Fig 1.
Selection and screening of AChEI drugs.
AChEI drugs were screened based on negative CDocker energy and IC50 values.
Fig 2.
A. Sequence alignment of HuPON2 on Chi-PON1 (PDB:1V04) shows 64.7% sequence identity and 82.6% sequence similarity. B. Crystal structure of HuPON1 (PDB:1V04); magenta color-coded. C. Homology model of HuPON2, prepared from HuPON1 template (PDB:1V04); violet color-coded. D. Superimposition of HuPON1 (magenta) and HuPON2 (violet), shows the close similarity of both the structures, RMSD value of 0.543. The analysis was performed in Discovery Studio 4.0.
Fig 3.
Docking of drug compounds in HuPON2.
A. In silico docking of the 3D structure of HuPON2 with DHC and B. In silico docking of the 3D structure of HuPON2 with PB. Various interacting residues are shown and the type of their interaction is color-coded. Discovery studio software was used for the visualization of the results and the creation of images.
Fig 4.
Comparison of AChE activity in the presence of inhibitors and enzymes.
A. DHC, in the presence and absence of HuPON2, B. PB, in the presence and absence of HuPON2. Inhibition is shown for AChE activity in the absence of inhibitors and HuPON2.
Fig 5.
Comparative activities of HuPON2-WT and catalytic site mutants and corresponding inhibition of selected AChEIs.
A. Esterase activity of HuPON2-WT compared with catalytic site mutants, B. Lactonase activity of HuPON2-WT compared with catalytic site mutants. The catalytic activity was followed in intervals ranging from 0 to 30 minutes. The activities were reported as product formed in terms of initial and final absorbance measured. Inhibition of AChEI by C. DHC and D. PB respectively in the presence of purified HuPON2-WT and mutant proteins. The well without HuPON2 protein was used as a control. Inhibition was reported as a percentage to control wells read. Results are the average of triplicates and error bars show the standard deviation.
Fig 6.
Comparative activities of HuPON2-WT and polymorphic site mutants and corresponding inhibition of selected AChEIs.
A. Esterase activity of HuPON2-WT compared with polymorphic site mutants, B. Lactonase activity of HuPON2-WT compared with polymorphic site mutants. The catalytic activity was followed in intervals ranging from 0 to 30 minutes. The activities were reported as product formed in terms of initial and final absorbance measured. Inhibition of AChEI by polymorphic HuPON2. C. DHC and D. PB in the presence of purified HuPON2-WT and polymorphic site mutant proteins. The wells without HuPON2 protein were used as control. AChEI drug inhibition was reported as a fold change to WT. Results are the average of triplicates and error bars show the standard deviation.
Table 1.
AChE enzyme kinetics.
Table 2.
Enzyme kinetics for WT and mutant HuPON2 with Phenylacetate as substrate.