Small-Molecule Cdc25A Inhibitors Protect Neuronal Cells from Death Evoked by NGF Deprivation and 6‑Hydroxydopamine

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases that are presently incurable. There have been reports of aberrant activation of cell cycle pathways in neurodegenerative diseases. Previously, we have found that Cdc25A is activated in models of neurodegenerative diseases, including AD and PD. In the present study, we have synthesized a small library of molecules targeting Cdc25A and tested their neuroprotective potential in cellular models of neurodegeneration. The Buchwald reaction and amide coupling were crucial steps in synthesizing the Cdc25A-targeting molecules. Several of these small-molecule inhibitors significantly prevented neuronal cell death induced by nerve growth factor (NGF) deprivation as well as 6-hydroxydopamine (6-OHDA) treatment. Lack of NGF signaling leads to neuron death during development and has been associated with AD pathogenesis. The NGF receptor TrkA has been reported to be downregulated at the early stages of AD, and its reduction is linked to cognitive failure. 6-OHDA, a PD mimic, is a highly oxidizable dopamine analogue that can be taken up by the dopamine transporters in catecholaminergic neurons and can induce cell death by reactive oxygen species (ROS) generation. Some of our newly synthesized molecules inhibit Cdc25A phosphatase activity, block loss of mitochondrial activity, and inhibit caspase-3 activation caused by NGF deprivation and 6-OHDA. Hence, it may be proposed that Cdc25A inhibition could be a therapeutic possibility for neurodegenerative diseases and these Cdc25A inhibitors could be effective treatments for AD and PD

Cdk4 inhibitors protect primary rat cortical neurons against Aβ induced death.

<p>Primary rat cortical neurons (5DIV) were exposed to Aβ (1.5 µM) in presence and absence of Cdk4 inhibitors (Cdk5I1 & 8A) for 48 h. Percentage of viable cells were estimated by intact nuclear counting assay. (A) Graphical representation of percentage of viable cells. Data represented as mean ± SD of two independent experiments. The asterisks denote statistically significant differences between indicated class:*p<0.05. (B) Representative phrase contrast micrographs show retention of neuronal processes in presence of Aβ by Cdk4 inhibitors.</p

Cdk4 inhibitors block phosphorylation of Rb protein in response to NGF deprivation in neuronal PC12 cells.

<p>Cells were subjected to NGF deprivation in presence and absence of Cdk4 inhibitors for 20(A) Representative images show that Cdk4 inhibitors (Cdk4I1 & 8A) blocks NGF deprivation induced elevation of phospho-Rb level. (B) Quantification of the immunostained cells. Intensity of 10–15 cells from each field of 5 random fields was quantified using NIH ImageJ. Data represented as mean ± SEM of 3 independent experiments.</p

The structures of synthesized Cdk4 inhibitors.

<p>The structures of synthesized Cdk4 inhibitors.</p

Specificity of Cdk4 inhibitors.

<p>(A) Kinase assay of Cdk4 (10 ng) was performed with Cdk4 inhibitors 8A and 8B at different doses as indicated. Data represented as mean ± SEM of 3 independent experiments. The asterisks denote statistically significant differences between indicated classes:*p<0.05. (B) Kinase assay was performed with Cdk4 inhibitors 8A and 8B (10 µM each) with Cdk4, Cdk2 and Cdk5 (10 ng each). Data represented as mean ± STDEV of 2 independent experiments performed in duplicate. The asterisks denote statistically significant differences between indicated classes:*p<0.05. (C) Differentiated PC12 cells were subjected to NGF deprivation in presence or absence inhibitors (8A, 5 µM and 8B, 1 µM) for 8 h. The cells were lysed and equal amount of protein from cell lysate were subjected to immunoprecipitation with Cdk4 antibody. The immunoprecipitated protein was then subjected to kinase assay. Data represented as mean ± SEM of 3 independent experiments. The asterisks denote statistically significant differences between indicated classes:*p<0.05.</p

General scheme for synthesis of compound 13.

<p>Reagent and conditions: (a) Conc. H₂SO₄, MeOH, 0°C to rt, 6 hrs. (b) palladium(II) acetate (0.05 equiv.), xantphos (0.1 equiv.) and cesium carbonate (3 equiv.), 80°C, 4 hrs. (c) Lithium hydroxide (1.5 equiv.), MeOH-water (5∶1), 0°C to rt, 1.5 hrs. (d) EDC.HCl (1.5 equiv.), HOBT (1.2 equiv.), TEA (3 equiv.), 0°C to rt, 7.0 hrs. (e) 4(M) HCl in 1,4 dioxane, 0°C to rt, 2.0 hrs.</p

Commercial inhibitors of Cdk4 protect neuronally differentiated PC12 cells against NGF deprivation.

<p>Cells were subjected to NGF deprivation in presence and absence of commercially available small molecule inhibitors of Cdk4 for 20(A) graphical representation of percentage of viable cells following NGF deprivation in presence of indicated concentrations of commercially available Cdk4 inhibitor (Cdk4I1). Data represented as mean ± SEM of three independent experiments performed in duplicates. Theasterisks and hash denote statistically significant differences between indicated class:#p<0.05; *p<0.01. (B) Graphical representation of percentage of viable cells following NGF deprivation in presence of indicated concentrations of commercially available Cdk4 inhibitor Cdk4I2. Data represented as mean ± SEM of three independent experiments performed in duplicates. The asterisks denote statistically significant differences between indicated class:*p<0.01. (C) Representative phase contrast micrographs show retention of neuronal processes even after NGF deprivation in presence of commercially available Cdk4 inhibitors (Cdk4I1 & Cdk4I2).</p

Synthesized inhibitors of Cdk4 protect neuronally differentiated PC12 cells against NGF deprivation.

<p>Cells were subjected to NGF deprivation in presence and absence of synthesized small molecule inhibitors of Cdk4 for 20(A) Graphical representation of percentage of viable cells following NGF deprivation in presence of synthesized small molecule Cdk4 inhibitors. Doses of each molecule are 8A: 5 µM; 8B: 1 µM; 8C: 1 µM; 8D: 5 µM; 12E: 1 µM; 12F: 5 µM; 12G: 1 µM; 13H: 1 µM; 12I: 1 µM; 13J: 1 µM. Data represented as mean ± SEM of three independent experiments performed in duplicates. The asterisks denote statistically significant differences between indicated class: **p<0.01, *p<0.05. (B) Dose kinetics of synthesized small molecule inhibitor 8A in differentiated PC12 cells in presence and absence of NGF. Data represented as mean ± SEM of three independent experiments. The asterisks denote statistically significant differences between indicated classes: *p<0.05. (C) Dose kinetics of synthesized small molecule inhibitor 8B in differentiated PC12 cells in presence and absence of NGF. Data represented as mean ± SEM of three independent experiments. The asterisks denote statistically significant differences between indicated classes: *p<0.05. (D) Representative phase contrast micrographs show retention of neuronal processes even after NGF deprivation in presence of synthesized Cdk4 inhibitors (8A & 13H).</p

Synthesized inhibitors of Cdk4 protect primary sympathetic neurons against NGF deprivation.

<p>Primary cultures of sympathetic neurons (5DIV) were subjected to NGF deprivation in presence and absence of synthesized small molecule inhibitors of Cdk4 for overnight. (A) Representative phase contrast micrographs show retention of neuronal processes even after NGF deprivation in presence of synthesized Cdk4 inhibitors (8A & 8B). (B) Graphical representation of percentage of viable cells following NGF deprivation in presence of synthesized small molecule Cdk4 inhibitors (8A: 5 µM; 8B: 1 µM). Data represented as mean ± SD of two independent experiments performed in duplicates. The asterisks denote statistically significant differences between indicated class: *p<0.05.</p

Cdk4 inhibitors block elevation of Bim and active caspase3 levels in response to NGF deprivation in neuronal PC12 cells.

<p>Cells were subjected to NGF deprivation in presence and absence of Cdk4 inhibitors for 20(A) Representative immunoblot showing reduction in NGF deprivation induced Bim level by Cdk4 inhibitors (Cdk4I1 & Cdk4I2). (B) Representative images of immunostained cells. Result shows Cdk4 inhibitors (Cdk4I1 & 8A) effectively blocks NGF deprivation induced elevation of active caspase3 level.</p