Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 μM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

Significance Dimerization of HIV-1 protease (PR) plays a critical role in the replication of HIV-1. Darunavir (DRV) inhibits not only proteolytic activity but also PR dimerization. The present study shows that PR dimerization process undergoes two steps and that DRV inhibits the first step of PR dimerization by binding to PR monomers in a one-to-one molar ratio. The present study also demonstrates that DRV binds to a transframe precursor PR protein, indicating that DRV’s monomer binding is involved in the Gag-Pol autoprocessing inhibition. To our knowledge, the present report represents the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV, which should help design further, more potent novel PR inhibitors.

We assessed various newly generated compounds that target the main protease (Mpro) 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 Mpro, 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 (Mpro) 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 Mpro, 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.

Human immunodeficiency virus type 1 (HIV-1) Gag protein is the principal structural component of the HIV particle. Localization of the Pr55(Gag) protein to the plasma membrane initiates virus assembly. Recent studies indicated that d-myo-phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) regulates Pr55(Gag) localization and assembly. We determined the binding affinity between Pr55(Gag) or its N-terminal MA domain and various phosphoinositide derivatives using a highly sensitive surface plasmon resonance (SPR) sensor and biotinylated inositol phosphate. The equilibrium dissociation constants obtained using this approach reflected the distinct magnitude of acyl group-based and phosphate group-based interactions. The dissociation constant (K(D)) for Pr55(Gag) complexed with 1,4,5-IP3 (an inositol with divalent phosphate groups and devoid of lipid groups) was 2170 microM, while the K(D) for di-C(8)-PI (a lipid-containing inositol devoid of divalent phosphate groups) was 186 microM, and the K(D) for di-C(8)-PI(4,5)P2 (an inositol with both lipid and divalent phosphate groups) was 47.4 microM. The same trend in affinity was observed when these phosphoinositides were complexed with MA. Our results suggest that the contribution of hydrophobic acyl chains is greater than negatively charged inositol phosphates in Pr55(Gag)/MA binding. Furthermore, each inositol phosphate (devoid of lipid groups) tested showed a distinct Pr55(Gag)-binding affinity depending on the position and number of phosphate groups. However, the position and number of phosphate groups had no effect on MA-binding affinity.