Reverse transcription-quantitative PCR (RT-qPCR)-based tests are widely used to diagnose coronavirus disease 2019 (COVID-19). As a result that these tests cannot be done in local clinics where RT-qPCR testing capability is lacking, rapid antigen tests (RATs) for COVID-19 based on lateral flow immunoassays are used for rapid diagnosis. However, their sensitivity compared with each other and with RT-qPCR and infectious virus isolation has not been examined. Here, we compared the sensitivity among four RATs by using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates and several types of COVID-19 patient specimens and compared their sensitivity with that of RT-qPCR and infectious virus isolation. Although the RATs read the samples containing large amounts of virus as positive, even the most sensitive RAT read the samples containing small amounts of virus as negative. Moreover, all RATs tested failed to detect viral antigens in several specimens from which the virus was isolated. The current RATs will likely miss some COVID-19 patients who are shedding infectious SARS-CoV-2.

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 (M^pro). 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 M^pro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead M^pro 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.

: 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 (M^pro) 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 M^pro, apparently promoting M^pro dimerization. X-ray crystallographic analysis shows that both compounds bind to M^pro’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.

We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M ^pro ) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2 ^WK521 _WT in VeroE6 ^TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2 ^WK521 _E166V ), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2 _E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M ^pro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1’-Glu166 interactions. TKB245 stayed bound to M ^pro _E166V , whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M ^pro , and compromised the enzymatic activity; the Ki values of recombinant M ^pro _E166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to M ^pro _E166V than nirmatrelvir. SARS-CoV-2 ^WK521 _WT selected with 5h acquired A191T substitution in M ^pro (SARS-CoV-2 ^WK521 _A191T ) and better replicated in the presence of 5h, than SARS-CoV-2 ^WK521 _WT . However, no significant enzymatic or structural changes in M ^pro _A191T were observed. The replicability of SARS-CoV-2 ^WK521 _E166V proved to be compromised compared to SARS-CoV-2 ^WK521 _WT but predominated over SARS-CoV-2 ^WK521 _WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2 ^WK521 _A191T surpassed that of SARS-CoV-2 ^WK521 _WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2’s drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.