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

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.

scholarly journals Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E

2021 ◽  
Author(s):  
Yue Zhu ◽  
Frank Scholle ◽  
Samantha C. Kisthardt ◽  
Deyu Xie
Keyword(s):  
Docking Simulation ◽  
Binding Pocket ◽  
Developed Countries ◽  
Host Cells ◽  
Human Coronavirus ◽  
Main Protease ◽  
Ic50 Values ◽  
In Vitro Inhibition ◽  
Human Coronavirus 229E

Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus, however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercetin, and rutin could bind to at least two subsites (S1, S1', S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from -8.8 to -7.4. Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from -7.1 to -7.8. In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 uM. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.

scholarly journals Human liver cathepsin L

1985 ◽  
Vol 226 (1) ◽  
pp. 233-241 ◽  
Author(s):  
R W Mason ◽  
G D J Green ◽  
A J Barrett
Keyword(s):  
Active Site ◽  
Human Liver ◽  
Cathepsin L ◽  
Cysteine Residue ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Active Site Cysteine ◽  
Apparent Homogeneity ◽  
Polypeptide Chains ◽  
Active Site Cysteine Residue

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.

scholarly journals The impact of calcitriol and estradiol on the SARS-CoV-2 biological activity: a molecular modeling approach

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Alireza Mansouri ◽  
Rasoul Kowsar ◽  
Mostafa Zakariazadeh ◽  
Hassan Hakimi ◽  
Akio Miyamoto
Keyword(s):  
Active Site ◽  
Clinical Investigation ◽  
Host Cells ◽  
Spike Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Risk Of Death ◽  
Main Protease ◽  
Computational Data ◽  
Novel Coronavirus ◽  
The Impact

AbstractThe novel coronavirus disease (COVID-19) is currently a big concern around the world. Recent reports show that the disease severity and mortality of COVID-19 infected patients may vary from gender to gender with a very high risk of death for seniors. In addition, some steroid structures have been reported to affect coronavirus, SARS-CoV-2, function and activity. The entry of SARS-CoV-2 into host cells depends on the binding of coronavirus spike protein to angiotensin converting enzyme-2 (ACE2). Viral main protease is essential for the replication of SARS-CoV-2. It was hypothesized that steroid molecules (e.g., estradiol, progesterone, testosterone, dexamethasone, hydrocortisone, prednisone and calcitriol) could occupy the active site of the protease and could alter the interaction of spike protein with ACE2. Computational data showed that estradiol interacted more strongly with the main protease active site. In the presence of calcitriol, the binding energy of the spike protein to ACE2 was increased, and transferring Apo to Locked S conformer of spike trimer was facilitated. Together, the interaction between spike protein and ACE2 can be disrupted by calcitriol. Potential use of estradiol and calcitriol to reduce virus invasion and replication needs clinical investigation.

scholarly journals The N-Terminal Carbamate is Key to High Cellular and Antiviral Potency for Boceprevir-Based SARS-CoV-2 Main Protease Inhibitors

2021 ◽  
Author(s):  
Yugendar R. Alugubelli ◽  
Zhi Zachary Geng ◽  
Kai Yang ◽  
Namir Shaabani ◽  
Kaustav Khatua ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Hydrophobic Interactions ◽  
Complex Structure ◽  
Host Cells ◽  
Side Chain ◽  
Active Site Cysteine ◽  
Main Protease ◽  
Control Compound ◽  
Inhibition Potency

Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and human host cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a β-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the war-head with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 opal residue, P2 dime-thylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed MPro in human 293T cells. All inhibitors including PF-07321332 with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC50 values around 1 μM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

scholarly journals Acrylamide Fragment Inhibitors that Induce Unprecedented Conformational Distortions in Enterovirus 71 3C and SARS-CoV-2 Main Protease

2020 ◽  
Author(s):  
Bo Qin ◽  
Gregory B. Craven ◽  
Pengjiao Hou ◽  
Xinran Lu ◽  
Emma S. Child ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Catalytic Triad ◽  
Active Site Cysteine ◽  
Dimeric Unit ◽  
Main Protease ◽  
Selective Agents ◽  
Catalytic Cysteine

ABSTRACTRNA viruses are critically dependent upon virally encoded proteases that cleave the viral polyproteins into functional mature proteins. Many of these proteases are structurally conserved with an essential catalytic cysteine and this offers the opportunity to irreversibly inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that selectively target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we effectively re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold as well as similar catalytic-triad. We demonstrate that the hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. In addition, we provide the first demonstration that targeting the active site cysteine of Mpro can also have profound allosteric effects, distorting secondary structures required for formation of the active dimeric unit of Mpro. These new data provide novel mechanistic insights into the design of EV71 3Cpro and SARS-CoV-2 Mpro inhibitors and identify acrylamide-tagged pharmacophores for elaboration into more selective agents of therapeutic potential.

Localization of Rat Cathepsin K in Osteoclasts and Resorption Pits: Inhibition of Bone Resorption and Cathepsin K-Activity by Peptidyl Vinyl Sulfones

1999 ◽  
Vol 380 (6) ◽  
Author(s):  
L. Xia ◽  
J. Kilb ◽  
H. Wex ◽  
Z. Li ◽  
A. Lipyansky ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Cathepsin L ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Cathepsin K ◽  
Selective Inhibition ◽  
Specific Substrate ◽  
Dependent Manner ◽  
Protease Inhibition ◽  
K Activity

AbstractWe have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment.Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MβNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MβNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MβNA, was prevented by both types of inhibitors.The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.

Reactivity-guided de novo molecular design and high throughput virtual screening of a targeted library of peptidomimetic compounds reveals charge-based structure-activity relationship of potential covalent inhibitors of the main protease of SARS-CoV-2

2020 ◽  
Vol 9 (2) ◽  
Author(s):  
Stephanie Sun ◽  
Kavya Anand ◽  
Ishani Ashok ◽  
Bhavesh Ashok ◽  
Ayush Bajaj ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Small Molecules ◽  
High Throughput ◽  
Active Site ◽  
De Novo ◽  
Cysteine Residue ◽  
Host Cells ◽  
Main Protease ◽  
Covalent Inhibitors ◽  
High Throughput Virtual Screening

In December of 2019, a novel coronavirus was first identified in Wuhan, China, and has since spread around the world, leaving a largely unsolved biomedical problem in its wake. Upon entry into host cells, the main protease is essential for the replication of viral RNA, which is what allows the virus to replicate inside humans. Inhibition of the main protease has been investigated as a potential strategy for inhibition of the viral replication cycle. Here, we designed a combinatorial library of small molecules and performed high-throughput virtual screening to identify a series of hit compounds that may serve as potential inhibitors of the main protease. In our design of covalent inhibitors of the coronavirus protease, we modeled a library of 361 peptidomimetic Michael acceptor small molecules, which are designed to engage the nucleophilic cysteine residue in the active site of the protease in an irreversible 1,4-conjugate addition. We then employed a variety of computational tools to determine the binding affinity of our designed compounds when bound to the protease active site, where we determined that cationic side chains are potentially beneficial for inhibition of SARS-CoV-2.   

scholarly journals The P3 O-Tert-Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors

2021 ◽  
Author(s):  
Yuying Ma ◽  
Kai Yang ◽  
Zhi Zachary Geng ◽  
Yugendar R. Alugubellia ◽  
Namir Shaabani ◽  
...  
Keyword(s):  
Active Site ◽  
Unnatural Amino Acids ◽  
Chemical Compositions ◽  
Structural Variations ◽  
Covalent Interaction ◽  
Tert Butyl ◽  
Main Protease ◽  
Ic50 Values ◽  
Inhibition Potency

As an essential enzyme to SARS-CoV-2, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of MPro inhibitors that contain 𝛽-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC50 values that range from 4.8 to 650 nM. The determined IC50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro MPro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the 𝛼-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

scholarly journals A Speedy Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors

2020 ◽  
Author(s):  
Kai S. Yang ◽  
Xinyu R. Ma ◽  
Yuying Ma ◽  
Yugendar R. Alugubelli ◽  
Danielle A. Scott ◽  
...  
Keyword(s):  
Chemical Space ◽  
A549 Cells ◽  
Covalent Bond ◽  
Structural Rearrangement ◽  
High Potency ◽  
Active Site Cysteine ◽  
Virus Inhibition ◽  
Main Protease ◽  
Reversible Covalent ◽  
Cytopathogenic Effect

ABSTRACTThe COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

scholarly journals Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2

2022 ◽  
Author(s):  
Lucianna H. Santos ◽  
Thales Kronenberger ◽  
Renata G Almeida ◽  
Elany Barbosa da Silva ◽  
Rafael E O Rocha ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Targets ◽  
Inhibitory Concentration ◽  
Binding Modes ◽  
Fluorogenic Substrate ◽  
Biochemical Assays ◽  
Main Protease ◽  
Ic50 Values ◽  
Biochemical Approach ◽  
Dynamics Simulations

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 μM and 66 μM. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 μM to 46 μM. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

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