Figure 1.
A. Heat map analysis of binding constants of 140 FDA approved nervous system drugs screened against Aβ, AChE and β-secretase by Autodock tool 4.2. In the gradient ruler, red colour indicated strong binding (ΔG<−6 kcal/mol), while green colour indicate weak binding (ΔG>−3 kcal/mol) and the five drugs showing higher affinity to all the above mentioned targets were zoomed. B. Chemical structures of the five drugs. All are tricyclic anti-depressant drugs.
Figure 2.
Protriptyline inhibits AChE activity.
A. Determination of IC50 values of five drugs for AChE by using 0.05–0.8 mM concentration range of all the drugs B. Estimation of the kinetic constants by Lineweaver–Burk analysis. AChE inhibition by protriptyline showed competitive inhibition. C. Isothermal Titration Calorimetric analysis of protriptyline – AChE interactions. The upper panel shows the raw data in the form of heat effect during titration and the lower panel shows corresponding thermogram representing the best fit curve D. Snapshot of drug binding with catalytic subsite of AChE E. snapshot of drug binding with anionic subsite of AChE F. Distribution of Protriptyline –anionic subsite (solid line) and Protriptyline –esteratic subsite (broken line) nonbonded (nonb) interaction energy; data are averaged over last 20 ns G. Fluorescence quenching of AChE by protriptyline H. CD spectra of binding of protriptyline to AChE and I. CD pro analysis to study the conformational change J. Evolution of the backbone RMSD for the Protriptylline bound (solid line) and free (broken line) AChE active sites from MD trajectories K. SASA distributions of active sites for Pro-bound (solid line) and free (broken line) AChE active sites from MD trajectories L. Measurement of AChE activity after treatment of neuro2a cells with 25 µM and 60 µM protriptyline for 15 h.
Figure 3.
Inhibition of Aβ aggregation by protriptyline.
Aβ13–22 aggregation in the absence and presence of protriptyline was investigated by A. Thioflavin T assay B. Light scattering C. CD analysis D. Atomic Force Microscopy images [i and iv] of aggregated amyloid and Amyloid + protriptyline (0.010 mM) in 10×10 µm2 and 20×20 µm2 surface area respectively. It is also represented in Line profile [ii and v] and 3D images [iii and vi].
Figure 4.
Destabilization of amyloid dimer by protriptyline.
A. Evolution of monomer-monomer interaction strength over time for free dimer (broken line) and Protriptylline-bound dimer (solid line). Inset. Distributions of the interactions from multiple trajectories, and the dimer interactions with Protriptylline (in brown) B. Distributions of the asphericity for free (in broken line) and Protriptylline-bound (solid line) dimer C. Representative snapshot of most populated cluster of free, and D. Protriptylline-bound dimer [16–20 region in blue colour with 19–20 showed in line representation; protriptyline in red colour and two Aβ peptides are in cyan and limon colour respectively] E. Residue-residue contact probabilities for free dimer, and F. Protriptylline-bound dimer G. Residue-wise Beta sheet percentages for free dimer (in red) and Protriptylline-bound dimer (in blue) H. Residue-wise helical percentages for free dimer (in red) and Pro-bound dimer (in blue).
Figure 5.
BACE-1 inhibition by protriptyline.
A. Determination of IC50 of BACE-1 by using various concentrations of protriptyline. The sigmoidal curve indicates the best fit for the percentage inhibition data obtained B. Lineweaver-Burk analysis to estimate the kinetic constants. It showed competitive inhibition. C. Snapshot of drug binding with active site of BACE-1. Active site residues in BACE-1 are in line representation. D. Active site of BACE-1. The structures from unbound (green) and ligand bound (orange) simulations are shown after all - atom superimposition. Snapshots are generated using PyMol.
Figure 6.
Protriptyline inhibits glycation.
A. Fluorescence emmission of Aβ and glycated Aβ in presence of various concentration of protriptyline B. Light scattering and C. Kinetics of amyloid aggregation by Thioflavin T of Aβ13–22 and glycated Aβ13–22 in the absence and presence of protriptyline.
Figure 7.
Protriptyline Does Not Affect Other Proteases.
Effect of protriptyline on A. Trypsin B. ADAM 17activity. Specific synthetic substrate BApNA and fluorogenic peptide was used for analyzing activity on trypsin and ADAM 17, respectively. Trypsin activity was unaffected, while ADMA 17 showed weak inhibition in presence of protriptyline.
Figure 8.
Cell viability in neuro2a cells.
Effect of various concentrations of protriptyline (25–500 µM) on cell viability was assessed by MTT assay. Cells were 90% viable up to 150 µM protriptyline concentration.
Figure 9.
The scheme represents that protriptyline (at the center) is able to inhibit key targets of AD pathogenesis such as AChE, BACE-1, Amyloid aggregation and glycation induced amyloid aggregation.