A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells

Mellott, Drake M.; Tseng, Chien Te K.; Drelich, Aleksandra; Fajtová, Pavla; Chenna, Balachandra; Kostomiris, Demetrios H.; Hsu, Jason C.; Zhu, Jiyun; Taylor, Zane W.; Kocurek, Klaudia I.; Tat, Vivian; Katzfuss, Ardala; Li, Linfeng; Giardini, Miriam A.; Skinner, Danielle; Hirata, Ken; Yoon, Michael C.; Beck, Sungjun; Carlin, Aaron F.; Clark, Alex E.; Beretta, Laura; Maneval, Daniel C.; Hook, Vivian; Frueh, Felix W.; Hurst, Brett L.; Wang, Hong; Raushel, Frank M.; O’Donoghue, Anthony J.; Siqueira-Neto, Jair L.; Meek, Thomas D.; McKerrow, James H.

doi:10.1021/acschembio.0c00875

Cited by 83 publications

(97 citation statements)

References 42 publications

(100 reference statements)

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“…Our findings are in line with previous studies [ 33 , 34 , 35 , 36 , 37 ]. Barnard et al [ 33 ] reported that calpain inhibitors, including calpeptin, could inhibit SARS-CoV replication in vitro.…”

Section: Discussionsupporting

confidence: 94%

“…We propose that calpeptin is supposed to mainly inhibit viral release rather than viral replication, as evidenced by the IC50 values of calpeptin based on the viral output studies in both Vero E6 and Calu-3 cells that were >2-fold lower than the IC50 values based on the percentage of infected cells ( Figure 3 and Figure 4 ). Nonetheless, it should be emphasized that this cysteine protease inhibitor may also inhibit coronaviral entry via inhibition of host cysteine protease [ 35 , 36 ]. Recently, Mediouni et al [ 37 ] found that calpeptin may exert dual effects of SARS-CoV-2 inhibition at the viral entry and post-entry processes.…”

Section: Discussionmentioning

confidence: 99%

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Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

et al. 2021

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The coronavirus disease 2019 (COVID-19) pandemic severely impacts health, economy, and society worldwide. Antiviral drugs against SARS-CoV-2 are urgently needed to cope with this global crisis. It has been found that the biogenesis and release mechanisms of viruses share a common pathway with extracellular vesicles (EVs). We hypothesized that small molecule inhibitors of EV biogenesis/release could exert an anti-SARS-CoV-2 effect. Here, we screened 17 existing EV inhibitors and found that calpeptin, a cysteine proteinase inhibitor, exhibited the most potent anti-SARS-CoV-2 activity with no apparent cytotoxicity. Calpeptin demonstrated the dose-dependent inhibition against SARS-CoV-2 viral nucleoprotein expression in the infected cells with a half-maximal inhibitory concentration (IC50) of 1.44 µM in Vero-E6 and 26.92 µM in Calu-3 cells, respectively. Moreover, calpeptin inhibited the production of infectious virions with the lower IC50 of 0.6 µM in Vero E6 cells and 10.12 µM in Calu-3 cells. Interestingly, a combination of calpeptin and remdesivir, the FDA-approved antiviral drug against SARS-CoV-2 viral replication, significantly enhanced the anti-SARS-CoV-2 effects compared to monotherapy. This study discovered calpeptin as a promising candidate for anti-SARS-CoV-2 drug development. Further preclinical and clinical studies are warranted to elucidate the therapeutic efficacy of calpeptin and remdesivir combination in COVID-19.

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Section: Discussionsupporting

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

et al. 2021

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“…K777 is a known CtsL inhibitor with high potency in inhibiting SARS-CoV-2 replication in human cell host. 23…”

Section: Mpi1-7 Mpi9 Gc376 and 11amentioning

confidence: 99%

Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics

Cao

Cho

Geng

et al. 2021

Preprint

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As an essential enzyme of SARS-CoV-2, the pathogen of COVID-19, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor with cellular potency. By coupling this toxicity alleviation with the expression of an MPro-eGFP fusion protein in a human cell host for straightforward characterization with fluorescent flow cytometry, we developed an effective method that allows bulk analysis of cellular potency of MPro inhibitors. In comparison to an antiviral assay in which MPro inhibitors may target host proteases or other processes in the SARS-CoV-2 life cycle to convene strong antiviral effects, this novel assay is more advantageous in providing precise cellular MPro inhibition information for assessment and optimization of MPro inhibitors. We used this assay to analyze 30 literature reported MPro inhibitors including MPI1-9 that were newly developed aldehyde-based reversible covalent inhibitors of MPro, GC376 and 11a that are two investigational drugs undergoing clinical trials for the treatment of COVID-19 patients in United States, boceprevir, calpain inhibitor II, calpain inhibitor XII, ebselen, bepridil that is an antianginal drug with potent anti-SARS-CoV-2 activity, and chloroquine and hydroxychloroquine that were previously shown to inhibit MPro. Our results showed that most inhibitors displayed cellular potency much weaker than their potency in direct inhibition of the enzyme. Many inhibitors exhibited weak or undetectable cellular potency up to 10 μM. On contrary to their strong antiviral effects, 11a, calpain inhibitor II, calpain XII, ebselen, and bepridil showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle to convene potent antiviral effects. characterization of these molecules on their antiviral mechanisms will likely reveal novel drug targets for COVID-19. Chloroquine and hydroxychloroquine showed close to undetectable cellular potency to inhibit MPro. Kinetic recharacterization of these two compounds rules out their possibility as MPro inhibitors. Our results also revealed that MPI5, 6, 7, and 8 have high cellular and antiviral potency with both IC50 and EC50 values respectively below 1 μM. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Given its strong cellular and antiviral potency, we cautiously suggest that MPI8 is ready for preclinical and clinical investigations for the treatment of COVID-19.

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“…[12][13][14] Some of them including camostat and K777 are under clinical trials for COVID-19. 16,17 An Mpro inhibitor that dually inhibits a host protease for the virus replication and pathogenesis potentiates improved overall antiviral potency and efficacy. To explore potential dual inhibition of a critical host protease, we tested inhibition potency of fourteen previously reported Mpro inhibitors including eleven peptidomimetic aldehydes and three diaryl esters against furin, TMPRSS2 and cathepsin L. To explore selectivity of these Mpro inhibitors against other host cysteine proteases, we tested their inhibition of cathepsins B and K as well.…”

Section: Introductionmentioning

confidence: 99%

MPI8 is Potent Against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L

Xinyu

Alugubelli

et al. 2021

Preprint

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A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10-3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.

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A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells

Cited by 83 publications

References 42 publications

Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics

MPI8 is Potent Against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L

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