Background:
The binding potency of M4K compounds to ALK2 has been assayed in biochemical assay and cellular assays in HEK293 or C2C12 myoblast cell lines. However, the potency of ALK2 inhibition by M4K compounds has not determined directly in DIPG patient-derived cell lines. While no major deviation from existing assay data is expected, direct experimental evidence is essential.
Experimental design:
DIPG cells will be trypsinized and resuspended in TSM-base medium without any growth factor for one-hour starvation. Subsequently, equal volume of TSM-base with 2X Activin A (200ng/mL) and M4K compounds or DMSO vehicle control will be added. After one-hour treatment, the cells will be pelleted and lysed in buffer with protease and phosphatase inhibitors for Western Blot analysis.
Results:
Chemical structures of the compounds tested
Figure 1. Inhibition of ALK2 phosphorylation of SMAD1/5 by M4K lead compounds in SU-DIPG-IV DIPG patient-derived cell line. Phosphorylated proteins were detected in Western Blot using specific antibodies.
Figure 2. Inhibition of ALK2 phosphorylation of SMAD1/5 by M4K lead compounds in HSJD-DIPG-007 DIPG patient-derived cell line. Phosphorylated proteins were detected in Western Blot using specific antibodies.
Figure 3. Inhibition of ALK2 phosphorylation of SMAD1/5 by M4K lead compounds in HSJD-DIPG-018 DIPG patient-derived cell line. Phosphorylated proteins were detected in Western Blot using specific antibodies.
Figure 4. Inhibition of ALK2 phosphorylation of SMAD1/5 by M4K lead compounds in SU-DIPG-XXI DIPG patient-derived cell line. Phosphorylated proteins were detected in Western Blot using specific antibodies.
Findings:
1) All M4K lead compounds inhibited ALK2 kinase activity in DIPG cells.
2) M4K3078 appeared to be less potent even though its potency was highest when measured in biochemical assay and cellular target engagement assay in HEK293. Additional experiments are necessary to verify this result before hypothesis of the underlying reason can be developed.
In a nut shell:
On target ALK2 inhibition in DIPG patient-derived cell lines by M4K lead compounds had been successfully verified using Western Blot.
For detailed experimental information, please refer to my Zenodo post.