Thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus

Abstract The papain-like protease of severe acute respiratory syndrome coronavirus (PLpro) (EC 3.4.22.46) is essential for the viral life cycle and therefore represents an important antiviral target. We have identified 6MP and 6TG as reversible and slow-binding inhibitors of SARS-CoV PLpro, which is the first report about small molecule reversible inhibitors of PLpro. The inhibition mechanism was investigated by kinetic measurements and computer docking. Both compounds are competitive, selective, and reversible inhibitors of the PLpro with K is values ∼10 to 20μM. A structure–function relationship study has identified the thiocarbonyl moiety of 6MP or 6TG as the active pharmacophore essential for these inhibitions, which has not been reported before. The inhibition is selective because these compounds do not exert significant inhibitory effects against other cysteine proteases, including SARS-CoV 3CLpro and several cathepsins. Thus, our results present the first potential chemical leads against SARS-CoV PLpro, which might be used as lead compounds for further optimization to enhance their potency against SARS-CoV. Both 6MP and 6TG are still used extensively in clinics, especially for children with acute lymphoblastic or myeloblastic leukemia. In light of the possible inhibition against subset of cysteine proteases, our study has emphasized the importance to study in depth these drug actions in vivo.

[1]  G. Elion,et al.  The purine path to chemotherapy , 1989, In Vitro Cellular & Developmental Biology.

[2]  H. Hsieh,et al.  Antiviral drug discovery against SARS-CoV. , 2006, Current medicinal chemistry.

[3]  W. Hong,et al.  Understanding the accessory viral proteins unique to the severe acute respiratory syndrome (SARS) coronavirus , 2006, Antiviral Research.

[4]  S. Diamond,et al.  Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[5]  René Bernards,et al.  A Genomic and Functional Inventory of Deubiquitinating Enzymes , 2005, Cell.

[6]  E. De Clercq,et al.  Strategies in the design of antiviral drugs , 2002, Nature reviews. Drug discovery.

[7]  Chao-Hsiung Lin,et al.  Quaternary structure of the severe acute respiratory syndrome (SARS) coronavirus main protease. , 2004, Biochemistry.

[8]  W. Evans,et al.  Altered mercaptopurine metabolism, toxic effects, and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphocytic leukemia. , 1991, The Journal of pediatrics.

[9]  R. Baric,et al.  Animal models and vaccines for SARS-CoV infection , 2007, Virus Research.

[10]  R. Ménard,et al.  The Papain-Like Protease from the Severe Acute Respiratory Syndrome Coronavirus Is a Deubiquitinating Enzyme , 2005, Journal of Virology.

[11]  Y. Tsunetsugu-Yokota,et al.  Severe acute respiratory syndrome (SARS) coronavirus: application of monoclonal antibodies and development of an effective vaccine , 2006, Reviews in medical virology.

[12]  Yigong Shi,et al.  Structure and mechanisms of the proteasome‐associated deubiquitinating enzyme USP14 , 2005, The EMBO journal.

[13]  W. Syu,et al.  Purification of papain by affinity chromatography. , 1983, Journal of Chromatography A.

[14]  R. Weinshilboum,et al.  Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia , 1990, The Lancet.

[15]  Ralph Baric,et al.  A Mouse-Adapted SARS-Coronavirus Causes Disease and Mortality in BALB/c Mice , 2007, PLoS pathogens.

[16]  Muyang Li,et al.  Crystal Structure of a UBP-Family Deubiquitinating Enzyme in Isolation and in Complex with Ubiquitin Aldehyde , 2002, Cell.

[17]  A. Danchin,et al.  The Severe Acute Respiratory Syndrome , 2003 .

[18]  V. Turk,et al.  Bovine stefin C, a new member of the stefin family. , 1993, Journal of Biological Chemistry.

[19]  M. Otto,et al.  Inhibition of Severe Acute Respiratory Syndrome-Associated Coronavirus (SARSCoV) by Calpain Inhibitors and β-D-N4-Hydroxycytidine , 2004, Antiviral chemistry & chemotherapy.

[20]  Herman Tse,et al.  Identification of Novel Small-Molecule Inhibitors of Severe Acute Respiratory Syndrome-Associated Coronavirus by Chemical Genetics , 2004, Chemistry & Biology.

[21]  Paul Garner,et al.  SARS: Systematic Review of Treatment Effects , 2006, PLoS medicine.

[22]  R. Weinshilboum.  Human pharmacogenetics: Introduction. , 1984, Federation proceedings.

[23]  Jie Zhou,et al.  The Adamantane-Derived Bananins Are Potent Inhibitors of the Helicase Activities and Replication of SARS Coronavirus , 2005, Chemistry & Biology.

[24]  Ching-Hon Pui,et al.  Molecular Diagnosis of Thiopurine S-Methyltransferase Deficiency: Genetic Basis for Azathioprine and Mercaptopurine Intolerance , 1997, Annals of Internal Medicine.

[25]  M. Drebot,et al.  Potent and selective inhibition of SARS coronavirus replication by aurintricarboxylic acid , 2004, Biochemical and Biophysical Research Communications.

[26]  A. Haas,et al.  Deubiquitinating Function of Adenovirus Proteinase , 2002, Journal of Virology.

[27]  I. A. Rose,et al.  Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin. , 1986, The Journal of biological chemistry.

[28]  W. Hong,et al.  Characterization of viral proteins encoded by the SARS-coronavirus genome , 2004, Antiviral Research.

[29]  Bryan L. Roth,et al.  Finding New Tricks For Old Drugs: An Efficient Route For Public-Sector Drug Discovery , 2005, Nature Reviews Drug Discovery.

[30]  R. Hyslop,et al.  Metabolism of 6-thiopurines. I. Irreversible binding of a metabolite of 6-thiopurine to mammalian hepatic protein in vitro. , 1981, The Journal of pharmacology and experimental therapeutics.

[31]  V. Turk,et al.  Autocatalytic processing of recombinant human procathepsin B is a bimolecular process , 1999, FEBS letters.

[32]  J. Weber.  Adenovirus endopeptidase and its role in virus infection. , 1995, Current topics in microbiology and immunology.

[33]  Raymond C Stevens,et al.  Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Ming Yan,et al.  Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection , 2004, Nature Medicine.

[35]  V. Turk,et al.  Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli. , 1996, European journal of biochemistry.

[36]  Zhongbin Chen,et al.  The Papain-Like Protease of Severe Acute Respiratory Syndrome Coronavirus Has Deubiquitinating Activity , 2005, Journal of Virology.

[37]  John L. Sullivan,et al.  Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus , 2003, Nature.

[38]  C. Walsh,et al.  The behavior and significance of slow-binding enzyme inhibitors. , 2006, Advances in enzymology and related areas of molecular biology.

[39]  Xin Chen,et al.  Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition. , 2005, Biochemistry.

[40]  R. Hyslop,et al.  Metabolism of 6-thiopurines. II. Covalent binding of a 6-thiopurine metabolite to mammalian tissue protein in vivo. , 1981, The Journal of pharmacology and experimental therapeutics.

[41]  Zihe Rao,et al.  Structural insights into SARS coronavirus proteins , 2005, Current Opinion in Structural Biology.

[42]  D. Brömme,et al.  Production and activation of recombinant papain-like cysteine proteases. , 2004, Methods.

[43]  A. Hershko,et al.  Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[44]  B. Turk.  Targeting proteases: successes, failures and future prospects , 2006, Nature Reviews Drug Discovery.

[45]  Lirong Chen,et al.  Small Molecules Blocking the Entry of Severe Acute Respiratory Syndrome Coronavirus into Host Cells , 2004, Journal of Virology.