Discovery of imidazole-based GSK-3 β inhibitors for transdifferentiation of human mesenchymal stem cells to neurons: A potential single-molecule neurotherapeutic foresight

Abstract

The transdifferentiation of human mesenchymal stem cells (hMSC) to functional neurons is crucial for the development of future neuro-regenerative therapeutics. Currently, transdifferentiation of hMSCs to neurons requires a "chemical cocktail" along with neural growth factors. The role of the individual molecules present in a "chemical cocktail" is poorly understood and may cause unwanted toxicity or adverse effects. Toward, this goal, we have showcased the discovery of an imidazole-based "single-molecule" transdifferentiation initiator SG-145C. This discovery was achieved via screening of a small molecule library through extensive in silico studies to shortlist the best-fitting molecules. This discovery evolved through a careful selection to target Glycogen synthase kinase-3β (GSK-3β), which is one of the important proteins responsible for neurogenesis. Rigorous computational experiments, as well as extensive biological assays, confirmed that SG-145C has significant potential to transdifferentiate hMSCs to neurons. Interestingly, our results suggest that SG-145C can inhibit the proteasomal degradation of phosphorylated β-catenin, in turn promoting transdifferentiation of hMSCs into neurons via the Wnt pathway.

Keywords: GSK-3β; hMSC; imidazole; neurodegeneration; transdifferentiation; β-III tubulin; β-catenin.

Figure 1

Establishment of the pharmacophoric model with GSK-3β and screening of lead molecules. (A) Projecting imidazole moiety into the GSK-3β pocket to modulate the scaffold at defined positions. (B) Among the top 10 screened molecule SG-145C stands out to be the best. (C) 3D-Pharmacophoric model showing a correlation coefficient of 0.915. (D) 3D coordinates of ligands with respect to pharmacophoric interactions (E,F) Pharmacophore mapping with the 3D pharmacophore model depicting SG-145C to be the best-fitted molecule. (G,H) Pharmacophore mapping with the 3D pharmacophore model depicting SG-138C to be the 2nd best-fitted molecule.

Figure 2

MTT and immunocytochemistry assay for identification of SG-145C as a lead molecule. (A) Percentage of cell survival after the treatment with the molecules in an increasing gradient of doses (3.125–100 μM) for 24 h. n ≥ 3 in all data sets and significance was calculated using one-way Anova. (B) MSCs were treated with the small molecules for 7 days and neuron specific markers were observed using β-III Tubulin (AF-488) and Map2 (AF-543) via immunocytochemistry. Images were captured in 20× magnification where the scale bar corresponds to 70 μm. Significance was calculated using students unpaired t-test *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Evaluation of optimum dose of SG-145C for transdifferentiation. An increasing gradient of SG-145C was used to treat MSCs and immunofluorescence study of neuronal markers β-III Tubulin (AF-488) and Map2 (AF-543) performed to elucidate the optimal concentration. Images were captured in 20× magnification where the scale bar corresponds to 70 μm.

Figure 4

Characterization of the differentiated neurons. (A). Immunoblot analysis to check the expression of neuronal marker proteins at various time points after treatment with 5 μM SG-145C followed by its (B) densitometric analysis. (C) qPCR analysis reveals the expression pattern of the neuronal marker genes in the transdifferentiated neurons after the treatment with SG-145C. (D) FACS analysis reveals the reduction in mesenchymal stem cell marker CD73 after differentiation. Significance was calculated using one-way Anova for Western Blots and student’s unpaired t-test for qPCR. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Cell proliferation and survival. (A) BrdU proliferation assay to assess the actively proliferating cells after the treatment of SG-145C in a dose-dependent manner. n > 3 in all data sets and significance was calculated using one-way Anova. (B) Immunocytochemistry to check the BrdU positive cells. Images were captured in 20X magnification where the scale bar corresponds to 70 μm. (C) Immunoblots of apoptotic and survival genes to check the homeostasis status of differentiated neurons followed by its (D) densitometric analysis. (E) Live-Dead cell assay using Calcein AM and PI. Images were captured in 40X magnification where the scale bar corresponds to 20 μm. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

GSK-3β Inhibition by SG-145C results in increased β-Catenin levels. (A,B) HOMO-LUMO isosurface showing localization of orbitals. (C) Kinase-Glo luminescent assay to check the inhibition of GSK-3β revealed a direct dose-dependent inhibition. (D) Immunocytochemistry assay reveals the expression of GSK-3β (AF-488) and β-Catenin (AF-647) in differentiated neurons. Images were captured in 40× magnification where the scale bar corresponds to 20 μm. (E,F) Immunoblot and densitometric analysis and (G) qPCR to check the expression of two key regulators of Wnt Pathway, GSK-3β, and β-Catenin in response to SG-145C treatment. n = 3 for all sets of experiments and significance was calculated using student’s unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7

SG-145C induced trans-differentiation proceeds via the Wnt pathway. (A) qPCR and (B,C) immunoblot analysis comparing the expression of neuron-specific genes in response to SG-145C in combination with Wnt pathway inhibitor NSC668036 and commercially available GSK-3β inhibitor CHIR99021. (D) ICC staining was performed to compare the level of trans-differentiation in response to the mentioned inhibitors in combination with SG-145C. β-III Tubulin (AF-488) was used as neuronal marker proteins for visualization. Images were captured in 20X magnification where the scale bar corresponds to 70 µm. n = 3 for all sets of experiments and significance was calculated using one-way Anova *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

Design and development of Imidazole-based small molecule library which has the ability to transdifferentiate Human Mesenchymal Stem Cells into neurons by altering the components of canonical Wnt pathway that is inhibition of activation of GSK-3β. Because of the inhibition of GSK-3β, its activity of phosphorylating and degrading β-catenin is also inhibited. As a result of which β-catenin is stabilized, which allows it to translocate to the nucleus and transcribe the downstream neuronal genes after interacting with the TCF/LEF family of transcription factors.