Assessing compound binding to the Eg5 motor domain using a thermal shift assay

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Eg5 kinesin is a mitotic motor protein and a validated cancer therapeutic target.
• Inhibition of Eg5 leads to cell cycle arrest, making it a focus for anti-cancer drug development.

Purpose of the Study:

• To investigate the interaction of pyrrolotriazine-4-one inhibitors with the Eg5 motor domain, particularly in the presence of adenosine 5'-diphosphate (ADP).
• To evaluate the utility of the thermal shift assay (TSA) for assessing direct binding and potency of Eg5 inhibitors.

Main Methods:

• Circular dichroism (CD) and isothermal titration calorimetry (ITC) were used to study inhibitor binding to Eg5.
• Thermal shift assay (TSA) was employed to measure the thermal melting temperature (T(m)) of Eg5 in the presence of inhibitors and ADP.
• Microtubule-dependent ATPase and cell-based cytotoxicity assays were performed to determine inhibitor potency (IC50 values).

Main Results:

• Pyrrolotriazine-4-one inhibitors demonstrated enhanced binding to Eg5 in the presence of ADP.
• TSA showed an increase in Eg5 T(m) of up to 7°C for potent inhibitors (IC50 values of 60-130 nM).
• Inhibitor potency correlated with the thermal stability enhancement of ADP-Eg5, and TSA confirmed direct binding across various chemotypes.

Conclusions:

• The thermal shift assay is a robust biophysical method for evaluating direct binding of Eg5 inhibitors.
• TSA complements traditional enzymatic and cell-based assays, providing a reliable alternative for determining inhibitor potency.
• This study validates TSA for screening and characterizing potential anti-cancer agents targeting Eg5.