A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent.

Area of Science:

• Oncology
• Molecular Biology
• Pharmacology

Background:

• Rigosertib is a novel anticancer agent investigated for its RAS-mimetic properties.
• A recent study suggested rigosertib's mechanism of action involves microtubule binding.
• The source of rigosertib used in prior studies was a commercial vendor.

Purpose of the Study:

• To compare the purity of clinical-grade and commercially sourced rigosertib.
• To investigate the potential impact of impurities on rigosertib's observed mechanism of action.
• To clarify rigosertib's interaction with tubulin and cellular response in different cell lines.

Main Methods:

• High-performance liquid chromatography (HPLC) was used to assess rigosertib purity.
• Tubulin polymerization assays were performed.
• Cell proliferation and phenotype assays were conducted using wild-type and mutant β-tubulin cell lines.

Main Results:

• Commercially sourced rigosertib contained approximately 5% of ON01500, a potent tubulin polymerization inhibitor.
• Clinical-grade rigosertib, free of ON01500, did not exhibit tubulin-binding activity.
• Cells with mutant β-tubulin showed reduced proliferation but developed a senescence-like phenotype, not resistance.

Conclusions:

• The microtubule-binding activity previously attributed to rigosertib is likely due to the ON01500 impurity.
• Clinical-grade rigosertib's mechanism of action is distinct from tubulin polymerization inhibition.
• Short-term cultures may misinterpret rigosertib-induced senescence as resistance in mutant cell lines.