Exploration of P1 and P4 modifications of nirmatrelvir: Design, synthesis, biological evaluation, and X-ray structural studies of SARS-CoV-2 Mpro inhibitors.

We report the synthesis, biological evaluation, and X-ray structural studies of a series of SARS-CoV-2 Mpro inhibitors based upon the X-ray crystal structure of nirmatrelvir, an FDA approved drug that targets the main protease of SARS-CoV-2. The studies involved examination of various P4 moieties, P1 five- and six-membered lactam rings to improve potency. In particular, the six-membered P1 lactam ring analogs exhibited high SARS-CoV-2 Mpro inhibitory activity. Several compounds effectively blocked SARS-CoV-2 replication in VeroE6 cells. One of these compounds maintained good antiviral activity against variants of concern including Delta and Omicron variants. A high-resolution X-ray crystal structure of an inhibitor bound to SARS-CoV-2 Mpro was determined to gain insight into the ligand-binding properties in the Mpro active site.

A lipid index for risk of hyperlipidemia caused by anti-retroviral drugs.

HIV-associated lipodystrophy has been reported in people taking anti-retroviral therapy (ART). Lipodystrophy can cause cardiovascular diseases, affecting the quality of life of HIV-infected individuals. In this study, we propose a pharmacological lipid index to estimate the risk of hyperlipidemia caused by anti-retroviral drugs. Lipid droplets were stained in cells treated with anti-retroviral drugs and cyclosporin A. Signal intensities of lipid droplets were plotted against the drug concentrations to obtain an isodose of 10 µM of cyclosporin A, which we call the Pharmacological Lipid Index (PLI). The PLI was then normalized by EC50. PLI/EC50 values were low in early proteinase inhibitors and the nucleoside reverse transcriptase inhibitor, d4T, indicating high risk of hyperlipidemia, which is consistent with previous findings of hyperlipidemia. In contrast, there are few reports of hyperlipidemia for drugs with high PLI/EC50 scores. Data suggests that PLI/EC50 is a useful index for estimating the risk of hyperlipidemia.

Exploratory Studies of Effective Inhibitors against the SARS-CoV-2 Main Protease by Halogen Incorporation and Amide Bond Replacement.

In the development of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs, its main protease (Mpro), which is an essential enzyme for viral replication, is a promising target. To date, the Mpro inhibitors, nirmatrelvir and ensitrelvir, have been clinically developed by Pfizer Inc. and Shionogi & Co., Ltd., respectively, as orally administrable drugs to treat coronavirus disease of 2019 (COVID-19). We have also developed several potent inhibitors of SARS-CoV-2 Mpro that include compounds 4, 5, TKB245 (6), and TKB248 (7), which possesses a 4-fluorobenzothiazole ketone moiety as a reactive warhead. In compounds 5 and TKB248 (7) we have also found that replacement of the P1-P2 amide of compounds 4 and TKB245 (6) with the corresponding thioamide improved their pharmacokinetics (PK) profile in mice. Here, we report the design, synthesis and evaluation of SARS-CoV-2 Mpro inhibitors with replacement of a digestible amide bond by surrogates (9-11, 33, and 34) and introduction of fluorine atoms in a metabolically reactive methyl group on the indole moiety (8). As the results, these compounds showed comparable or less potency compared to the corresponding parent compounds, YH-53/5h (2) and 4. These results should provide useful information for further development of Mpro inhibitors.

Neutralization against Omicron sublineages (BA.2/BA.5/BQ.1.1/XBB/XBB.1.5) in bivalent BNT162b2-vaccinated HCWs with or without risk factors, or following BT infection with Omicron.

SARS-CoV-2-BA.4/5-adapted-bivalent-BNT162b2-vaccine (bvBNT), developed in response to the recent emergence of immune-evasive Omicron-variants, has been given to individuals who completed at least 2-doses of the monovalent-BNT162b2-vaccine (mvBNT). In the present cohort study, we evaluated neutralization-titers (NT50s) against Wuhan-strain (SCoV2Wuhan) and Omicron-sublineages including BA.2/BA.5/BQ.1.1/XBB/XBB.1.5, and vaccine-elicited S1-binding-IgG in sera from participants-vaccinated with 5th-bvBNT following 4th-mvBNT. The 5th-bvBNT-dose elicited good protective-activity against SCoV2Wuhan with geometric-mean (gMean)-NT50 of 1966-2091, higher than the peak-values post-4th-mvBNT with no statistical significance, and favorable neutralization-activity against not only BA.5 but also BA.2, with ~ 3.2-/~ 2.2-fold greater gMean-NT50 compared to the peak-values post-4th-mvBNT-dose, in participants with or without risk factors. However, neutralization-activity of sera post-5th-bvBNT-dose was low against BQ.1.1/XBB/XBB.1.5. Interestingly, participants receiving bvBNT following breakthrough (BT) infection during Omicron-wave had significantly enhanced neutralization-activity against SCoV2Wuhan/BA.2/BA.5 with ~ 4.6-/~ 6.3-/~ 8.1-fold greater gMean-NT50, respectively, compared to uninfected participants receiving bvBNT. Sera from BT-infected-participants receiving bvBNT had enhanced neutralization-activity against BQ.1.1/XBB/XBB.1.5 by ~ 3.8-fold compared to those from the same participants post-4th-mvBNT-dose, and had enhanced gMean-NT50 ~ 5.4-fold greater compared to those of uninfected-participants' sera post-bvBNT. These results suggest that repeated stimulation brought about by exposure to BA.5's-Spike elicit favorable cross-neutralization-activity against various SARS-CoV-2-variants.

Structure-Activity Relationship Studies of SARS-CoV-2 Main Protease Inhibitors Containing 4-Fluorobenzothiazole-2-carbonyl Moieties.

The main protease (Mpro) of SARS-CoV-2 is an attractive target for the development of drugs to treat COVID-19. Here, we report the design, synthesis, and structure-activity relationship (SAR) studies of highly potent SARS-CoV-2 Mpro inhibitors including TKB245 (5)/TKB248 (6). Since we have previously developed Mpro inhibitors (3) and (4), several hybrid molecules of these previous compounds combined with nirmatrelvir (1) were designed and synthesized. Compounds such as TKB245 (5) and TKB248 (6), containing a 4-fluorobenzothiazole moiety at the P1' site, are highly effective in the blockade of SARS-CoV-2 replication in VeroE6 cells. Replacement of the P1-P2 amide with the thioamide surrogate in TKB248 (6) improved its PK profile in mice compared to that of TKB245 (5). A new diversity-oriented synthetic route to TKB245 (5) derivatives was also developed. The results of the SAR studies suggest that TKB245 (5) and TKB248 (6) are useful lead compounds for the further development of Mpro inhibitors.

SARS-CoV-2 papain-like protease (PLpro) inhibitory and antiviral activity of small molecule derivatives for drug leads.

We report here the synthesis and biological evaluation of a series of small molecule SARS-CoV-2 PLpro inhibitors. We compared the activity of selected compounds in both SARS-CoV-1 and SARS-CoV-2 PLpro inhibitory and antiviral assays. We have synthesized and evaluated several new structural variants of previous leads against SARS-CoV-2 PLpro. The replacement of the carboxamide functionality with sulfonamide derivatives resulted in PLpro inhibitors with potent PLpro inhibitory and antiviral activity in VeroE6 cells similar to GRL0617. To obtain molecular insight, we created an optimized model of a potent sulfonamide derivative in the SARS-CoV-2 PLpro active site.

GRL-142 binds to and impairs HIV-1 integrase nuclear localization signal and potently suppresses highly INSTI-resistant HIV-1 variants.

Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIVKGD) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIVKGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC50 of 130 femtomolar. When cells were exposed to HIVKGD IN-containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS's putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142-bound HIVKGD's PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant-harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.

Synthesis of novel entecavir analogues having 4'-cyano-6''-fluoromethylenecyclopentene skeletons as an aglycone moiety as highly potent and long-acting anti-hepatitis B virus agent.

Encouraged by our recent findings that 4'-cyano-deoxyguanosine (2), entecavir analogues 4 and 5 are highly potent anti-hepatitis B virus (HBV) agents, we designed and synthesized 6 having a hybridized structure of 4 and 5. The chiral quaternary carbon portion at the 4'-position, which is substituted by cyano- and 5'-hydroxymethyl groups, was stereospecifically constructed by radical-mediated 5-exo-dig mode cyclization of 10. The introduction of the fluorine atom into the 6''-position was achieved by radical-mediated stannylation of sulfide (E)-11 and subsequent electrophilic fluorination of (E)-12. The desired (E)-6 was obtained after the introduction of the guanine base into (E)-18 under Mitsunobu conditions and following global deprotection. The stereoisomer (Z)-6 was also prepared by the same procedure using (Z)-12. Compound (E)-6 showed highly potent anti-HBV activity (EC50 = 1.2 nM) with favorable cytotoxicity (CC50 = 93 µM).

A novel anti-HBV agent, E-CFCP, restores Hepatitis B virus (HBV)-induced senescence-associated cellular marker perturbation in human hepatocytes.

Cellular senescence is a cellular state with a broad spectrum of age-related physiological conditions that can be affected by various infectious diseases and treatments. Therapy of hepatitis B virus (HBV) infection with nucleos(t)ide analogs [NA(s)] is well established and benefits many HBV-infected patients, but requires long-term, perhaps lifelong, medication. In addition to the effects of HBV infection, the effects of NA administration on hepatocellular senescence are still unclear. This study investigated how HBV infection and NA treatment influence cellular senescence in human hepatocytes and humanized-liver chimeric mice chronically infected with live HBV. HBV infection upregulates or downregulates multiple cellular markers including senescence-associated ß-galactosidase (SA-ß-Gal) activity and cell cycle regulatory proteins (e.g., p21CIP1) expression level in hepatocellular nuclei and humanized-mice liver. A novel highly potent anti-HBV NA, E-CFCP, per se did not have significant disturbance on markers evaluated. Besides, E-CFCP treatment restored HBV-infected cells to their physiological phenotypes that are comparable to the HBV-uninfected cells. The results reported here demonstrate that, regardless of the mechanism(s), chronic HBV infection perturbates multiple senescence-associated markers in human hepatocytes and humanized-mice liver, but E-CFCP can restore this phenomenon.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

A transgenic reporter mouse model for in vivo assessment of retinoic acid receptor transcriptional activation.

Background: Vitamin A is essential for a wide range of life processes throughout embryogenesis to adult life. With the aim of developing an in vivo model to monitor retinoic acid receptor (RAR) transactivation real-time in intact animals, we generated transgenic mice carrying a luciferase (luc) reporter gene under the control of retinoic acid response elements (RAREs) consisting of three copies of a direct repeat with five spacing nucleotides (DR5). Methods: Transgenic mice carrying a RARE dependent luciferase reporter flanked with insulator sequence were generated by pronuclear injection. RARE dependent luciferase activity was detected by in vivo imaging or in tissue extracts following manipulations with RAR/retinoid X receptor (RXR) agonists, RAR antagonists or in vitamin A deficient mice. Results: We found a strong induction of luciferase activity in a time and dose dependent manner by retinoic acid as well as RAR agonists, but not by the RXR agonist (using n=4-6 per group; 94 mice). In addition, luciferase activity was strongly reduced in vitamin A-deficient mice (n=6-9; 30 mice). These observations confirm that luciferase activity was controlled by RAR activation in the RARE-luc mouse. Luciferase activity was detectable in various organs, with high activity especially in brain and testis, indicating strong retinoid signalling in these tissues. Conclusion: The RARE-luc transgenic mice, which enabled real-time in vivo assessment of RAR activation, will be useful in understanding the normal physiology of vitamin A, the role of retinoid signalling in pathologies as well as to evaluate pharmacological ligands for RARs.

Potent and biostable inhibitors of the main protease of SARS-CoV-2.

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection in vitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.

Effects of a novel hepatitis B anti-viral drug E-CFCP in renal organic acid transporters.

Currently, the emergence of drug resistance is an important issue in the treatment of hepatitis B virus (HBV). Recently, our collaborating group developed a novel long-acting anti-HBV drug, E-CFCP. However, until this study, the effects of E-CFCP in the kidney have remained unclarified. Using cell viability and uptake assays, we examined the effects of E-CFCP on the function of renal organic anion transporters (OATs). No cytotoxicity was shown related to the E-CFCP in the renal OATs in either assay. Thus, this study suggested that E-CFCP may be a novel, excellent candidate drug for the treatment of drug-resistant HBV.

Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

We newly generated two human induced pluripotent stem cell (hiPSC)-derived spheroid lines, termed Spheroids_4MACE2-TMPRSS2 and Spheroids_15M63ACE2-TMPRSS2, both of which express angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are critical for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Both spheroids were highly susceptible to SARS-CoV-2 infection, and two representative anti-SARS-CoV-2 agents, remdesivir and 5h (an inhibitor of SARS-CoV-2's main protease), inhibited the infectivity and replication of SARS-CoV-2 in a dose-dependent manner, suggesting that these human-derived induced spheroids should serve as valuable target cells for the evaluation of anti-SARS-CoV-2 activity. IMPORTANCE The hiPSC-derived spheroids we generated are more expensive to obtain than the human cell lines currently available for anti-SARS-CoV-2 drug evaluation, such as Calu-3 cells; however, the spheroids have better infection susceptibility than the existing human cell lines. Although we are cognizant that there are human lung (and colonic) organoid models for the study of SARS-CoV-2, the production of those organoids is greatly more costly and time consuming than the generation of human iPSC-derived spheroid cells. Thus, the addition of human iPSC-derived spheroids for anti-SARS-CoV-2 drug evaluation studies could provide the opportunity for more comprehensive interpretation of the antiviral activity of compounds against SARS-CoV-2.

Droplet digital PCR assay provides intrahepatic HBV cccDNA quantification tool for clinical application.

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.

Design, Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Substituted Tricyclic Hexahydro-Furofuran Derivatives as P2 Ligands.

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2' ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2' ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.

Highly Neutralizing COVID-19 Convalescent Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters.

Despite various attempts to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients with COVID-19 convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly neutralizing COVID-19 convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing activity-determined convalescent plasmas, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in a significant reduction of viral titers in lungs by up to 32-fold compared to the viral titers in hamsters receiving control nonneutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs more than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using microcomputerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19 convalescent plasmas variably contained antibodies against SARS-CoV-2 components, including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent neutralizing activity-confirmed COVID-19 convalescent plasmas would be efficacious in treating patients with COVID-19. IMPORTANCE Convalescent plasmas obtained from patients who recovered from a specific infection have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized controlled clinical trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94 to 95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly neutralizing effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally induced lung lesions. The present data provide a proof of concept that the presence of highly neutralizing antibody in COVID-19 convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.

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.