Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 uM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Angs., showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

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Are Carbonic Anhydrases Suitable Targets to Fight Protozoan Parasitic Diseases?

Current Medicinal Chemistry ◽

10.2174/0929867325666180326160121 ◽

2019 ◽

Vol 25 (39) ◽

pp. 5266-5278 ◽

Cited By ~ 9

Author(s):

Katia D'Ambrosio ◽

Claudiu T. Supuran ◽

Giuseppina De Simone

Keyword(s):

Drug Resistance ◽

Animal Models ◽

Carbonic Anhydrase ◽

Drug Target ◽

Treatment Options ◽

Parasitic Diseases ◽

Carbonic Anhydrases ◽

Extensive Drug Resistance ◽

Protozoan Infections

Protozoans belonging to Plasmodium, Leishmania and Trypanosoma genera provoke widespread parasitic diseases with few treatment options and many of the clinically used drugs experiencing an extensive drug resistance phenomenon. In the last several years, the metalloenzyme Carbonic Anhydrase (CA, EC 4.2.1.1) was cloned and characterized in the genome of these protozoa, with the aim to search for a new drug target for fighting malaria, leishmaniasis and Chagas disease. P. falciparum encodes for a CA (PfCA) belonging to a novel genetic family, the η-CA class, L. donovani chagasi for a β-CA (LdcCA), whereas T. cruzi genome contains an α-CA (TcCA). These three enzymes were characterized in detail and a number of in vitro potent and selective inhibitors belonging to the sulfonamide, thiol, dithiocarbamate and hydroxamate classes were discovered. Some of these inhibitors were also effective in cell cultures and animal models of protozoan infections, making them of considerable interest for the development of new antiprotozoan drugs with a novel mechanism of action.

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The Repurposed Drugs Suramin and Quinacrine Cooperatively Inhibit SARS-CoV-2 3CLpro In Vitro

Viruses ◽

10.3390/v13050873 ◽

2021 ◽

Vol 13 (5) ◽

pp. 873

Author(s):

Raphael J. Eberle ◽

Danilo S. Olivier ◽

Marcos S. Amaral ◽

Ian Gering ◽

Dieter Willbold ◽

...

Keyword(s):

Binding Mode ◽

Drug Candidates ◽

Main Protease ◽

Ic50 Values ◽

Repurposed Drugs ◽

Antiparasitic Drugs ◽

Inhibitory Potential ◽

Dynamics Simulations ◽

Micromolar Range

Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) the WHO reported more than 148 million confirmed cases and 3.1 million losses globally up to now. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide, resulting in a pandemic of unprecedented magnitude. To date, several clinically safe and efficient vaccines (e.g., Pfizer-BioNTech, Moderna, Johnson & Johnson, and AstraZeneca COVID-19 vaccines) as well as drugs for emergency use have been approved. However, increasing numbers of SARS-Cov-2 variants make it imminent to identify an alternative way to treat SARS-CoV-2 infections. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is repurposing clinically developed drugs, e.g., antiparasitic drugs. The results described in this study demonstrated the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CLpro). Quinacrine and suramin molecules presented a competitive and noncompetitive inhibition mode, respectively, with IC50 values in the low micromolar range. Surface plasmon resonance (SPR) experiments demonstrated that quinacrine and suramin alone possessed a moderate or weak affinity with SARS-CoV-2 3CLpro but suramin binding increased quinacrine interaction by around a factor of eight. Using docking and molecular dynamics simulations, we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin, in combination with quinacrine, showed promising synergistic efficacy to inhibit SARS-CoV-2 3CLpro. We suppose that the identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease and repurposable drug candidates offer fast therapeutic breakthroughs, mainly in a pandemic moment.

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A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication

Nature Communications ◽

10.1038/s41467-021-20900-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shin-ichiro Hattori ◽

Nobuyo Higashi-Kuwata ◽

Hironori Hayashi ◽

Srinivasa Rao Allu ◽

Jakka Raghavaiah ◽

...

Keyword(s):

Small Molecule ◽

Covalent Bond ◽

Amino Acid Residues ◽

X Ray ◽

Viral Breakthrough ◽

Main Protease ◽

Small Molecule Compound ◽

Polar Interactions ◽

High Concentrations

AbstractExcept 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 (Mpro). 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 Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.

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Identification of Small Molecule Inhibitors of the Deubiquitinating Activity of the SARS-CoV-2 Papain-Like Protease: in silico Molecular Docking Studies and in vitro Enzymatic Activity Assay

Frontiers in Chemistry ◽

10.3389/fchem.2020.623971 ◽

2020 ◽

Vol 8 ◽

Author(s):

Eleni Pitsillou ◽

Julia Liang ◽

Katherine Ververis ◽

Kah Wai Lim ◽

Andrew Hung ◽

...

Keyword(s):

Molecular Docking ◽

Enzymatic Activity ◽

Small Molecules ◽

Binding Site ◽

Protein Docking ◽

Activity Assay ◽

Epicatechin Gallate ◽

Inhibitor Binding ◽

Enzymatic Activity Assay

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin >> rutin, cyanidin-3-O-glucoside > epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.

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

10.1101/2021.07.01.450756 ◽

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.

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Structure of the SAS-6 cartwheel hub from Leishmania major

eLife ◽

10.7554/elife.01812 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 43

Author(s):

Mark van Breugel ◽

Rainer Wilcken ◽

Stephen H McLaughlin ◽

Trevor J Rutherford ◽

Christopher M Johnson

Keyword(s):

Small Molecules ◽

Self Assembly ◽

Leishmania Major ◽

Cell Organelles ◽

Cylindrical Cell ◽

X Ray ◽

Cilia And Flagella ◽

Proof Of Principle

Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible.

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PLEKHA4 Promotes Wnt/β-catenin Signaling-Mediated G1/S Transition and Proliferation in Melanoma

10.1101/2020.07.29.225516 ◽

2020 ◽

Author(s):

Adnan Shami Shah ◽

Xiaofu Cao ◽

Andrew C. White ◽

Jeremy M. Baskin

Keyword(s):

Small Molecules ◽

Ubiquitin Ligase ◽

Targeted Therapies ◽

Drug Target ◽

Binding Partners ◽

Xenograft Models ◽

Melanoma Patients ◽

Cell Cycle Transition ◽

Braf Mutant

ABSTRACTMelanoma patients incur substantial mortality, despite promising recent advances in targeted therapies and immunotherapies. In particular, inhibitors targeting BRAF-mutant melanoma can lead to resistance, and no targeted therapies exist for NRAS-mutant melanoma, motivating the search for additional therapeutic targets and vulnerable pathways. Here, we identify a regulator of Wnt/β-catenin signaling, PLEKHA4, as a factor required for melanoma proliferation and survival. PLEKHA4 knockdown in vitro leads to lower Dishevelled levels, attenuated Wnt/β-catenin signaling, and a block of progression through the G1/S cell cycle transition. In mouse xenograft models, inducible PLEKHA4 knockdown attenuated tumor growth in BRAF- and NRAS-mutant melanomas and synergized with the clinically used inhibitor encorafenib in a BRAF-mutant model. As an E3 ubiquitin ligase regulator with both lipid and protein binding partners, PLEKHA4 presents several opportunities for targeting with small molecules. Our work identifies PLEKHA4 as a promising drug target for melanoma and clarifies a controversial role for Wnt/β-catenin signaling in the control of melanoma proliferation.

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Millet Derived Flavonoids as Potential SARS-CoV-2 Main Protease Inhibitors: A Computational Approach

10.26434/chemrxiv.12733622 ◽

2020 ◽

Author(s):

Abhisek Mishra ◽

Sobha Chnadra Rath ◽

Iswar Baitharu ◽

Bhawani Prasad Bag

Keyword(s):

Drug Target ◽

Hiv Protease ◽

Binding Affinities ◽

Autodock Vina ◽

Major Health ◽

Main Protease ◽

Negative Effect ◽

Mutagenic Property

The on-going pandemic COVID-19 has emerged as a major health threat across the globe. At present, anti-viral drug discoveries are of great importance in combating the pandemic. Millets are known to contain numerous flavonoids with potential anti-viral properties. However, their anti-viral efficacy against SARS-CoV-2 is yet to be studied. The study uses the SARS-CoV-2 main protease (Mpro) as the potential drug target and docks with eleven millet derived flavonoids taking HIV protease inhibiting drugs nelfinavir and saquinavir as control. AutoDock Vina was used for assessing the binding affinities and strength of binding of flavonoids present in millets with the target protein Mpro. Further, the drug-likeness and pharmacokinetics properties of these flavonoids were analyzed using admetSAR. The ADMET analysis showed that isoorientin, orientin, vitexin, meletin, catechin, and myricetin possess potential mutagenic property while daidzein could have a negative effect on reproductive making these compounds as poor candidates for drug development against SARS-CoV-2. Based on the docking result and positive ADMET properties, the present study infers that apigenin may be considered as a potential inhibitor of SARS-CoV-2 Mpro and may be further investigated to test its anti-viral activities using in-vitro and in-vivo study.

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Millet Derived Flavonoids as Potential SARS-CoV-2 Main Protease Inhibitors: A Computational Approach

10.26434/chemrxiv.12733622.v1 ◽

2020 ◽

Author(s):

Abhisek Mishra ◽

Sobha Chnadra Rath ◽

Iswar Baitharu ◽

Bhawani Prasad Bag

Keyword(s):

Drug Target ◽

Hiv Protease ◽

Binding Affinities ◽

Autodock Vina ◽

Major Health ◽

Main Protease ◽

Negative Effect ◽

Mutagenic Property

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Two New Cytotoxic Steroidal Alkaloids from Sarcococca Hookeriana

Molecules ◽

10.3390/molecules24010011 ◽

2018 ◽

Vol 24 (1) ◽

pp. 11 ◽

Cited By ~ 1

Author(s):

Shaojie Huo ◽

Jichun Wu ◽

Xicheng He ◽

Lutai Pan ◽

Jiang Du

Keyword(s):

Cell Lines ◽

Human Cancer ◽

Cytotoxic Activities ◽

Steroidal Alkaloids ◽

X Ray ◽

Human Cancer Cell Lines ◽

X Ray Crystallography ◽

Ic50 Values ◽

Mcf 7

Two new steroidal alkaloids, named hookerianine A (1) and hookerianine B (2) were isolated from the stems and roots of Sarcococca hookeriana Baill., along with two known compounds, sarcorucinine G (3) and epipachysamine D (4). On the basis of spectroscopic methods and by comparison with literature data, their structures were determined. As well as X-ray crystallography was performed to confirm compound 4. To identify novel antitumor inhibitors, all compounds were performed a CCK-8 assay against five human cancer cell lines SW480, SMMC-7721, PC3, MCF-7 and K562 in vitro. Compound 2 exhibited moderate cytotoxic activities to all cell lines with IC50 values in the range of 5.97–19.44 μM. Compound 3 was the most effective one against SW480 and K562 cell lines with IC50 values of 5.77 and 6.29 μM, respectively.

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