew W Artenstein
- Center for Biodefense and Emerging Pathogens, Department of Medicine, Memorial Hospital of Rhode Island, Pawtucket 02860, USA.
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Weldon JE, Pastan I. A guide to taming a toxin--recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J 2011; 278:4683-700. [PMID: 21585657 PMCID: PMC3179548 DOI: 10.1111/j.1742-4658.2011.08182.x] [Citation(s) in RCA: 181] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Pseudomonas exotoxin A (PE) is a highly toxic protein secreted by the opportunistic pathogen Pseudomonas aeruginosa. The modular structure and corresponding mechanism of action of PE make it amenable to extensive modifications that can redirect its potent cytotoxicity from disease to a therapeutic function. In combination with a variety of artificial targeting elements, such as receptor ligands and antibody fragments, PE becomes a selective agent for the elimination of specific cell populations. This review summarizes our current understanding of PE, its intoxication pathway, and the ongoing efforts to convert this toxin into a treatment for cancer.
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Affiliation(s)
- John E Weldon
- Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA
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40
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Couture F, D'Anjou F, Day R. On the cutting edge of proprotein convertase pharmacology: from molecular concepts to clinical applications. Biomol Concepts 2011; 2:421-438. [PMID: 22308173 DOI: 10.1515/bmc.2011.034] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
There is increasing interest in the therapeutic targeting of proteases for the treatment of important diseases. Additionally new protein-based therapeutic strategies have the potential to widen the available treatments against these pathologies. In the last decade, accumulated evidence has confirmed that the family of proteases known as proprotein convertases (PCs) are potential targets for viral infections, osteoarthritis, cancer and cardiovascular disease, among others. Nevertheless, there are still many unanswered questions about the relevance of targeting PCs in a therapeutic context, especially regarding the anticipated secondary effects of treatment, considering the observed embryonic lethality of some PC knockout mice. In this review, the benefits of PCs as pharmacological targets will be discussed, with focus on concepts and strategies, as well as on the state of advancement of actual and future inhibitors.
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Affiliation(s)
- Frédéric Couture
- Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada
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41
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Ozawa A, Lick AN, Lindberg I. Processing of proaugurin is required to suppress proliferation of tumor cell lines. Mol Endocrinol 2011; 25:776-84. [PMID: 21436262 DOI: 10.1210/me.2010-0389] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Augurin is a secretory molecule produced in pituitary, thyroid, and esophagus and implicated in a wide array of physiological processes, from ACTH release to tumor suppression. However, the specific proaugurin-derived peptides present in various cell types are not yet known. In order to shed light on the posttranslational modifications required for biological activity, we here describe the posttranslational processing of proaugurin in AtT-20 and Lovo cells and identify proaugurin-derived products generated by convertases. In vitro cleavage of proaugurin with proprotein convertases produced multiple peptides, including a major product with a mass of 9.7 kDa by mass spectrometry. Metabolic labeling of C-terminally tagged proaugurin in AtT-20 and AtT-20/PC2 cells resulted in a major 15-kDa tagged form on SDS-PAGE, which likely corresponds to the 9.7-kDa in vitro fragment, with the added tag, its linker, and posttranslational modification(s). The secretion of neither proaugurin nor this cleavage product was stimulated by forskolin, indicating its lack of storage in regulated secretory granules and lack of cleavage by PC2. Incubation of cells with the furin inhibitor nona-d-arginine resulted in impaired cleavage of proaugurin, whereas metalloprotease inhibitors did not affect proaugurin proteolysis. These data support the idea that proaugurin is cleaved by furin and secreted via the constitutive secretory pathway. Interestingly, proaugurin was sulfated during trafficking; sulfation was completely inhibited by brefeldin A. Proliferation assays with three different tumor cell lines demonstrated that only furin-cleaved proaugurin could suppress cell proliferation, suggesting that proteolytic cleavage is a posttranslational requirement for proaugurin to suppress cell proliferation.
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Affiliation(s)
- Akihiko Ozawa
- Department of Anatomy and Neurobiology, University of Maryland-Baltimore, 20 Penn Street, Baltimore, Maryland 21201, USA
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42
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Effects of retroviral envelope-protein cleavage upon trafficking, incorporation, and membrane fusion. Virology 2010; 405:214-24. [PMID: 20591459 DOI: 10.1016/j.virol.2010.06.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2010] [Revised: 03/01/2010] [Accepted: 06/01/2010] [Indexed: 11/20/2022]
Abstract
Retroviral envelope glycoproteins undergo proteolytic processing by cellular subtilisin-like proprotein convertases at a polybasic amino-acid site in order to produce the two functional subunits, SU and TM. Most previous studies have indicated that envelope-protein cleavage is required for rendering the protein competent for promoting membrane fusion and for virus infectivity. We have investigated the role of proteolytic processing of the Moloney murine leukemia virus envelope-protein through site-directed mutagenesis of the residues near the SU-TM cleavage site and have established that uncleaved glycoprotein is unable either to be incorporated into virus particles efficiently or to induce membrane fusion. Additionally, the results suggest that cleavage of the envelope protein plays an important role in intracellular trafficking of protein via the cellular secretory pathway. Based on our results it was concluded that a positively charged residue located at either P2 or P4 along with the arginine at P1 is essential for cleavage.
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43
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Remacle AG, Gawlik K, Golubkov VS, Cadwell GW, Liddington RC, Cieplak P, Millis SZ, Desjardins R, Routhier S, Yuan XW, Neugebauer WA, Day R, Strongin AY. Selective and potent furin inhibitors protect cells from anthrax without significant toxicity. Int J Biochem Cell Biol 2010; 42:987-95. [PMID: 20197107 PMCID: PMC2862824 DOI: 10.1016/j.biocel.2010.02.013] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2010] [Accepted: 02/22/2010] [Indexed: 01/08/2023]
Abstract
Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions.
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Affiliation(s)
| | | | | | | | | | - Piotr Cieplak
- Burnham Institute for Medical Research, La Jolla, CA 92037
| | | | - Roxane Desjardins
- Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé and Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
| | - Sophie Routhier
- Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé and Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
| | - Xue Wen Yuan
- Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé and Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
| | - Witold A. Neugebauer
- Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé and Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
| | - Robert Day
- Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé and Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4
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44
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Morlon-Guyot J, Méré J, Bonhoure A, Beaumelle B. Processing of Pseudomonas aeruginosa exotoxin A is dispensable for cell intoxication. Infect Immun 2009; 77:3090-9. [PMID: 19380469 PMCID: PMC2708563 DOI: 10.1128/iai.01390-08] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2008] [Revised: 01/07/2009] [Accepted: 04/13/2009] [Indexed: 01/09/2023] Open
Abstract
Exotoxin A is a major virulence factor of Pseudomonas aeruginosa. This toxin binds to a specific receptor on animal cells, allowing endocytosis of the toxin. Once in endosomes, the exotoxin can be processed by furin to generate a C-terminal toxin fragment that lacks the receptor binding domain and is retrogradely transported to the endoplasmic reticulum for retrotranslocation to the cytosol through the Sec61 channel. The toxin then blocks protein synthesis by ADP ribosylation of elongation factor 2, thereby triggering cell death. A shorter intracellular route has also been described for this toxin. It involves direct translocation of the entire toxin from endosomes to the cytosol and therefore does not rely on furin-mediated cleavage. To examine the implications of endosomal translocation in the intoxication process, we investigated whether the toxin required furin-mediated processing in order to kill cells. We used three different approaches. We first fused to the N terminus of the toxin proteins with different unfolding abilities so that they inhibited or did not inhibit endosomal translocation of the chimera. We then assayed the amount of toxin fragments delivered to the cytosol during cell intoxication. Finally we used furin inhibitors and examined the fate and intracellular localization of the toxin and its receptor. The results showed that exotoxin cytotoxicity results largely from endosomal translocation of the entire toxin. We found that the C-terminal fragment was unstable in the cytosol.
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45
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Rholam M, Fahy C. Processing of peptide and hormone precursors at the dibasic cleavage sites. Cell Mol Life Sci 2009; 66:2075-91. [PMID: 19300906 PMCID: PMC11115611 DOI: 10.1007/s00018-009-0007-5] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2008] [Revised: 02/11/2009] [Accepted: 02/17/2009] [Indexed: 01/31/2023]
Abstract
Many functionally important cellular peptides and proteins, including hormones, neuropeptides, and growth factors, are synthesized as inactive precursor polypeptides, which require post-translational proteolytic processing to become biologically active polypeptides. This is achieved by the action of a relatively small number of proteases that belong to a family of seven subtilisin-like proprotein convertases (PCs) including furin. In view of this, this review focuses on the importance of privileged secondary structures and of given amino acid residues around basic cleavage sites in substrate recognition by these endoproteases. In addition to their participation in normal cell functions, PCs are crucial for the initiation and progress of many important diseases. Hence, these proteases constitute potential drug targets in medicine. Accordingly, this review also discusses the approaches used to shed light on the cleavage preference and the substrate specificity of the PCs, a prerequisite to select which PCs are promising drug targets in each disease.
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Affiliation(s)
- Mohamed Rholam
- Interfaces, Traitements, Organisation et Dynamique des Systrèmes, Université Paris Diderot (Paris 7), CNRS UMR 7086, Bâtiment Lavoisier, 15 rue Jean-Antoine de Baïf, 75205, Paris Cedex 13, France.
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46
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Wang S, Han J, Wang Y, Lu W, Chi C. Design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2. Acta Biochim Biophys Sin (Shanghai) 2008. [PMID: 18850049 PMCID: PMC7110110 DOI: 10.1111/j.1745-7270.2008.00470.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
The mammalian proprotein convertase furin has been found to play an important role in diverse physiological and pathological events, such as the activation of viral glycoproteins and bacterial exotoxins. Small, non‐toxic and highly active, furin inhibitors are considered to be attractive drug candidates for diseases caused by virus and bacteria. In this study, a series of peptide inhibitors were designed and synthesized based on the C‐terminal fragment of histone H1.2, which has an inhibitory effect on furin. Replacing the reactive site of inhibitors with the consensus substrate recognition sequence of furin has been found to increase inhibitory activity greatly. The most potent inhibitor, I4, with 14 amino acid residues has a Ki value of 17 nM for furin. Although most of the synthesized peptides were temporary inhibitors, the inhibitor I5, with nine amino acids, retained its full potency, even after a 3 h incubation period with furin at 37 °C. These inhibitors may potentially lead to the development of anti‐viral and antibacterial drug compounds.
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Affiliation(s)
- Suming Wang
- School of Life Sciences, University of Science and Technology of China, Anhui 230027, China
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47
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Chrétien M, Seidah NG, Basak A, Mbikay M. Proprotein convertases as therapeutic targets. Expert Opin Ther Targets 2008; 12:1289-300. [DOI: 10.1517/14728222.12.10.1289] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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Yedavalli VS, Zhang N, Cai H, Zhang P, Starost MF, Hosmane RS, Jeang KT. Ring expanded nucleoside analogues inhibit RNA helicase and intracellular human immunodeficiency virus type 1 replication. J Med Chem 2008; 51:5043-51. [PMID: 18680273 PMCID: PMC2537995 DOI: 10.1021/jm800332m] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
A series of ring expanded nucleoside (REN) analogues were synthesized and screened for inhibition of cellular RNA helicase activity and human immunodeficiency virus type 1 (HIV-1) replication. We identified two compounds, 1 and 2, that inhibited the ATP dependent activity of human RNA helicase DDX3. Compounds 1 and 2 also suppressed HIV-1 replication in T cells and monocyte-derived macrophages. Neither compound at therapeutic doses was significantly toxic in ex vivo cell culture or in vivo in mice. Our findings provide proof-of-concept that a cellular factor, an RNA helicase, could be targeted for inhibiting HIV-1 replication.
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Affiliation(s)
- Venkat S.R.K Yedavalli
- Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
| | - Ning Zhang
- Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
| | - Hongyi Cai
- Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
| | - Peng Zhang
- Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
| | - Matthew F. Starost
- Veterinary Pathologist, Division of Veterinary Resources, National Institutes of Health, Bethesda, Maryland 20892
| | - Ramachandra S. Hosmane
- Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
| | - Kuan-Teh Jeang
- Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
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Remacle AG, Shiryaev SA, Oh ES, Cieplak P, Srinivasan A, Wei G, Liddington RC, Ratnikov BI, Parent A, Desjardins R, Day R, Smith JW, Lebl M, Strongin AY. Substrate cleavage analysis of furin and related proprotein convertases. A comparative study. J Biol Chem 2008; 283:20897-906. [PMID: 18505722 DOI: 10.1074/jbc.m803762200] [Citation(s) in RCA: 110] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R downward arrow and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R downward arrow motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.
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Affiliation(s)
- Albert G Remacle
- Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
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Bontemps Y, Scamuffa N, Calvo F, Khatib A. Potential opportunity in the development of new therapeutic agents based on endogenous and exogenous inhibitors of the proprotein convertases. Med Res Rev 2007; 27:631-48. [PMID: 17019676 PMCID: PMC7168524 DOI: 10.1002/med.20072] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The proprotein convertases (PCs) are responsible for the endoproteolytic processing of various protein precursors (e.g., growth factors, receptors, adhesion molecules, and matrix metalloproteinases) implicated in several diseases such as obesity, diabetes, atherosclerosis, cancer, and Alzheimer disease. The potential clinical and pharmacological role of the PCs has fostered the development of various PC‐inhibitors. In this review we summarized the recent findings on PCs inhibitors, their mode of actions and potential use in the therapy of various diseases. © 2006 Wiley Periodicals, Inc. Med Res Rev, 27, No. 5, 631–648, 2007
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Affiliation(s)
- Yannick Bontemps
- INSERM, U 716, Equipe AVENIR, Institut de Génétique Moléculaire, Paris 75010, France
- Université Paris 7, Paris 75251, France
| | - Nathalie Scamuffa
- INSERM, U 716, Equipe AVENIR, Institut de Génétique Moléculaire, Paris 75010, France
- Université Paris 7, Paris 75251, France
| | - Fabien Calvo
- INSERM, U 716, Equipe AVENIR, Institut de Génétique Moléculaire, Paris 75010, France
- Université Paris 7, Paris 75251, France
| | - Abdel‐Majid Khatib
- INSERM, U 716, Equipe AVENIR, Institut de Génétique Moléculaire, Paris 75010, France
- Université Paris 7, Paris 75251, France
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