In vitro activity: In the screening study, ML390 was identified as the most potent compound against the engineered ERHOX-GFP cell line. Moreover, the addition of uridine to the cell culture media could abrogate the differentiation effects of ML390, demonstrating further evidences that ML390’ effects were due to their inhibition of DHODH-catalyzed pyrimidine synthesis. In addition, ML390 was found to be not able to inhibit DHODH in the P. falciparum parasite, which is the causative agent of malaria. Furthermore, the X-ray structure indicated that the binding of ML390 to the enzyme might be increased by modifying ML390 with a ring in its central portion to lock the molecule into its binding conformation with the amide substituents.

Kinase Assay: ML390 is a novel potent inhibitor of human DHODH (Dihydroorotate dehydrogenase) that induces differentiation in acute myeloid leukemia (AML) with EC50 of 1.8μM, 8.8μM, 6.5μM, and 0.56μM in ER-HOX-GFP, U937, THP-1 cells and DHODH enzyme, respectively. ML390 was identified as the most potent compound against the engineered ERHOX-GFP cell line. Moreover, the addition of uridine to the cell culture media could abrogate the differentiation effects of ML390, demonstrating further evidences that ML390’ effects were due to their inhibition of DHODH-catalyzed pyrimidine synthesis. Therefore, ML390 may offer insight into the mechanism of overcoming differentiation arrest, and has the potential to used as a treatment for patients with AML.

Cell Assay: Lys-GFP-ER-HoxA9 cells were treated in culture with 10 μM ML390 for 48 hr. The cells were washed three times in normal saline, and metabolites were extracted into 80% ice-cold methanol. Metabolites were analyzed. Negative ionization mode analyses of polar metabolites were acquired using an LC-MS system comprising an Acquity UPLC System (Waters) and a 5500 QTrap triple quadrupole mass spectrometer (AB SCIEX). Samples for negative ion mode analyses of polar metabolites were achieved using the HILIC (hydrophilic interaction chromatography) method under basic conditions as described previously, and MS data were acquired over m/z 70-750. MS data were processed using MultiQuant.