The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a significant global health threat. While vaccines have been developed rapidly and shown high efficacy, the emergence of variants with mutations in the spike-encoding region has raised concerns about vaccine efficacy. Currently available anti-SARS-CoV-2 therapeutics, including remdesivir, nirmatrelvir, and molnupiravir, have limitations in antiviral potency. Nirmatrelvir, for example, can lead to SARS-CoV-2 replication rebound and relapse symptoms. The emergence of drug-resistant variants further underscores the need for more potent and effective therapeutics. This research aimed to identify novel, potent inhibitors of SARS-CoV-2 Mpro, a crucial enzyme for viral replication, building upon the knowledge gained from previously studied inhibitors such as GRL1720, GRL0920, and 5h. The researchers hypothesized that novel compounds incorporating a 4-fluorinated benzothiazole moiety could achieve significantly improved antiviral activity and pharmacokinetic properties compared to existing treatments. Fluorination is known to enhance lipophilicity, cell membrane penetration, and oral bioavailability, crucial factors for effective antiviral drugs. The study’s importance lies in the urgent need for more effective treatments to combat the evolving SARS-CoV-2 variants and address the limitations of current antiviral therapies.

The existing literature highlights the limitations of current antiviral treatments for COVID-19. Studies have demonstrated the emergence of SARS-CoV-2 variants resistant to existing drugs like nirmatrelvir, partly due to insufficient drug potency and the virus's rapid replication rate. The need for improved therapies is further emphasized by reports of virologic rebound following nirmatrelvir-ritonavir treatment, indicating insufficient viral suppression. Previous research explored benzothiazole-containing Mpro inhibitors, showing promise but also limitations. The researchers built upon this prior work, focusing on modifications to improve potency and pharmacokinetic properties.

The researchers designed and synthesized a series of fluorinated analogs of a known benzothiazole-containing Mpro inhibitor (5h). They introduced fluorine atoms to the phenyl ring, aiming to improve lipophilicity and bioavailability. The synthesized compounds were evaluated for their antiviral activity against various SARS-CoV-2 strains, including alpha, beta, gamma, delta, kappa, and omicron variants, using cell-based assays employing VeroE6, HeLa-ACE2-TMPRSS2, and A549-ACE2-TMPRSS2 cells. Antiviral activity was assessed by measuring viral RNA levels using RT-qPCR. Cytotoxicity was evaluated using a WST-8 assay. Enzymatic activity inhibition was measured using a fluorescence resonance energy transfer (FRET) assay. The study also evaluated pharmacokinetic parameters, including half-life (T1/2), maximum concentration (Cmax), and oral bioavailability (F) in mice. The binding modes of the most promising compounds, TKB245 and TKB248, to Mpro were investigated using native mass spectrometry and X-ray crystallography. The in vivo efficacy of TKB245 and TKB248 was assessed in human ACE2-knocked-in mice infected with SARS-CoV-2 Omicron BA.1 and Delta variants. Viral titers in lung tissues were determined using plaque assays, and the body weights and survival rates of the mice were monitored. The study also investigated the intracellular concentrations of TKB245, TKB248, and nirmatrelvir in VeroE6 and HeLa-ACE2-TMPRSS2 cells using LC/MS. Statistical analyses, including repeated ANOVA and Wilcoxon rank-sum test, were used to compare viral loads and other parameters between groups. X-ray crystallographic analysis provided detailed structural information on the binding interactions between the inhibitors and Mpro.

Two novel compounds, TKB245 and TKB248, were identified as potent inhibitors of SARS-CoV-2 Mpro. TKB245 demonstrated exceptionally potent antiviral activity (EC50 values around 30 nM in cell-based assays), significantly surpassing the activity of molnupiravir and nirmatrelvir. Both TKB245 and TKB248 exhibited comparable activity against various SARS-CoV-2 variants, including alpha, beta, gamma, delta, kappa, and omicron strains. TKB245 showed potent activity across various cell lines (VeroE6, HeLa-ACE2-TMPRSS2, and A549-ACE2-TMPRSS2), outperforming ensitrelvir and molnupiravir. Intracellular concentration studies revealed that TKB245 and TKB248 achieved significantly higher intracellular concentrations than nirmatrelvir. In vivo studies using human ACE2-knocked-in mice demonstrated that TKB245 significantly reduced viral titers in the lungs of mice infected with Omicron BA.1 and Delta variants. TKB248 showed comparable efficacy against Omicron BA.1 and BA.2 variants. Native mass spectrometry indicated that TKB245 and nirmatrelvir promote Mpro dimerization, while TKB248 showed a lesser effect at lower concentrations. X-ray crystallography revealed that both TKB245 and TKB248 bind to the Mpro active site, forming a covalent bond with Cys145, and their 4-fluorobenzothiazole moiety plays a crucial role in their potent activity. The pharmacokinetic parameters of TKB245 and TKB248 were improved compared to 5h, exhibiting longer half-lives and better oral bioavailability in mice. No significant acute toxicity was observed in the mice treated with TKB245 and TKB248.

The findings of this study address the urgent need for more effective antiviral therapies against SARS-CoV-2 by identifying two potent and orally bioavailable Mpro inhibitors, TKB245 and TKB248. TKB245 demonstrates significantly higher potency than existing clinical treatments, and both compounds show activity against multiple SARS-CoV-2 variants. The mechanistic studies suggest that the 4-fluorobenzothiazole moiety contributes significantly to their potent activity. The superior intracellular concentrations and in vivo efficacy observed with TKB245 and TKB248 compared to nirmatrelvir are noteworthy. The findings highlight the potential of these compounds as promising candidates for the development of novel and improved COVID-19 therapeutics. The mechanism of action, particularly the promotion of Mpro dimerization, provides insights into potential strategies for drug optimization.

This study successfully identified two novel, potent SARS-CoV-2 Mpro inhibitors, TKB245 and TKB248, with superior activity compared to current clinical treatments. TKB245's exceptional potency, broad-spectrum activity, favorable pharmacokinetic profile, and efficacy in vivo highlight its potential as a lead compound for the development of improved COVID-19 therapies. Further research should focus on optimizing these compounds for clinical application and evaluating their long-term safety and efficacy. The insights gained into the structure-activity relationships and mechanism of action provide a strong foundation for future drug discovery efforts.

The study's in vivo efficacy assessment was conducted in human ACE2-knocked-in mice, which may not fully recapitulate the complexities of human COVID-19 pathogenesis. While no significant acute toxicity was observed, long-term toxicity studies are needed before clinical translation. The study focused primarily on antiviral activity and pharmacokinetic parameters, and further investigations into pharmacodynamic parameters and potential drug-drug interactions are warranted.