Related Experiment Video
Updated: Mar 3, 2026
Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
Published on: October 23, 2019
Identification of Human UMP/CMP Kinase 1 as Doxorubicin Binding Target Using Protein Microarray
Shuxian Chen1, Xu Wang1, Xianghui Ye1
11 School of Pharmaceutical Science, Jiangnan University, Wuxi, Jiangsu, China.
Abstract:
Doxorubicin (DOX) is a leading anthracycline drug with exceptional efficacy; however, little is known about the molecular mechanisms of its side effects, which include heart muscle damage, noncancerous cell death, and drug resistance. A total of 17,950 human proteins expressed in HEK293 cells were screened and yielded 14 hits. Competitive and binding experiments further verified the binding of DOX to UMP/CMP kinase 1 (CMPK1), and microscale thermophoresis showed that DOX binds to CMPK1 with a Kd of 1216 nM. In addition, we observed that the binding of DOX to CMPK1 activated the phosphorylation of CMP, dCMP, and UMP. A significant activation was observed at the concentration of 30 µM DOX and reached plateau at the concentration of DOX 30 µM, 150 µM, and 100 µM, respectively. DOX would add up stimulation of CMPK1 by DTT and overcome inhibition of CMPK1 by NaF, EDTA. In summary, we showed that DOX might bind to the nonactive site of CMPK1 and regulate its activity with magnesium.
Insights
Doxorubicin (DOX) binds to UMP/CMP kinase 1 (CMPK1), potentially explaining its side effects. This interaction activates CMPK1, influencing cellular processes and drug resistance mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Doxorubicin (DOX) is a crucial anthracycline chemotherapy agent.
- The molecular mechanisms underlying DOX's side effects, including cardiotoxicity, apoptosis, and drug resistance, remain largely uncharacterized.
- Understanding DOX's off-target interactions is vital for mitigating adverse effects.
Purpose of the Study:
- To identify novel molecular targets of Doxorubicin (DOX) in human cells.
- To elucidate the binding interaction between DOX and its identified targets.
- To investigate the functional consequences of DOX binding to its targets.
Main Methods:
- High-throughput screening of 17,950 human proteins expressed in HEK293 cells to identify DOX-binding proteins.
- Competitive and binding assays to confirm DOX-CMPK1 interaction.
- Microscale thermophoresis to quantify the binding affinity (Kd) of DOX to CMPK1.
- Enzyme activity assays to assess the effect of DOX on CMPK1 phosphorylation.
Main Results:
- Fourteen human proteins were identified as potential DOX binders, with UMP/CMP kinase 1 (CMPK1) being a key hit.
- Doxorubicin (DOX) binds to CMPK1 with a dissociation constant (Kd) of 1216 nM.
- DOX binding activates CMPK1-mediated phosphorylation of CMP, dCMP, and UMP in a dose-dependent manner.
- DOX modulates CMPK1 activity, enhancing stimulation by DTT and overcoming inhibition by NaF and EDTA, suggesting interaction at a non-active site involving magnesium.
Conclusions:
- Doxorubicin (DOX) directly binds to UMP/CMP kinase 1 (CMPK1).
- DOX binding to CMPK1 activates its enzymatic activity, potentially contributing to DOX's known side effects and resistance mechanisms.
- The findings suggest a novel mechanism for DOX's action and highlight CMPK1 as a potential target for therapeutic intervention.
