Indole Chloropyridinyl Ester-Derived SARS-CoV-2 3CLpro Inhibitors: Enzyme Inhibition, Antiviral Efficacy, Structure-Activity Relationship, and X-ray Structural Studies.

Here, we report the synthesis, structure-activity relationship studies, enzyme inhibition, antiviral activity, and X-ray crystallographic studies of 5-chloropyridinyl indole carboxylate derivatives as a potent class of SARS-CoV-2 chymotrypsin-like protease inhibitors. Compound 1 exhibited a SARS-CoV-2 3CLpro inhibitory IC50 value of 250 nM and an antiviral EC50 value of 2.8 µM in VeroE6 cells. Remdesivir, an RNA-dependent RNA polymerase inhibitor, showed an antiviral EC50 value of 1.2 µM in the same assay. Compound 1 showed comparable antiviral activity with remdesivir in immunocytochemistry assays. Compound 7d with an N-allyl derivative showed the most potent enzyme inhibitory IC50 value of 73 nM. To obtain molecular insight into the binding properties of these molecules, X-ray crystal structures of compounds 2, 7b, and 9d-bound to SARS-CoV 3CLpro were determined, and their binding properties were compared.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

Biochemical and Structural Properties of Entecavir-Resistant Hepatitis B Virus Polymerase with L180M/M204V Mutations.

Entecavir (ETV) is a widely used anti-hepatitis B virus (HBV) drug. However, the emergence of resistant mutations in HBV reverse transcriptase (RT) results in treatment failure. To understand the mechanism underlying the development of ETV resistance by HBV RT, we analyzed the L180M, M204V, and L180M/M204V mutants using a combination of biochemical and structural techniques. ETV-triphosphate (ETV-TP) exhibited competitive inhibition with dGTP in both wild-type (wt) RT and M204V RT, as observed using Lineweaver-Burk plots. In contrast, RT L180M or L180M/M204V did not fit either competitive, uncompetitive, noncompetitive, or typical mixed inhibition, although ETV-TP was a competitive inhibitor of dGTP. Crystallography of HIV RTY115F/F116Y/Q151M/F160M/M184V, mimicking HBV RT L180M/M204V, showed that the F115 bulge (F88 in HBV RT) caused by the F160M mutation induced deviated binding of dCTP from its normal tight binding position. Modeling of ETV-TP on the deviated dCTP indicated that a steric clash could occur between ETV-TP methylene and the 3'-end nucleoside ribose. ETV-TP is likely to interact primarily with HBV RT M171 prior to final accommodation at the deoxynucleoside triphosphate (dNTP) binding site (Y. Yasutake, S. Hattori, H. Hayashi, K. Matsuda, et al., Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9). Therefore, in HBV RT L180M/M204V, ETV-TP may be stuck at M171, a residue that is conserved in almost all HBV isolates, leading to the strange inhibition pattern observed in the kinetic analysis. Collectively, our results provide novel insights into the mechanism of ETV resistance of HBV RT caused by L180M and M204V mutations. IMPORTANCE HBV infects 257 million people in the world, who suffer from elevated risks of liver cirrhosis and cancer. ETV is one of the most potent anti-HBV drugs, and ETV resistance mutations in HBV RT have been extensively studied. Nevertheless, the mechanisms underlying ETV resistance have remained elusive. We propose an attractive hypothesis to explain ETV resistance and effectiveness using a combination of kinetic and structural analyses. ETV is likely to have an additional interaction site, M171, beside the dNTP pocket of HBV RT; this finding indicates that nucleos(t)ide analogues (NAs) recognizing multiple interaction sites within RT may effectively inhibit the enzyme. Modification of ETV may render it more effective and enable the rational design of efficient NA inhibitors.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

SARS-CoV-2 encodes main protease (Mpro), an attractive target for therapeutic interventions. We show Mpro is susceptible to glutathionylation leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that a single modification with glutathione is sufficient to block dimerization and loss of activity. Proteolytic digestions of Mpro revealed Cys300 as a primary target of glutathionylation, and experiments using a C300S Mpro mutant revealed that Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of Cys300 and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to human disease and the pathophysiology of SARS-CoV-2 in bats, which develop oxidative stress during flight.

A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication.

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.

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

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.

7-Deaza-7-fluoro modification confers on 4'-cyano-nucleosides potent activity against entecavir/adefovir-resistant HBV variants and favorable safety.

We designed, synthesized and identified a novel nucleoside derivative, 4'-C-cyano-7-deaza-7-fluoro-2'-deoxyadenosine (CdFA), which exerts potent anti-HBV activity (IC50 ~26 nM) with favorable hepatocytotoxicity (CC50 ~56 µM). Southern blot analysis using wild-type HBV (HBVWT)-encoding-plasmid-transfected HepG2 cells revealed that CdFA efficiently suppresses the production of HBVWT (IC50 = 153.7 nM), entecavir (ETV)-resistant HBV carrying L180M/S202G/M204V substitutions (HBVETVR; IC50 = 373.2 nM), and adefovir dipivoxil (ADV)-resistant HBV carrying A181T/N236T substitutions (HBVADVR; IC50=192.6 nM), whereas ETV and ADV were less potent against HBVETVR and HBVADVR (IC50: >1,000 and 4,022.5 nM, respectively). Once-daily peroral administration of CdFA to human-liver-chimeric mice over 14 days (1 mg/kg/day) comparably blocked HBVWT and HBVETVR viremia by 0.7 and 1.2 logs, respectively, without significant changes in body-weight or serum human-albumin levels, although ETV only slightly suppressed HBVETVR viremia (CdFA vs ETV; p = 0.032). Molecular modeling suggested that ETV-TP has good nonpolar interactions with HBVWT reverse transcriptase (RTWT)'s Met204 and Asp205, while CdFA-TP fails to interact with Met204, in line with the relatively inferior activity against HBVWT of CdFA compared to ETV (IC50: 0.026 versus 0.003 nM). In contrast, the 4'-cyano of CdFA-TP forms good nonpolar contacts with RTWT's Leu180 and RTETVR's Met180, while ETV-TP loses interactions with RTETVR's Met180, explaining in part why ETV is less potent against HBVETVR than CdFA. The present results show that CdFA exerts potent activity against HBVWT, HBVETVR and HBVADVR with enhanced safety and that 7-deaza-7-fluoro modification confers potent activity against drug-resistant HBV variants and favorable safety, shedding light to further design more potent and safer anti-HBV nucleoside analogs.

CMCdG, a Novel Nucleoside Analog with Favorable Safety Features, Exerts Potent Activity against Wild-Type and Entecavir-Resistant Hepatitis B Virus.

We designed, synthesized, and characterized a novel nucleoside analog, (1S,3S,5S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4'-cyano-methylenecarbocyclic-2'-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG's in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBVWTCe) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBVETV-RL180M/S202G/M204V). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC50], ∼30 nM) and HBVWTCe plasmid-transfected Huh7 cells (IC50, 206 nM) and efficiently suppressed ETV-resistant HBVETV-RL180M/S202G/M204V (IC50, 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 µM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBVWTCe-infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of HBVETV-RL180M/S202G/M204V viremia by ∼1 log in HBVETV-RL180M/S202G/M204V-infected human liver-chimeric mice, while ETV (1 mg/kg/day q.d.) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body weights or serum human albumin levels. Structural analyses using homology modeling, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBVWTCe reverse transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBVETV-RL180M/S202G/M204V RT complex. However, CMCdG-TP retains good contacts with both the HBVWTCe RT and HBVETV-RL180M/S202G/M204V RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed light on the further development of more potent and safer anti-HBV agents.

Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors incorporating aminothiochromane and aminotetrahydronaphthalene carboxamide derivatives as the P2 ligands.

We describe the design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors with carboxamide derivatives as the P2 ligands. We have specifically designed aminothiochromane and aminotetrahydronaphthalene-based carboxamide ligands to promote hydrogen bonding and van der Waals interactions in the active site of HIV-1 protease. Inhibitors 4e and 4j have shown potent enzyme inhibitory and antiviral activity. High resolution X-ray crystal structures of 4d- and 4k-bound HIV-1 protease revealed molecular insights into the ligand-binding site interactions.

GRL-079, a Novel HIV-1 Protease Inhibitor, Is Extremely Potent against Multidrug-Resistant HIV-1 Variants and Has a High Genetic Barrier against the Emergence of Resistant Variants.

We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2' cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC50s) were 2.5 to 30 nM against wild-type HIV-1NL4-3, 0.3 to 6.7 nM against HIV-2EHO, and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIVMDR). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIVDRVrp51), with EC50s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC50s of >1,000 nM) against HIVDRVrp51 Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2' Cp-Abt group forms strong hydrogen bonds with Asp30'. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIVMDR strains (HIV11MIX), HIV11MIX became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV11MIX failed to replicate at >0.08 µM and >0.2 µM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2' Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1NL4-3 and a wide spectrum of HIVMDR strains.