ABSTRACT
The haloalkane dehalogenase LinB is a well-known enzyme that contains buried active site and is used for many modelling studies. Using classical molecular dynamics simulations of enzymes and substrates, we searched for transient binding sites on the surface of the LinB protein by calculating maps of enzyme-ligand interactions that were then transformed into sparse matrices. All residues considered as functionally important for enzyme performance (e.g., tunnel entrances) were excluded from the analysis to concentrate rather on non-obvious surface residues. From a set of 130 surface residues, twenty-six were proposed as a promising improvement of enzyme performance. Eventually, based on rational selection and filtering out the potentially unstable mutants, a small library of ten mutants was proposed to validate the possibility of fine-tuning the LinB protein. Nearly half of the predicted mutant structures showed improved activity towards the selected substrates, which demonstrates that the proposed approach could be applied to identify non-obvious yet beneficial mutations for enzyme performance especially when obvious locations have already been explored.
Subject(s)
Hydrolases , Molecular Dynamics Simulation , Binding Sites , Hydrolases/metabolism , Catalytic DomainABSTRACT
Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.
Subject(s)
Coronavirus M Proteins/antagonists & inhibitors , Factor Xa Inhibitors/chemistry , SARS-CoV-2/metabolism , Benzamides/chemistry , Benzamides/metabolism , Binding Sites , COVID-19/virology , Coronavirus M Proteins/metabolism , Factor Xa Inhibitors/metabolism , Humans , Molecular Dynamics Simulation , Protein Binding , Protein Stability , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyridines/chemistry , Pyridines/metabolism , Pyridones/chemistry , Pyridones/metabolism , Rivaroxaban/chemistry , Rivaroxaban/metabolism , SARS-CoV-2/isolation & purification , COVID-19 Drug TreatmentABSTRACT
In the present study we tested, using the microscale thermophoresis technique, a small library of thionocarbamates, thiolocarbamates, sulfide and disulfide as potential lead compounds for SARS-CoV-2 Mpro drug design. The successfully identified binder is a representative of the thionocarbamates group with a high potential for future modifications aiming for higher affinity and solubility. The experimental analysis was extended by computational studies that show insufficient accuracy of the simplest and widely applied approaches and underline the necessity of applying more advanced methods to properly evaluate the affinity of potential SARS-CoV-2 Mpro binders.