GRL-0920, an Indole Chloropyridinyl Ester, Completely Blocks SARS-CoV-2 Infection

Abstract

We assessed various newly generated compounds that target the main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and various previously known compounds reportedly active against SARS-CoV-2, employing RNA quantitative PCR (RNA-qPCR), cytopathicity assays, and immunocytochemistry. Here, we show that two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, exerted potent activity against SARS-CoV-2 in cell-based assays performed using VeroE6 cells and TMPRSS2-overexpressing VeroE6 cells. While GRL-0820 and the nucleotide analog remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred. No significant anti-SARS-CoV-2 activity was found for several compounds reportedly active against SARS-CoV-2 such as lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychroloquine. In contrast, GRL-0920 exerted potent activity against SARS-CoV-2 (50% effective concentration [EC₅₀] = 2.8 μM) and dramatically reduced the infectivity, replication, and cytopathic effect of SARS-CoV-2 without significant toxicity as examined with immunocytochemistry. Structural modeling shows that indole and chloropyridinyl of the derivatives interact with two catalytic dyad residues of M^pro, Cys145 and His41, resulting in covalent bonding, which was verified using high-performance liquid chromatography-mass spectrometry (HPLC/MS), suggesting that the indole moiety is critical for the anti-SARS-CoV-2 activity of the derivatives. GRL-0920 might serve as a potential therapeutic for coronavirus disease 2019 (COVID-19) and might be optimized to generate more-potent anti-SARS-CoV-2 compounds.IMPORTANCE Targeting the main protease (M^pro) of SARS-CoV-2, we identified two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, active against SARS-CoV-2, employing RNA-qPCR and immunocytochemistry and show that the two compounds exerted potent activity against SARS-CoV-2. While GRL-0820 and remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred as examined with immunocytochemistry. In contrast, GRL-0920 completely blocked the infectivity and cytopathic effect of SARS-CoV-2 without significant toxicity. Structural modeling showed that indole and chloropyridinyl of the derivatives interacted with two catalytic dyad residues of M^pro, Cys145 and His41, resulting in covalent bonding, which was verified using HPLC/MS. The present data should shed light on the development of therapeutics for COVID-19, and optimization of GRL-0920 based on the present data is essential to develop more-potent anti-SARS-CoV-2 compounds for treating COVID-19.

Keywords: COVID-19; SARS-CoV-2; antiviral agents; main protease.

FIG 1

Sequence conservation and structural comparison of four of coronaviruses’ main proteases. (A) The amino acid sequences of SARS-CoV-2 M^pro and SARS-CoV M^pro have 96% identity (12 amino acids [highlighted in blue] of 306 amino acids differ between the two). Conserved amino acids among the four M^pro sequences are shown in red. Distant coronavirus variants MERS-CoV and CoV-NL63 exhibit much less sequence conservation. Sequences were aligned using ENDscript server. (B) Structures of Mpro of SARS-CoV-2 (PDB entry 6LU7; in green) superimposed on M^pro of SARS-CoV (2AMQ; in blue), MERS-CoV (5C3N; in salmon), and CoV-NL63 (5GWY; in yellow) are shown. Catalytic dyad residues Cys145 and His41 are indicated in stick mode. For clarity, only a monomer structure is shown and the covalent protease inhibitor N3 was omitted. The picture was generated using UCSF Chimera.

FIG 2

Antiviral activity of selected compounds against SARS-CoV-2. VeroE6 cells were exposed to SARS-CoV-2^WK-521. The viral copy numbers in the culture supernatants (left axis, open bars) and percent inhibition (right axis, red lines) of each compound in VeroE6 cells were determined using RT-qPCR. Each compound was tested on at least 3 different occasions. For details of the EC₅₀ and CC₅₀ values, see Table S1. The data illustrate representative ones and are shown as means ± standard deviations (SD). All compounds were tested and compared in one assay performed in duplicate.

FIG 3

GRL-0920 and remdesivir virtually completely block the infectivity and cytopathic effect of SARS-CoV-2^WK-521 in VeroE6 and VeroE6^TMPRSS2 cells. (A) GRL-0920 potently blocks the cytopathic effect of SARS-CoV-2 in VeroE6 cells. VeroE6 cells were exposed to SARS-CoV-2^WK-521, cultured in the presence of GRL-0920 or remdesivir, and examined under a microscope (magnification, ×200). (B) GRL-0920, GRL-0820, and remdesivir potently blocked the infectivity and cytopathic effect of SARS-CoV-2^WK-521 in VeroE6^TMPRSS2 cells, while lopinavir, nelfinavir, and favipiravir failed to block the infection. (C) GRL-0920 at 100 μM completely blocked SARS-CoV-2^WK-521 infection in VeroE6 cells, while nafamostat and hydroxychloroquine did not exert significant antiviral activity. Nafamostat did not exert significant toxicity to the cells, but it totally failed to block the infectivity of the virus. For immunocytochemistry, the IgG fraction of serum from a convalescent COVID-19 individual was employed as the primary antibody. The SARS-CoV-2 antigens, F-actin, and nuclei are shown in green, red, and blue, respectively. The image in each inset in panels B and C represents the merged image.

FIG 4

Molecular models of interactions of GRL-0920 and GRL-0820 with SARS-CoV-2 M^pro. The proposed mechanism is the formation of a covalent bond of GRL-0920 and GRL-0820 with Cys145 of SARS-CoV-2 M^pro (RCSB PDB ID 6Y2F) through acylation, followed by the departure of the chloropyridine moiety. The location and interactions of the active site residues with GRL-0920 prior to cleavage are shown in panel a and after the loss of the chloropyridine moiety are shown in panel b. Similar interactions of SARS-CoV-2 M^pro with GRL-0820 are shown in panels c and d. The reaction may be reversible with the existence of both forms. The carbons of GRL-0920 and GRL-0820 are shown in magenta, whereas the carbons of M^pro in gray. Nitrogen, oxygen, sulfur, chlorine, and polar hydrogens are shown in blue, red, yellow, dark green, and white, respectively. The covalent bond is shown with a green arrow. Hydrogen and halogen bonds are shown with yellow and magenta dashed lines, respectively.

FIG 5

Thermal stability of M^pro in the presence or absence of GRL-0920. Thermal stability of M^pro in the presence of GRL-0920 was analyzed using differential scanning fluorimetry (DSF) as described for experiment 1. Note that the T_m value of M^pro (5 μM) alone was 53.63°C, while the T_m values decreased to 51.04, 49.13, and 46.98°C in the presence of 5, 50, and 100 μM GRL-0920, respectively, suggesting that the M^pro get destabilized when GRL-0920 covalently bound to M^pro. Note that, as expected, the presence of HIV-1 protease inhibitor GRL-142 (5 μM) increased the thermal stability of HIV-1 protease (5 μM) as shown for experiment 2.