Virtual Ligand-Based Screening Reveals Purmorphamine Analogs with the Capacity to Induce the Osteogenic Differentiation of Human Mesenchymal Stem Cells

Abstract

Human mesenchymal stem cells (hMSCs) have extensive proliferative capacity, are able to self-renew and have the potential to differentiate into cells of the connective tissue lineages. These properties make them a putative cell type for tissue engineering applications, as well as a possible in vivo target for the pharmaceutical modulation of the differentiation processes.The aim of this study was to find one or more small-molecule substances that would enhance the osteogenic differentiation of hMSCs in vitro. The strategy used here was ligand-based virtual screening for substances similar to the previously suggested osteoinductive purmorphamine followed by an in vitro screening of the selected analogs in hMSCs isolated from bone marrow. We investigated the osteoinductive capacity of several purmorphamine analogs by determining the protein and gene expression of markers for osteogenic differentiation as well as the extracellular matrix (ECM) mineralization of these cells. Treatment with two candidate substances or purmorphamine resulted in increased levels of alkaline phosphatase (ALP) activity compared to the control. Other purmorphamine analogs demonstrated higher calcium deposition in the ECM after 5 weeks of osteogenic differentiation, compared to both purmorphamine and the control condition. The resulting substances, which had positive effects on the osteogenic differentiation, are promising as possible modes of treatment for bone-related diseases or defects that target and enhance the osteogenic differentiation of MSCs, in vitro or in vivo. Furthermore, the concept of combining the virtual ligand-based screening method with in vitro screening, using human adult stem cells as a possible strategy for drug discovery, is demonstrated.

Effect of Purmorphamine on Osteogenic Differentiation of Human Mesenchymal Stem Cells in a Three-Dimensional Dynamic Culture System

“…However, similar to our previous study on the 2D culture system, 14 there was no difference in mineralization of the extracellular matrix with or without purmorphamine. As with the current study, Grane´li et al 16 showed that calcium deposition in the extracellular matrix was more or less the same with or without purmorphamine during osteogenic differentiation of hMSCs and this molecule did not increase the quantity of the mineralized matrix.…”

Hedgehog Pathway Agonism: Therapeutic Potential and Small‐Molecule Development

“…Studies performed in human mesenchymal stem cells (hMSCs) have also demonstrated that purmorphamine treatment (2 μ M ) activates Hh signaling and initiates differentiation into osteoblasts 83. While traditional SAR studies based around the purmorphamine scaffold have not been disclosed, a recent report described a ligand‐based virtual screen designed to identify structurally related small molecules with similar osteoinductive properties 84. Three analogues, 38 – 40 (see Figure 7 and Table 2), exhibited promising preliminary osteoinductive properties in human mesenchymal stem cells; however, these results did not appear to result from direct activation of Hh signaling.…”