Identification of potential PKC inhibitors through pharmacophore designing, 3D-QSAR and molecular dynamics simulations targeting Alzheimer’s disease

<p>Protein kinases are ubiquitously expressed as Serine/Threonine kinases, and play a crucial role in cellular activities. Protein kinases have evolved through stringent regulation mechanisms. Protein kinases are also involved in tauopathy, thus are important targets for developing Anti-Alzheimer’s disease compounds. Structures with an indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors for human protein kinase C, here we report the generation of four point 3D geometric featured pharmacophore model. In order to identify novel and potent PKCθ inhibitors, the pharmacophore model was screened against 80,000,00 compounds from various chemical databases such as., ZINC, SPEC, ASINEX, which resulted in 127 compound hits, and were taken for molecular docking filters (HTVS, XP docking). After in-depth analysis of binding patterns, induced fit docking (flexible) was employed for six compounds along with the cocrystallized inhibitor. Molecular docking study reveals that compound 6F found to be tight binder at the active site of PKCθ as compared to the cocrystal and has occupancy of 90 percentile. MM-GBSA also confirmed the potency of the compound 6F as better than cocrystal. Molecular dynamics results suggest that compound 6F showed good binding stability of active sites residues similar to cocrystal 7G compound. Present study corroborates the pharmacophore-based virtual screening, and finds the compound 6F as a potent Inhibitor of PKC, having therapeutic potential for Alzheimer’s disease. Worldwide, 46.8 million people are believed to be living with Alzheimer’s disease. When elderly population increases rapidly and neurodegenerative burden also increases in parallel, we project the findings from this study will be useful for drug developing efforts targeting Alzheimer’s disease.</p

NMR chemical shift perturbations in Bm-Aspin due to pepsin interactions.

<p>Perturbations in the chemical shift position of the residues Y215 (a), I214 (b), and A213 (c) respectively in Bm-Aspin upon addition of increasing concentrations of pepsin. Ratios of Bm-Aspin to pepsin are: 1∶0 (red), 1∶0.1 (cyan), 1∶0.5 (green), and 1.1 (yellow).</p

Kinetics of aspartic protease inhibition by Bm-Aspin.

<p>Lineweaver-Burk Plots showing the variation (1/V with that of 1/S) of competitive inhibition of pepsin (A) and cathepsin-E (C), non-competitive for renin (B) and mixed inhibition for cathepsin-D (D) respectively. Assays were carried out in triplicates, with the fixed quantity of proteases (5 mM) and varying concentrations of Bm-Aspin (0 mM, 1 mM, 2.5 mM and 5 mM) The inhibition constants were determined using Graphpad Prism 2.0 (San Diego, CA).</p

NMR screening on Bm-Aspin with different detergents.

<p>¹⁵N HSQC spectra of Bm-Aspin at pH 7.0 with the addition of: (i) No detergent (ii) 0.5 M Urea and 1% glycerol (iii) 1% <i>n</i>-octyl-β-D-glucoside (OG), (iv) 100 mM n-Dodecyl β-D-Maltopyranoside (DDM) (v) 1% triton X-100, and (vi) 100 mM SDS.</p

Chemical shift perturbations upon addition of human aspartic proteases.

<p>(A) NMR Chemical shift Perturbations in Bm-Aspin due to different protease interactions at their saturated conditions. Chemical shift perturbations of the residues Y215 (i), I214 (ii), and A213 (iii) respectively, upon the addition of human aspartic proteases at their saturation levels. Free Bm-Aspin (Magenta), Bm-Aspin+Cathepsin-D (Yellow), Bm-Aspin+Cathepsin-E (Green), Bm-Aspin+Renin (Red), Bm-Aspin+Pepsin (Grey). (B) Comparison of Bm-Aspin chemical shift perturbation upon addition of human aspartic proteases. The bar diagram indicating the radial shift (calculated for each of the affected residues, by combining both the chemical shifts of ¹H and ¹⁵N, using the equation: Radial shift displacement (Δδ) = {(H_f−H_b) ²+[(N_f−N_b)/6] ²} ^1/2. A scaling factor of 6 was used to normalize the differences in the ¹H and ¹⁵N spectral widths. H_f, H_b, N_f, and N_b are the chemical shifts of each residue's amide ¹H and ¹⁵N in the <b>free</b> (Bm-Aspin alone) and <b>bound</b> (Bm-Aspin+protease complex) states, respectively) in ppm, observed due to NMR chemical shift perturbations in Bm-Aspin with the addition of proteases, (Bm-Aspin+Pepsin in blue, Bm-Aspin+Renin in red, Bm-Aspin+Cathepsin-E in green, Bm-Aspin+Cathepsin-D in magenta), observed for the following 10 residues: G16, G22, G82, G169, G190, A192, A204, A213, I214, and Y215.</p

Sequential residue connectivities using triple resonance NMR strip plot.

<p>Plot showing the strips of four triple resonance NMR spectra of Bm-Aspin in the following order: HNCOCA, HNCA, CBCACONH and HNCACB. The sequential ¹³Cα connectivities for the residues' stretch T189-V194 are indicated by the continuous line drawn between the adjacent Cα.</p

Bm-Aspin ¹⁵N HSQC spectra at varying concentrations of SDS.

<p>Comparison of Bm-Aspin ¹⁵N HSQC spectra in the presence of varying concentrations of SDS; (<b>i</b>) 50 mM SDS, (ii) 100 mM SDS, (iii) 150 mM SDS, and (iv) 200 mM SDS. The inset box indicates the well resolved glycine peaks for comparison to identify the optimum solvent conditions for a well behaved NMR spectrum.</p

Synthesis and Biological Evaluation of Calothrixins B and their Deoxygenated Analogues

A series of calothrixin B (<b>2</b>) analogues bearing substituents at the ‘E’ ring and their corresponding deoxygenated quinocarbazoles lacking quinone unit were synthesized. The cytotoxicities of calothrixins <b>1</b>, <b>2</b>, and <b>15b</b>–<b>p</b> and quinocarbazole analogues were investigated against nine cancer cell lines. The quinocarbazoles <b>21a</b> and <b>25a</b> inhibited the catalytic activity of human topoisomerase II. The plasmid DNA cleavage abilities of calothrixins <b>1</b>, <b>2</b>, and <b>15b</b>–<b>p</b> identified compound <b>15h</b> causing DNA cleavage comparable to that of calothrixin A (<b>1</b>). Calothrixin A (<b>1</b>), 3-fluorocalothrixin <b>15h</b> and 4-fluoroquinocarbazole <b>21b</b> induced extensive DNA damage followed by apoptotic cell death. Spectral and plasmid unwinding studies demonstrated an intercalative mode of binding for quinocarbazoles. We identified two promising drug candidates, the 3-fluorocalothrixin B <b>15h</b> with low toxicity in animal model and its deoxygenated derivative 4-fluoroquinocarbazole <b>21b</b> as having potent cytotoxicity against NCI-H460 cell line with a GI₅₀ of 1 nM