Virtual screening and in vitro assays of novel hits as promising DPP-4 inhibitors

Abstract The emergence of SARS/MERS drug-resistant SARS-CoV2 comes with higher rates of transmission and mortality. Like all coronaviruses, SARS-CoV-2 is a relatively large virus consisting of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs by identifying potential drugs that are predicted to effectively inhibit critical enzymes. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral multiplication cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2’-O-MT. For virtual screening, the energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard(Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs (n=5903) that are approved by worldwide regulatory bodies. The screening was performed against viral targets using three sequential docking modes (i.e. HTVS, SP, and XP). Our in-silico virtual screening identified ~290 potential drugs based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. Herein we report the evaluation of in-vitro efficacy of selected hit drug molecules on SARS-CoV-2 inhibition. Among eight molecules included in our evaluation, we found inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), as the potent inhibitor of SARS-CoV-2 in-vitro. Further, in-silico predicted target validation through enzymatic assays confirmed 3CLpro to be the target. Therefore, our data support advancing BIM IX for clinical evaluation as a potential treatment for COVID-19. This is the first study that has showcased the possibility of using bisindolylmaleimide IX to treat COVID-19 through this pipeline.

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Bisindolylmaleimide IX: A novel anti-SARS-CoV2 agent targeting viral main protease 3CLpro demonstrated by virtual screening and in vitro assays

10.21203/rs.3.rs-48709/v1 ◽

2020 ◽

Author(s):

Yash Gupta ◽

Dawid Maciorowski ◽

Samantha E. Zak ◽

Krysten A. Jones ◽

Rahul S. Kathayat ◽

...

Keyword(s):

Virtual Screening ◽

In Silico ◽

Binding Free Energy ◽

Dynamics Simulation ◽

In Vitro Assays ◽

Protein Preparation ◽

Drug Molecules ◽

Virus Inhibition ◽

Energy Perturbation

Abstract The emergence of SARS/MERS drug-resistant COVID-19 with high transmission and mortality has recently been declared a pandemic. Like all coronaviruses, SARS-CoV-2 is a relatively large virus consisting of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs by identifying potential drugs that are predicted to effectively inhibit critical enzymes. We targeted seven proteins with enzymatic activities known to be essential at different stages of the virus life cycle; PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2’-O-MT. For virtual screening, the energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard1. Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs (n=5903) that are approved by worldwide regulatory bodies. The screening was performed against viral targets using three sequential docking modes (i.e. HTVS, SP, and XP). Our in-silico virtual screening identified ~290 potential drugs based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. Top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. Herein we report the evaluation of in-vitro efficacy of selected hit drug molecules on SARS-CoV-2 virus inhibition. Among eight molecules included in our evaluation, we found the micromolar inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), as the most potent inhibitor of SARS-CoV-2 in-vitro. Further, in-silico predicted target validation through enzymatic assays confirmed its interaction with 3CLpro to be the target. Therefore, our data support advancing BIM IX for clinical evaluation as a potential treatment for COVID-19. This is the first study that has showcased the possibility of using bisindolylmaleimide IX to treat COVID-19 through this pipeline.

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Discovering new mTOR inhibitors for cancer treatment through virtual screening methods and in vitro assays

Scientific Reports ◽

10.1038/srep18987 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 17

Author(s):

Ling Wang ◽

Lei Chen ◽

Miao Yu ◽

Li-Hui Xu ◽

Bao Cheng ◽

...

Keyword(s):

Virtual Screening ◽

Cancer Treatment ◽

Mtor Inhibitors ◽

In Vitro Assays ◽

Screening Methods

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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Discovery of New AKT1 Inhibitors by Combination of In silico Structure Based Virtual Screening Approaches and Biological Evaluations

10.26434/chemrxiv.7591202 ◽

2019 ◽

Author(s):

Filip Fratev ◽

Denisse A. Gutierrez ◽

Renato J. Aguilera ◽

suman sirimulla

Keyword(s):

Virtual Screening ◽

Success Rate ◽

Protein Interactions ◽

In Silico ◽

Biological Data ◽

In Vitro Binding ◽

Vitro Binding ◽

Screening Protocol ◽

Screening Approaches

AKT1 is emerging as a useful target for treating cancer. Herein, we discovered a new set of ligands that inhibit the AKT1, as shown by in vitro binding and cell line studies, using a newly designed virtual screening protocol that combines structure-based pharmacophore and docking screens. Taking together with the biological data, the combination of structure based pharamcophore and docking methods demonstrated reasonable success rate in identifying new inhibitors (60-70%) proving the success of aforementioned approach. A detail analysis of the ligand-protein interactions was performed explaining observed activities.<br>

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In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207321666180220125406 ◽

2018 ◽

Vol 21 (3) ◽

pp. 215-221

Author(s):

Haroon Khan ◽

Muhammad Zafar ◽

Helena Den-Haan ◽

Horacio Perez-Sanchez ◽

Mohammad Amjad Kamal

Keyword(s):

In Silico ◽

Docking Studies ◽

In Vivo Studies ◽

In Vitro Assays ◽

Chronic Obstructive ◽

Lipoxygenase Inhibitors ◽

Lipoxygenase Inhibition ◽

In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

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