Human CSF1R Stable Cell Line - Ba/F3

Colony-stimulating factor-1 receptor (CSF1R) is a receptor tyrosine kinase that controls the differentiation and maintenance of most tissue-resident macrophages. Inhibition of CSF1R has been considered a potential therapeutic approach for a variety of human diseases. Here, researchers report the synthesis, development, and structure-activity relationship of a series of highly selective pyrrolo[2,3-d]pyrimidine compounds. These compounds exhibit sub-nanomolar inhibitory activity against this receptor and excellent selectivity for other kinases in the platelet-derived growth factor receptor (PDGFR) family. The crystal structures of the protein and compound 23 indicate that the protein's binding conformation is similar to DFG-out. Cellular activity analysis, pharmacokinetic analysis, and in vivo stability studies were performed on the most promising compounds in this series, suggesting their potential therapeutic value for various diseases. Furthermore, these compounds primarily inhibit the autoinhibitory form of the receptor, contrasting with the behavior of pexidartinib, which may explain the excellent selectivity of these structures.

Here, researchers performed two cell-based assays on some compounds: one using genetically engineered Ba/F3 cells dependent on CSF1R activation, and the other using mouse bone marrow-derived macrophages. Disappointingly, the potency of these compounds showed a low correlation with the potency observed in the CSF1R kinase activity assay. However, in the mouse bone marrow-derived macrophage activity assay, some compounds showed activity comparable to pexidartinib. The Ba/F3 cell activity assay showed superior activity of pexidartinib, while some compounds that performed well in the kinase activity assay showed no activity. Among these new inhibitors, the metabolically unstable 3-pyridyl compound 39 showed the best inhibitory effect on CSF1R-overexpressing Ba/F3 cells. Unfortunately, when interleukin-3 was administered concurrently as a toxicity control, both phenol 36 and 3-pyridyl compound 39 severely affected CSF1R-overexpressing Ba/F3 cell viability. The efflux ratio also clearly indicated that the carboxylic acid group was highly unfavorable, which may also be one of the reasons for their inactivity in the Ba/F3 assay.

Figure 1. Cellular potency of selected compounds toward CSF1R-overexpressing Ba/F3 cells, bone marrow-derived macrophages from mice, osteoclast differentiation, and the corresponding Caco2-Efflux ratio. (Aarhus T I, et al., 2023)