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

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.<b> </b>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<i>,</i> 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.

人间充质干细胞（human mesenchymal stem cells, hMSCs）具有旺盛的增殖能力，可自我更新，且具备向结缔组织谱系细胞分化的潜能。上述特性使其成为组织工程应用的潜在候选细胞类型，同时也可作为药物调控分化过程的体内潜在靶点。
本研究旨在筛选可在体外促进人间充质干细胞成骨分化的一种或多种小分子物质。本研究采用的研究策略为：首先基于配体，对与此前报道的成骨诱导性化合物普马福明（purmorphamine）结构相似的物质开展虚拟筛选；随后在骨髓分离的人间充质干细胞中，对筛选得到的类似物进行体外验证。本研究通过检测成骨分化标志物的蛋白与基因表达水平，以及细胞的细胞外基质（extracellular matrix, ECM）矿化程度，评估了多种普马福明类似物的成骨诱导能力。与对照组相比，经两种候选物质或普马福明处理的细胞，其碱性磷酸酶（alkaline phosphatase, ALP）活性水平均显著升高。在成骨分化培养5周后，其余普马福明类似物处理组的细胞外基质钙沉积量，均高于普马福明组与对照组。上述可有效促进成骨分化的活性物质，有望作为靶向并增强间充质干细胞成骨分化的治疗手段，用于骨相关疾病或骨缺损的临床治疗，其应用场景可覆盖体外实验与体内研究。此外，本研究验证了将基于配体的虚拟筛选与体外筛选相结合、以人类成体干细胞作为药物发现潜在策略的研究思路的可行性。