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.

Keywords: 6-Mercaptopurine; 6-Thioguanine; MERS-CoV papain-like protease; Mycophenolic acid; Synergistic inhibition.

Fig. 1

Inhibition of coronaviral PL^pro proteolytic activity by thiopurines, NEM, or mycophenolic acid. The proteolytic activity of MERS-CoV and SARS-CoV PL^pro, as well as the E168R mutant of SARS-CoV PL^pro in the presence of 6MP (8–50 μM), 6TG (6–50 μM), NEM (50–500 μM) or mycophenolic acid (50–2000 μM) were measured, respectively. The concentrations of peptidyl substrate (Dabcyl–FRLKGGAPIKGV–Edans) was 20 μM, while the concentration of MERS-CoV PL^pro, SARS-CoV PL^pro, and its E168R mutant was 1, 0.1, and 0.3 μM, respectively. MPA: mycophenolic acid.

Fig. 2

Inhibition of MERS-CoV PL^pro by 6MP (A), 6TG (B), or mycophenolic acid (C). The proteolytic activity of PL^pro was measured in the presence of different peptide substrate concentration (4–50 μM) and various inhibitor concentrations (0–50 μM for 6MP or 6TG and 0–500 μM for mycophenolic acid). The solid lines in panel A and B are the best fit by the competitive inhibition (Eq. (1)) and those in panel C are the best-fit by the noncompetitive inhibition (Eq. (2)). The R_sqr are 0.978, 0.979, and 0.988, respectively. All assays were repeated several times to ensure reproducibility. The kinetic parameters such as K_m, k_cat and K_is from the best fit were shown in Table 2.

Fig. 3

Mutual effect of the coronaviral PL^pro inhibitors. Two inhibitor patterns for mycophenolic acid with either 6MP (A) or 6TG (B) present, and those for NEM with either 6MP (C) or mycophenolic acid (D) present were performed. The concentrations of peptidyl substrate and enzyme were 15 and 1 μM, respectively. The points are the reciprocal of the experimental velocities, and the lines are the best fit of the data to Eq. (3). The results suggest that α value of the four experiments (A–D) is 0.73, 0.19, 2.7 × 10⁷ and 0.22, respectively.

Fig. 4

Docking of 6MP and mycophenolic acid with MERS-CoV PL^pro. The docking for 6MP (carbons colored by orange) and mycophenolic acid (magenta) with MERS-CoV PL^pro (cyan; PDB code: 4PT5) were performed with DS modeling 1.7 software (Accelrys). The docked models and that for 6MP with SARS-CoV PL^pro (gray) are then overlaid for comparison. The dotted lines show the hydrophilic interactions between PL^pro and inhibitors. 6MP is able to be targeted to the catalytic triad of MERS-CoV PL^pro. Instead, mycophenolic acid is located far from the catalytic triad and shows hydrogen bonding interactions with the residues Asp164 and Arg168, which are Gly164 and Glu168 in SARS-CoV PL^pro. According to the structure of SARS-CoV PL^pro in complex with Ub, the docked site of mycophenolic acid should be near the putative S3-S4 subsites, whereas 6MP may occupy S1’ subsite (Chou et al., 2014). The figure was produced using PyMol (http://www.pymol.org). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)