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.

4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus.

UNLABELLED: Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50 ] = 0.4 nM) and HIV-1 (IC50 = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50 = 0.7 nM), but is much less active against HIV-1 (IC50 = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA; IC50 = 0.3 nM) is less active against HBV (IC50 = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50 = 7.2 nM) and ETV-resistant HBV (IC50 = 69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 µM. Once-daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV-RL180M/S202G/M204V-infected mice, whereas ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. CONCLUSION: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients.

Lysocellin, a metabolite of the novel drug 'alopestatin', induces G1 arrest and prevents cytotoxicity induced by etoposide.

We report here that lysocellin, a polyether antibiotic from a streptomycete, induces G1 phase arrest in human osteosarcoma MG63 cells. Lysocellin up-regulates p21WAF1/Cip1 and down-regulates cyclin D1 at the mRNA level. In addition, cyclin D1 is down-regulated by the proteasome-dependent signal pathway in MG63 cells. In drug combination studies, we found that lysocellin treatment weakened the cytotoxic activity of etoposide in MG63 cells using a colony-formation assay. To study the in vivo efficacy of lysocellin, we isolated a novel compound related to lysocellin from the same streptomycete, and found that the novel drug is converted to lysocellin in vivo and decreases etoposide-induced alopecia in a neonatal rat model. We raise the possibility that this novel drug, named 'alopestatin', may be a promising agent against alopecia.

Impact of chymase inhibitor on cardiac function and survival after myocardial infarction.

OBJECTIVES: Recent studies have demonstrated that hamsters, like humans, possess both angiotensin converting enzyme (ACE)- and chymase-dependent angiotensin (Ang) II-forming pathways in cardiovascular tissues. We recently found that, after myocardial infarction (MI) in hamsters, cardiac chymase was significantly activated. In order to determine whether suppression of cardiac chymase activity could provide prognostic benefit after MI, we examined the effects of NK3201, a novel, orally active and specific chymase inhibitor, on cardiac function and survival during the acute phase of MI in hamsters. METHODS: Two hundred and ten male Syrian hamsters were used in the present study. The left coronary artery of each hamster was ligated to induce MI. NK3201 at a dose of 30 mg/kg per day was administered orally by gastric gavage, starting either 3 days before or 1 day after MI. RESULTS: ACE and chymase activities were significantly increased in the infarcted left ventricle 3 days after MI. NK3201 treatment starting 3 days before MI significantly inhibited the increase in cardiac chymase activity, while it did not affect ACE activity either in plasma or in heart 3 days after MI. A significant improvement in cardiac function was observed 3 and 14 days after MI in the group receiving NK3201. Compared with vehicle treatment, NK3201 treatment initiated either 3 days before or 1 day after MI significantly reduced the mortality rate during the 14 days of observation following MI. CONCLUSIONS: These findings indicate that cardiac chymase plays an important role after MI and this finding may provide a novel therapeutic target in post-MI treatment.

A novel chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[,4-dioxo-1-phenyl-7-(2-pyridyloxy)]2-heptyl]acetamide (NK3201), suppressed intimal hyperplasia after balloon injury.

In this study, we investigated whether an orally active chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2-heptyl]acetamide (NK3201), prevents intimal hyperplasia in carotid arteries injured by a balloon catheter in dog. Each dog was administered NK3201 (1 mg/kg per day, p.o.) or placebo beginning 5 days before balloon injury and continuing through the experiments. Four weeks after balloon injury, NK3201 did not affect the plasma renin and angiotensin-converting enzyme activities. The chymase activity was significantly increased in the injured arteries, whereas the angiotensin-converting enzyme activity was not. NK3201 significantly reduced the chymase activity in the injured arteries. The intimal area in the placebo- and NK3201-treated group and was 0.46 +/- 0.06 and 0.24 +/- 0.04 mm2, respectively, and this difference was significant. In this study, we demonstrated for the first time that a chymase inhibitor prevented the development of intimal hyperplasia in the balloon-injured arteries.

Development of the chymase inhibitor as an anti-tissue-remodeling drug: myocardial infarction and some other possibilities.

Chymase leading to tissue remodeling is expected to be a potent pharmaceutical target. Its functions in vivo are still unclear, because of lack of orally available inhibitors. Recently, however, the chymase inhibitor NK3201 (2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidin-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2-heptyl] acetamide) was demonstrated to have oral activity against neointimal hyperplasia in dog models (Takai S. et al., Life Sci 69, 1725 - 1732 (2001)). In this review, by showing the efficacy of NK3201 in some hamster models, chymase functions in vivo are summarized, and the potency of this chymase inhibitor is introduced. In vitro study, NK3201 showed potent chymase specific inhibitory activity, and Dixon plot analysis indicated competitive inhibition. Oral administration of NK3201 into normal rats resulted in rapid spread over every tissue except the brain, and sufficient activity to inhibit tissue chymase was detected even after 24 h. In passive cutaneous anaphylaxis, myocardial infarction and bleomycin-induced pulmonary fibrosis models, orally administered NK3201 showed potent inhibition of inflammatory response, tissue angiotensin II formation, and fibrosis, respectively. These data suggest that chymase has a vital role in tissue remodeling through promotion of the inflammatory response, tissue angiotensin II and tissue fibrosis. Our recent data indicated chymase participation in bladder fibrosis, like interstitial cystitis. Therefore, the orally active chymase inhibitor NK3201 may have protective effects on tissue remodeling in several diseases.