RESUMO
BACKGROUNDS: A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated. RESULTS: Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference. CONCLUSIONS: Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Proteínas Virais/química , Proteínas Virais/genética , Antivirais/química , Proteases 3C de Coronavírus , Cisteína Endopeptidases/biossíntese , Europa (Continente) , Regulação Viral da Expressão Gênica , Humanos , Íons/química , Metais/química , Processamento de Proteína Pós-Traducional , Proteólise , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/biossínteseRESUMO
Ubiquitin-specific protease 2 (USP2) belongs to the family of deubiquitinases that can rescue protein targets from proteasomal degradation by reversing their ubiquitination. In various cancers, including prostate cancer and ovarian carcinoma, upregulation of USP2 leads to an increase in the levels of deubiquitinated substrates such as fatty acid synthase, MDM2, cyclin D1 and Aurora-A. USP2 thus plays a critical role in tumor cells' survival and therefore represents a therapeutic target. Here a leukemia drug, 6-thioguanine, was found to be a potent inhibitor of USP2. Enzyme-kinetic and X-ray crystallographic data suggest that 6-thioguanine displays a noncompetitive and slow-binding inhibitory mechanism against USP2. Our study provides a clear rationale for the clinical evaluation of 6-thioguanine for USP2-upregulated cancers.
Assuntos
Endopeptidases/metabolismo , Tioguanina/farmacologia , Cristalografia por Raios X/métodos , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/metabolismo , Humanos , Cinética , Processamento de Proteína Pós-Traducional , Tioguanina/metabolismo , Tioguanina/farmacocinética , Ubiquitina Tiolesterase , Ubiquitinação/fisiologiaRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) is a new highly pathogenic human coronaviruses that emerged in Jeddah and Saudi Arabia and has quickly spread to other countries in Middle East, Europe and North Africa since 2012. Up to 17 December 2014, it has infected at least 938 people with a fatality rate of about 36% globally. This has resulted in an urgent need to identify antiviral drugs that are active against MERS-CoV. The papain-like protease (PL(pro)) of MERS-CoV represents an important antiviral target as it is not only essential for viral maturation, but also antagonizes interferon stimulation of the host via its deubiquitination activity. Here, we report the discovery that two SARS-CoV PL(pro) inhibitors, 6-mercaptopurine (6MP) and 6-thioguanine (6TG), as well as the immunosuppressive drug mycophenolic acid, are able to inhibit MERS-CoV PL(pro). Their inhibition mechanisms and mutually binding synergistic effect were also investigated. Our results identify for the first time three inhibitors targeting MERS-CoV PL(pro) and these can now be used as lead compounds for further antiviral drug development.