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

Viruses ◽  
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.

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

2020 ◽  
Author(s):  
Maria Kuzikov ◽  
Elisa Costanzi ◽  
Jeanette Reinshagen ◽  
Francesca Esposito ◽  
Laura Vangeel ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Small Molecules ◽  
Drug Target ◽  
Treatment Options ◽  
Cleavage Sites ◽  
X Ray ◽  
Binding Characteristics ◽  
Main Protease ◽  
Ic50 Values

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.

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 In Silico Identification and Docking-Based Drug Repurposing Against the Main Protease of SARS-CoV-2, Causative Agent of COVID-19

2020 ◽  
Author(s):  
Yogesh Kumar ◽  
Harvijay Singh
Keyword(s):  
Virtual Screening ◽  
Active Site ◽  
Viral Infections ◽  
Drug Repurposing ◽  
Docking Study ◽  
Docking Studies ◽  
Main Protease ◽  
Novel Coronavirus ◽  
Approved Drugs

<div>The rapidly enlarging COVID-19 pandemic caused by novel SARS-coronavirus 2 is a global</div><div>public health emergency of unprecedented level. Therefore the need of a drug or vaccine that</div><div>counter SARS-CoV-2 is an utmost requirement at this time. Upon infection the ssRNA genome</div><div>of SARS-CoV-2 is translated into large polyprotein which further processed into different</div><div>nonstructural proteins to form viral replication complex by virtue of virus specific proteases:</div><div>main protease (3-CL protease) and papain protease. This indispensable function of main protease</div><div>in virus replication makes this enzyme a promising target for the development of inhibitors and</div><div>potential treatment therapy for novel coronavirus infection. The recently concluded α-ketoamide</div><div>ligand bound X-ray crystal structure of SARS-CoV-2 Mpro (PDB ID: 6Y2F) from Zhang et al.</div><div>has revealed the potential inhibitor binding mechanism and the determinants responsible for</div><div>involved molecular interactions. Here, we have carried out a virtual screening and molecular</div><div>docking study of FDA approved drugs primarily targeted for other viral infections, to investigate</div><div>their binding affinity in Mpro active site. Virtual screening has identified a number of antiviral</div><div>drugs, top ten of which on the basis of their bending energy score are further examined through </div><div>molecular docking with Mpro. Docking studies revealed that drug Lopinavir-Ritonavir, Tipranavir</div><div>and Raltegravir among others binds in the active site of the protease with similar or higher</div><div>affinity than the crystal bound inhibitor α-ketoamide. However, the in-vitro efficacies of the drug</div><div>molecules tested in this study, further needs to be corroborated by carrying out biochemical and</div><div>structural investigation. Moreover, this study advances the potential use of existing drugs to be</div><div>investigated and used to contain the rapidly expanding SARS-CoV-2 infection.</div>

Dabigatran Inhibits Both Clot-Bound and Fluid Phase Thrombin In Vitro: Effects Compared to Heparin and Hirudin.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3998-3998 ◽  
Author(s):  
Joanne van Ryn ◽  
Norbert Hauel ◽  
Lisa Waldmann ◽  
Wolfgang Wienen
Keyword(s):  
Active Site ◽  
Platelet Rich Plasma ◽  
Fluid Phase ◽  
Irreversible Inhibitor ◽  
Thrombin Inhibition ◽  
Ic50 Values ◽  
Artificial Surface ◽  
In Vitro Effects ◽  
Targeted Inhibition

Abstract Thrombin bound to fibrin either in an existing thrombus or on an artificial surface is thought to be protected from inhibition by heparin/ATIII. Thus, this surface can remain “active” despite heparin / LMWH therapy. As long as anticoagulant levels are maintained, fluid phase thrombin inhibition is sufficient to prevent progression of thrombosis. However, during times of trough levels or upon discontinued therapy, this active thrombin could initiate further prothrombotic events. Dabigatran is a reversible, univalent, direct inhibitor of thrombin. Considering its specificity and selectivity for the active site, it is to be expected that it can inhibit thrombin equally, both when bound to a clot and in fluid phase. Thus, this study investigated the ability of dabigatran to inhibit thrombin bound to fibrin, or thrombin in plasma as compared to heparin and hirudin in vitro. Clot-bound thrombin: Clots were generated in human platelet rich plasma by adding CaCl2 and incubating 2 hrs at 37°C (1). Clots were then washed to remove trapped FPA. Washed clots were then transferred to 0.5 ml platelet poor plasma (PPP) containing either dabigatran, heparin, hirudin or buffer and incubated at 37°C. Fluid-phase thrombin: Thrombin (20 pM) was added to PPP containing either dabigatran, heparin, hirudin or buffer and incubated for 1 hr at 37°C. Fibrinopeptide A (FPA) Measurement: Uncleaved fibrinogen was precipitated and fibrin formation was measured as FPA release using ELISA. The IC50 (concentration required to inhibit FPA release by 50%) in fluid phase and clot-bound conditions was calculated using nonlinear curve-fitting. Dabigatran, hirudin and heparin inhibited fluid and clot-bound thrombin with increasing concentrations. Dabigatran inhibited both clot-bound and fluid phase thrombin with similar affinity, IC50 values of 200 nM and 186 nM, respectively. The IC50 of heparin-induced inhibition of clot-bound thrombin was 98.7 nM and in fluid phase 8.4 nM. This is consistent with published results indicating that heparin is not as efficient in inhibiting thrombin bound to a clot as in the fluid phase. Hirudin is a potent, irreversible inhibitor of thrombin and in this study it inhibited both free and bound thrombin equally in low nM ranges, the IC50 of clot-bound thrombin was 0.59 nM and fluid phase thrombin 1.63 nM. The IC50 values were converted into a ratio of clot-bound vs fluid phase thrombin to allow a more direct comparison and are listed in the table. Dabigatran Heparin Hirudin Ratio IC50 Clot-bound: Fluid Phase IIa 1.07 11.76 0.36 Dabigatran is equally effective in inhibiting both clot-bound and fluid phase thrombin. These data are consistent with targeted inhibition of the active site of thrombin by a small molecule, irrespective of whether thrombin is bound via the exosite to fibrin or is present as free enzyme in plasma. In contrast, heparin is less effective in inhibiting of clot-bound than fluid phase thrombin, consistent with its mechanism of thrombin inhibition via ATIII. Hirudin more potently inhibits clot-bound than free thrombin at lower concentrations, at higher concentrations inhibition of fluid phase thrombin is favoured. Thus these data suggest that dabigatran could also be effective in clinical situations where surface-bound thrombin could play a role, such as during catheterization procedures or the treatment of established thrombosis.

scholarly journals Chemical Composition and Enzymatic Screening of Micromeria fruticosa serpyllifolia Volatile Oils Collected from Three Different Regions of West Bank, Palestine

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Nihaya Salameh ◽  
Naser Shraim ◽  
Nidal Jaradat
Keyword(s):  
Plant Extracts ◽  
Biological Activities ◽  
West Bank ◽  
Chemical Components ◽  
Chemical Compositions ◽  
Therapeutic Effects ◽  
Volatile Oils ◽  
Ic50 Values ◽  
Micromeria Fruticosa

Introduction. Volatile oils (VOs) have been commonly used in cosmetics and food as fragrances, flavoring, and preservative agents or in alternative medicine for their therapeutic effects. This necessitates investigating those plants and their VOs. This study was conducted to investigate the chemical compositions of the VOs of Micromeria fruticosa serpyllifolia growing widely in three regions in Palestine (i.e., Hebron, Ramallah, and Nablus districts representing south, middle, and north of West Bank). Afterwards, VOs were subjected to in vitro screening and their enzymatic properties were compared. Methods. The analysis of chemical components of VOs was performed by gas chromatography coupled with mass spectrometry (GC-MS). The antilipase activity was evaluated using porcine pancreatic lipase and p-nitrophenyl butyrate. The antiamylase activity was assessed using porcine pancreatic α-amylase, starch, and 3,5-dinitrosalicylic. Results. Plant extracts yield range was 0.67 to 0.99 w/w%. GC-MS analysis showed the high percentages of oxygenated components in the range of 86.1-89.88% and nonoxygenated components in the range of 4.38-4.71%. Seven components were observed, pulegone was the most abundant component in the three samples in the range of 74.43-86.04%, and isomenthone was the second most abundant component with the range of 3.16-14.41%. The sample collected from Nablus showed the most potent antilipase and antiamylase activity with IC50 values of 39.81 μg/mL and 3.31 μg/mL, respectively. Conclusions. The study showed that Micromeria fruticosa serpyllifolia volatile oils samples from different regions in Palestine contained different proportions of phytochemicals which provided different potential biological activities such as antiobesity and antidiabetes activities that were in line with traditional uses of the plant extracts. The plant extracts showed higher antilipase and antiamylase potency than that of the relative references and there were significant differences in these activities compared to each other.

scholarly journals SARS-CoV-2 Main Protease Active Site Ligands in the Human Metabolome

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1409
Author(s):  
Anna Maria Sardanelli ◽  
Camilla Isgrò ◽  
Luigi Leonardo Palese
Keyword(s):  
Active Site ◽  
Therapeutic Strategy ◽  
Silybum Marianum ◽  
Antiviral Therapies ◽  
Global Pandemic ◽  
Main Protease ◽  
Toxicological Profile ◽  
Starting Point ◽  
Antiviral Activities

In late 2019, a global pandemic occurred. The causative agent was identified as a member of the Coronaviridae family, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we present an analysis on the substances identified in the human metabolome capable of binding the active site of the SARS-CoV-2 main protease (Mpro). The substances present in the human metabolome have both endogenous and exogenous origins. The aim of this research was to find molecules whose biochemical and toxicological profile was known that could be the starting point for the development of antiviral therapies. Our analysis revealed numerous metabolites—including xenobiotics—that bind this protease, which are essential to the lifecycle of the virus. Among these substances, silybin, a flavolignan compound and the main active component of silymarin, is particularly noteworthy. Silymarin is a standardized extract of milk thistle, Silybum marianum, and has been shown to exhibit antioxidant, hepatoprotective, antineoplastic, and antiviral activities. Our results—obtained in silico and in vitro—prove that silybin and silymarin, respectively, are able to inhibit Mpro, representing a possible food-derived natural compound that is useful as a therapeutic strategy against COVID-19.

scholarly journals Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro

2021 ◽  
Vol 57 (78) ◽  
pp. 10083-10086
Author(s):  
Love Panchariya ◽  
Wajahat Ali Khan ◽  
Shobhan Kuila ◽  
Kirtishila Sonkar ◽  
Sibasis Sahoo ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Viral Replication ◽  
Active Site ◽  
Main Protease

Zn2+ binds to the active site of the SARS-CoV-2 main protease (Mpro), and inhibits enzyme activity and viral replication in vitro.

scholarly journals DESIGN, SYNTHESIS AND COX1/2 INHIBITORY ACTIVITY OF NEW QUINAZOLINE-5-ONE DERIVATIVES

2017 ◽  
Vol 13 (1) ◽  
pp. 5923-5931
Author(s):  
Ahmed S. Aboraia ◽  
Mohammed A. Hara ◽  
Mostafa H. Abdelrahman ◽  
Mohamed M. Amin ◽  
Osama I. El-Sabbaghab
Keyword(s):  
Active Site ◽  
Docking Study ◽  
Selectivity Index ◽  
Inhibition Assay ◽  
Inhibitor Concentration ◽  
Design Synthesis ◽  
Ic50 Values ◽  
Cox 2 ◽  
Cox 1

A new series of 1-(4-Acetylphenyl)-7,7-dimethyl-3-(substitutedphenyl)-1,2,3,4,7,8-octahydroquinazolin-5(6H)-ones (6-15) were synthesized and tested against COX-1 and COX-2 enzymes. Those compounds exhibited strong interaction at the COX-2 binding site and poor interaction at the COX-1 active site. Subjected to in vitro cyclooxygenase COX-1/COX-2 inhibition assay; most of the compounds especially compounds 6, 7, 12, 13, and 16 exhibited potent anti-inflammatory effects, selective COX-2 inhibition, with half-maximal inhibitor concentration (IC50) values of 0.22–1.42 μM and selectivity index (SI) values of 6.16–14.18 compared with celecoxib (IC50 = 0.05 μM and COX-2 SI: 296), diclofenac (IC50 = 0.8 μM and COX-2 SI: 4.87), and indomethacin (IC50 = 0.49 μM and COX-2 SI: 0.08) as reference drugs. Docking study has been carried out to confirm the binding affinity and selectivity of the most active compound (compound 6) to COX-2 enzyme.

scholarly journals In Silico Identification and Docking-Based Drug Repurposing Against the Main Protease of SARS-CoV-2, Causative Agent of COVID-19

2020 ◽  
Author(s):  
Yogesh Kumar ◽  
Harvijay Singh
Keyword(s):  
Virtual Screening ◽  
Active Site ◽  
Viral Infections ◽  
Drug Repurposing ◽  
Docking Study ◽  
Docking Studies ◽  
Main Protease ◽  
Novel Coronavirus ◽  
Approved Drugs

<div>The rapidly enlarging COVID-19 pandemic caused by novel SARS-coronavirus 2 is a global</div><div>public health emergency of unprecedented level. Therefore the need of a drug or vaccine that</div><div>counter SARS-CoV-2 is an utmost requirement at this time. Upon infection the ssRNA genome</div><div>of SARS-CoV-2 is translated into large polyprotein which further processed into different</div><div>nonstructural proteins to form viral replication complex by virtue of virus specific proteases:</div><div>main protease (3-CL protease) and papain protease. This indispensable function of main protease</div><div>in virus replication makes this enzyme a promising target for the development of inhibitors and</div><div>potential treatment therapy for novel coronavirus infection. The recently concluded α-ketoamide</div><div>ligand bound X-ray crystal structure of SARS-CoV-2 Mpro (PDB ID: 6Y2F) from Zhang et al.</div><div>has revealed the potential inhibitor binding mechanism and the determinants responsible for</div><div>involved molecular interactions. Here, we have carried out a virtual screening and molecular</div><div>docking study of FDA approved drugs primarily targeted for other viral infections, to investigate</div><div>their binding affinity in Mpro active site. Virtual screening has identified a number of antiviral</div><div>drugs, top ten of which on the basis of their bending energy score are further examined through </div><div>molecular docking with Mpro. Docking studies revealed that drug Lopinavir-Ritonavir, Tipranavir</div><div>and Raltegravir among others binds in the active site of the protease with similar or higher</div><div>affinity than the crystal bound inhibitor α-ketoamide. However, the in-vitro efficacies of the drug</div><div>molecules tested in this study, further needs to be corroborated by carrying out biochemical and</div><div>structural investigation. Moreover, this study advances the potential use of existing drugs to be</div><div>investigated and used to contain the rapidly expanding SARS-CoV-2 infection.</div>

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