Stable benzotriazole esters as mechanism-based inactivators of the severe acute respiratory syndrome 3CL protease

Abstract

Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 fatalities in 2003. Currently, there is no effective treatment for this epidemic. SARS-3CL(pro) has been shown to be essential for replication and is thus a target for drug discovery. Here, a class of stable benzotriazole esters was reported as mechanism-based inactivators of 3CL(pro), and the most potent inactivator exhibited a k(inact) of 0.0011 s(-1) and a K(i) of 7.5 nM. Mechanistic investigation with kinetic and mass spectrometry analyses indicates that the active site Cys145 is acylated, and that no irreversible inactivation was observed with the use of the C145A mutant. In addition, a noncovalent, competitive inhibition became apparent by using benzotriazole ester surrogates in which the bridged ester-oxygen group is replaced with carbon.

Figure 1

Microtiter Plate-Based Reactions and In Situ Screening (A) Reactions of 1 or 2 with 90 acids in microtiter plates, followed by in situ screening for inhibitors of SARS-CoV 3CL^pro. (B) Reactions of HBTU and 90 acids in microtiter plates, followed by in situ screening for inhibitors of SARS-CoV 3CL^pro. (C) Molecular structures of inhibitors 3–10 against SARS-CoV 3CL^pro.

Figure 2

Kinetic Studies of Inhibitor 4 and SARS-CoV 3CL^pro (A) The progress curves in the presence of 0.3–3.0 μM inhibitor for reactions initiated by adding enzyme (final concentration of 0.05 μM) into a mixture of substrate (6 μM) and inhibitor 4. Over the entire 5 min time window, the uninhibited enzyme displayed a linear progress curve, whereas the inhibited enzyme with a different concentration of inhibitor showed a time-dependent reduction of activity. (B) The same experiments as performed in (A), but with 1 mM DTT in the preincubation mixture. (C) Preincubation time dependence of the fractional velocity of the protease-catalyzed reaction in the presence of 0.02–0.2 μM time-dependent inhibitor 4. (D) Kitz and Wilson replot of the half-life (t_1/2) of enzyme inactivation as a function of the reciprocal of the slow inactivator concentration. The k_inact is 0.0013 s⁻¹ and K_i is 17.4 nM for the time-dependent inactivator 4 based on the kinetic data.

Figure 3

MALDI Spectrum of Inhibitor 4 and SARS 3CL^pro (A) MALDI spectrum of tryptic 3CL^pro. (B) MALDI spectrum of tryptic acylated 3CL^pro. (C) MALDI MS/MS spectra of T15 and acylated T15 peptides (GSFLNGSC^∗GSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGK) showed a mass shift of 147.2 Da (4149.6 Da versus 4296.8 Da) on b₃₉, indicating that Cys145 (C^∗) is the acylation site.

Figure 4

Proposed Mechanism for Inhibition of SARS-CoV 3CL^pro by Acylation with Benzotriazole Esters

Figure 5

Synthesis of Noncovalent Inhibitors (A) Synthesis of compounds 13–20 and their IC₅₀s or K_is for SARS-CoV 3CL^pro. (B) The equilibrium structures of the benzotriazole compounds in solution.

Figure 6

A Modeling Complex of SARS 3CL^pro and Benzotriazole Esters Binding modes of compounds 3 (yellow), 4 (red), 8 (blue), and 9 (green) in the active site of SARS-CoV 3CL^pro (PDB luk4). Models were generated by Autodock and displayed by MGLTOOLS (MGL, Scripps).