Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (M^pro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against M^pro (K_i: 1-10 μM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC₅₀: 1-12 μM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the M^pro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.

Fig. 1. Synthetic route for the preparation of the nitroalkene compounds used in this study.

Inhibitors FGA145, FGA146 and FGA147 were prepared through a common synthetic route starting from Boc-L-glutamic acid. FGA77, FGA86 and FGA159 were prepared through a synthetic route starting from Boc-L-homophenyl alaninal.

Fig. 2. Inhibition of SARS-CoV-2 infection in Huh-7-ACE2 cell by FGA145, FGA146 and FGA147.

Cytotoxicity assays for the three compounds (left column), all of them presented a CC₅₀ greater than 100 μM. Effect of the three compounds on the virus titer (center and right columns); FGA146 was the most potent inhibitor with an EC₅₀ of 0.9 μM, followed by FGA147 and FGA145 with EC₅₀ of 1.9 and 11.7 μM, respectively. All graphs show means ± s.d.; asterisks indicate p values (*p ≤ 0.05) obtained by two-tailed unpaired t tests.

Fig. 3. Crystal structures of SARS-CoV-2 M^pro in complex with inhibitors.

M^pro is shown in ribbon and the inhibitors in ball and stick representation. 2Fo-Fc electron density map contoured at 1σ (shown in gray mesh) of FGA146 (a) and FGA147 (b) bound covalently to the catalytic cysteine (Cys145). c Electrostatic surface representation of the active site of M^pro with bound FGA146 (violet) and FGA147 (light blue). Red indicates negative charge and blue positive charge.

Fig. 4. Conformational changes in M^pro upon binding of the inhibitors.

a Most significant changes in the active site and domain I are located at P2 helix and the P5 loop. Smaller changes can be observed at the P4 β-hairpin flap, loop I and loop II. b Superposition of M^pro in complex with FGA146 and FGA147. Monomer A and B from the complex with FGA146 are shown in light green and yellow orange, respectively, and from the complex with FGA147 are shown in salmon and light blue, respectively. Significant displacements of some of the helices from the dimerization domain (Domain III, circles) of monomer A can be observed, while the same domain from monomer B does not show these displacements. c Superposition of monomer A (light green) and B (yellow orange) of the M^pro in complex with FGA146. The helical dimerization domain (Domain III) shows differences in the relative positions of some of the helices. Four of them show significant displacements (α-6, α-7, α-8 and α-9) while the las helix (α-10) does not show any significant displacement.

Fig. 5. Proposed Mechanism of SARS-CoV-2 M^pro Cysteine Protease Inhibition by nitroalkene compounds.

The mechanism has three steps: entry of the inhibitor into the active site, then addition of the cysteine thiolate, and finally protonation of the nitronate.

Fig. 6. Structures of the states. Detail of the M06-2X/6-31 + G(d,p)/MM optimized structures of the states located along the inhibition reaction of M^pro by FGA146 (top panels) and FGA147 (bottom panels).

Carbon atoms of the inhibitor are shown in yellow, and those of the catalytic residues C145 and H41 are shown in cyan. Key distances are in Å.

Fig. 7. Effect of the inhibitors on the thermal stability of M^pro.

a Thermal stability of M^pro in the presence of FGA146 using circular dichroism. b Thermal stability of M^pro in the presence of FGA147 using circular dichroism. The T_m value of M^pro in the absence of inhibitors (black squares) was 49.2 °C, while in the presence of 25 (blue circles) and 100 μM (green triangles) of FGA146, the values decreased to 45.0 and 42.8 °C, respectively. In the case of FGA147, these values decreased to 46.2 and 45.0 °C, respectively. c Change of the T_m in the presence of the inhibitors. FGA86 shows a slight increase in stability at the lower concentration (25 μM) and a decrease in stability at the higher concentration (100 μM). There is no significant change in stability in the presence of FGA177; while in the presence of FGA145, FGA146 and FGA147 shows a significant decrease in stability, suggesting that they could bind covalently to the protein. The presence of FGA159 increased the stability of the protein. The error bars represent the fitting error of the denaturation data.

Fig. 8. Binding of inhibitors to M^pro.

a Binding isotherm of FGA145 (black squares) and FGA146 (blue circles and green triangles) to M^pro. The concentration of FGA145 was measured by absorption, and that of FGA146 was measured by absorption (blue circles) and fluorescence (green triangles). All data are mean values ± standard deviation of two technical replicates. b ITC binding profile of FGA147 to M^pro.

Fig. 9. Reversibility of the binding of the inhibitor FGA146 to M^pro.

In black a DMSO control with no inhibitor added is shown, in green Nirmatrelvir as a reversible control and in blue the compound FGA146. All samples were measured as triplicates. Due to substrate depletion over time only the first 1000 s are shown.

Fig. 10. Chemical structure of LM188 and Nirmatrelvir.

Both were used as controls in the reversibility experiments: inhibitor LM188 was used as an irreversible inhibitor and Nirmatrelvir as a reversible inhibitor.