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Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus.

ABSTRACT: 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.

SUBMITTER: Cheng KW

PROVIDER: S-EPMC7113672 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus.

Cheng Kai-Wen KW Cheng Shu-Chun SC Chen Wei-Yi WY Lin Min-Han MH Chuang Shang-Ju SJ Cheng I-Hsin IH Sun Chiao-Yin CY Chou Chi-Yuan CY

Antiviral research 20141224

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 ...[more]

PMID: 25542975

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Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant ongoing threat to global human health. As part of its viral genome, MERS-CoV encodes a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize type I interferon (IFN-I) immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon-stimulated gene product 15 (hISG15) was probed with isothermal calorimetry, which suggests that the C-terminal domain of hISG15 is principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 Å in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs.IMPORTANCE Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact on its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe the pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.

Project description:UnlabelledThe papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition.ImportanceUnlocking the secrets of how coronavirus (CoV) papain-like proteases (PLpros) perform their multifunctional roles during viral replication entails a complete mechanistic understanding of their substrate recognition and enzymatic activities. We show that the PLpro domains from the MERS and SARS coronaviruses can recognize and process the same substrates, but with different catalytic efficiencies. The differences in substrate recognition between these closely related PLpros suggest that neither enzyme can be used as a generalized model to explain the kinetic behavior of all CoV PLpros. As a consequence, decoding the mechanisms of PLpro-mediated antagonism of the host innate immune response and the development of anti-CoV PLpro enzyme inhibitors will be a challenging undertaking. The results from this study provide valuable information for understanding how MERS-CoV PLpro-mediated antagonism of the host innate immune response is orchestrated, as well as insight into the design of inhibitors against MERS-CoV PLpro.

Dataset Information

Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus.

Publications

Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus.

Similar Datasets

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