Front Cover: MPI8 is Potent against SARS‐CoV‐2 by Inhibiting Dually and Selectively the SARS‐CoV‐2 Main Protease and the Host Cathepsin L (ChemMedChem 1/2022)

AbstractThe main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1’ pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1’ pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1’, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the α-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

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Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture

10.1101/2020.10.30.362335 ◽

2020 ◽

Author(s):

Yanmei Hu ◽

Chunlong Ma ◽

Tommy Szeto ◽

Brett Hurst ◽

Bart Tarbet ◽

...

Keyword(s):

Cell Culture ◽

Antiviral Activity ◽

Cathepsin L ◽

Antiviral Drug ◽

Antiviral Effect ◽

Neutralization Assay ◽

Drug Candidates ◽

Main Protease ◽

Human Coronaviruses ◽

Calpain Inhibitors

AbstractAs the COVID-19 pandemic continues to fold out, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. Despite the weaker enzymatic inhibition of calpain inhibitors II and XII against Mpro compared to GC-376, calpain inhibitors II and XII had more potent cellular antiviral activity. This observation promoted us to hypothesize that the cellular antiviral activity of calpain inhibitors II and XII might also involve the inhibition of cathepsin L in addition to Mpro. To test this hypothesis, we tested calpain inhibitors II and XII in the SARS-CoV-2 pseudovirus neutralization assay in Vero E6 cells and found that both compounds significantly decreased pseudoviral particle entry into cells, indicating their role in inhibiting cathepsin L. The involvement of cathepsin L was further confirmed in the drug time-of-addition experiment. In addition, we found that these four compounds not only inhibit SARS-CoV-2, but also SARS-CoV, MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift binding assay and enzymatic FRET assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 are not only promising antiviral drug candidates against existing human coronaviruses, but also might work against future emerging CoVs.

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Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro/3CLpro) and Human Cathepsin-L

International Journal of Molecular Sciences ◽

10.3390/ijms222111779 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11779

Author(s):

Elisa Costanzi ◽

Maria Kuzikov ◽

Francesca Esposito ◽

Simone Albani ◽

Nicola Demitri ◽

...

Keyword(s):

Biochemical Characterization ◽

Cathepsin L ◽

Covalent Binding ◽

Covalent Bond ◽

Binding Mode ◽

Space Groups ◽

Main Protease ◽

Catalytic Cysteine ◽

Future Drug ◽

Human Cathepsin

After almost two years from its first evidence, the COVID-19 pandemic continues to afflict people worldwide, highlighting the need for multiple antiviral strategies. SARS-CoV-2 main protease (Mpro/3CLpro) is a recognized promising target for the development of effective drugs. Because single target inhibition might not be sufficient to block SARS-CoV-2 infection and replication, multi enzymatic-based therapies may provide a better strategy. Here we present a structural and biochemical characterization of the binding mode of MG-132 to both the main protease of SARS-CoV-2, and to the human Cathepsin-L, suggesting thus an interesting scaffold for the development of double-inhibitors. X-ray diffraction data show that MG-132 well fits into the Mpro active site, forming a covalent bond with Cys145 independently from reducing agents and crystallization conditions. Docking of MG-132 into Cathepsin-L well-matches with a covalent binding to the catalytic cysteine. Accordingly, MG-132 inhibits Cathepsin-L with nanomolar potency and reversibly inhibits Mpro with micromolar potency, but with a prolonged residency time. We compared the apo and MG-132-inhibited structures of Mpro solved in different space groups and we identified a new apo structure that features several similarities with the inhibited ones, offering interesting perspectives for future drug design and in silico efforts.

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Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against Mpro and cathepsin L

Science Advances ◽

10.1126/sciadv.abe0751 ◽

2020 ◽

Vol 6 (50) ◽

pp. eabe0751 ◽

Cited By ~ 7

Author(s):

Michael Dominic Sacco ◽

Chunlong Ma ◽

Panagiotis Lagarias ◽

Ang Gao ◽

Julia Alma Townsend ◽

...

Keyword(s):

Viral Entry ◽

Cathepsin L ◽

Antiviral Drug ◽

Side Chain ◽

Calpain Inhibitor ◽

Main Protease ◽

New Directions ◽

Dual Inhibitors ◽

Hydrophilic Residue ◽

Calpain Inhibitors

The main protease (Mpro) of SARS-CoV-2 is a key antiviral drug target. While most Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of Mpro in complex with calpain inhibitors II and XII and three analogs of GC-376. The structure of Mpro with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. The structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.

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

ChemMedChem ◽

10.1002/cmdc.202100456 ◽

2021 ◽

Author(s):

Wenshe Liu ◽

R. Xinyu ◽

Yugendar R. Alugubelli ◽

Yuying Ma ◽

Erol C. Vantasever ◽

...

Keyword(s):

Cathepsin L ◽

Main Protease

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

10.1101/2021.06.10.447950 ◽

2021 ◽

Author(s):

Xinyu R Ma ◽

Yugendar R Alugubelli ◽

Yuying Ma ◽

Erol Can Vatansever ◽

Danielle A Scott ◽

...

Keyword(s):

Active Site ◽

Cathepsin L ◽

Critical Role ◽

Cathepsin K ◽

Host Cells ◽

High Potency ◽

Active Site Cysteine ◽

Cellular Analysis ◽

Main Protease ◽

Ic50 Values

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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Natural Apocarotenoids and Their Synthetic Glycopeptide Conjugates Inhibit SARS-CoV-2 Replication

Pharmaceuticals ◽

10.3390/ph14111111 ◽

2021 ◽

Vol 14 (11) ◽

pp. 1111

Author(s):

Ilona Bereczki ◽

Henrietta Papp ◽

Anett Kuczmog ◽

Mónika Madai ◽

Veronika Nagy ◽

...

Keyword(s):

Cathepsin L ◽

Antiviral Effect ◽

Food Additive ◽

New Drugs ◽

Computational Studies ◽

Mechanistic Studies ◽

Cell Viability Assay ◽

Viability Assay ◽

Main Protease ◽

A Cell

The protracted global COVID-19 pandemic urges the development of new drugs against the causative agent SARS-CoV-2. The clinically used glycopeptide antibiotic, teicoplanin, emerged as a potential antiviral, and its efficacy was improved with lipophilic modifications. This prompted us to prepare new lipophilic apocarotenoid conjugates of teicoplanin, its pseudoaglycone and the related ristocetin aglycone. Their antiviral effect was tested against SARS-CoV-2 in Vero E6 cells, using a cell viability assay and quantitative PCR of the viral RNA, confirming their micromolar inhibitory activity against viral replication. Interestingly, two of the parent apocarotenoids, bixin and β-apo-8’carotenoic acid, exerted remarkable anti-SARS-CoV-2 activity. Mechanistic studies involved cathepsin L and B, as well as the main protease 3CLPro, and the results were rationalized by computational studies. Glycopeptide conjugates show dual inhibitory action, while apocarotenoids have mostly cathepsin B and L affinity. Since teicoplanin is a marketed antibiotic and the natural bixin is an approved, cheap and widely used red colorant food additive, these readily available compounds and their conjugates as potential antivirals are worthy of further exploration.

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ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model

10.1101/2021.02.14.431129 ◽

2021 ◽

Cited By ~ 1

Author(s):

Koen Vandyck ◽

Rana Abdelnabi ◽

Kusum Gupta ◽

Dirk Jochmans ◽

Andreas Jekle ◽

...

Keyword(s):

Infectious Virus ◽

Cathepsin L ◽

Cysteine Protease Inhibitor ◽

Hamster Model ◽

Main Protease ◽

Promising Target ◽

Promising Therapeutic Target ◽

Potential Safety ◽

Human Cathepsin

AbstractThere is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 μM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

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