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 approximately 10 to 20 microM. 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.

Fig. 1

Inhibition of PLpro deubiquitination activity by thiopurines and Zn²⁺. The percentages of PLpro activity remaining after treatment with 6MP, 6TG, or Zn²⁺ were measured. The fluorogenic substrate ubiquitin–AMC (1 μM) was used as the substrate.

Fig. 2

Docking and inhibition mechanism of 6MP and 6TG with PLpro. 6MP (colored blue) and 6TG (green) could be targeted to the active site of PLpro. The distances between the sulfur atom of the active-site Cys1651 and the sulfur atoms of 6MP or 6TG were all 3.4 Å.

Fig. 3

Competitive inhibition of PLpro by 6MP or 6TG. (A) 6MP; (B) 6TG. The inset figures represent [6MP] or [6TG] vs. slope replots. The enzymatic activity of PLpro was measured in the presence of different peptide substrate concentrations (5–50 μM) and various inhibitor concentrations: 0 (closed circles), 2 (open circles), 10 (closed triangles), 25 (open triangles), 35 (closed squares), 50 (open squares), 100 (closed diamonds) μM. The solid lines are the best fit by the competitive inhibition equation (Eq. (2)). R_sqr = 0.920 and 0.929, respectively. The same experiments were repeated at least three times. The kinetic parameters such as K_m, k_cat and K_is from the best fit were shown in Table 2.

Fig. 4

Time-dependent inactivation of PLpro by 6MP and 6TG. (A) Different concentrations of 6MP (2 μM, closed circles; 25 μM, triangles; 35 μM, diamonds; 50 μM, squares; 100 μM, invert triangles; and 200 μM, open circles) was incubated with PLpro for 2 h. E_t/E₀ indicates the residual enzymatic activity at time t. The solid lines show the best semi-log fits of the data at different 6MP concentrations by pseudo-first-order rate equation (Eq. (3)). (B) Different concentrations of 6TG (0 μM, closed circles; 5–100 μM, open circles) were incubated with PLpro for 10 min. The solid lines were the best-fit results according to the slow-binding equation (Eq. (4)). (C and D) The observed inactivation rate constants (k_inact) from panels A and B were replotted against 6MP or 6TG concentration. The solid line is the fit of the saturation equation (Eq. (5)) (R_sqr = 0.992 and 0.975, respectively). The best-fit parameters (K_inact and k_max) are shown in Table 2. The same experiments were repeated at least three times.

Fig. 5

Kinetic mechanism of inhibitors. The upper part denotes enzyme inhibition and inactivation by 6TG (or 6MP). The lower part shows the putative hydrolysis of peptide substrate pathway. The original peptide substrate Dabcyl-FRLKGGAPIKGV-Edans (or ubiquitin–AMC) was cleaved at the Gly–Ala (or Gly–AMC) peptide bond. The N-terminal half (or AMC) was released as the first product while the C-terminal half (or ubiquitin) acylated the active site Cys1651, which was then deacylated and completed the catalytic cycle.